Ex Parte Higgins

Patent Trial and Appeal Board

Feb 28, 2018

14210884 (P.T.A.B. Feb. 28, 2018)

United States Patent and Trademark Office
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O.Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
14/210,884 03/14/2014 Ross HIGGINS 114784-8030.US01 2324
113677 7590 03/02/2018
Perkins Pnie T T P - PHT freneral
EXAMINER
PO Box 1247 MARTINELL, JAMES
Seattle, WA 98111-1247
ART UNIT PAPER NUMBER
1634
NOTIFICATION DATE DELIVERY MODE
03/02/2018 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
patentprocurement @perkinscoie. com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte ROSS HIGGINS
Appeal 2017-004573
Application 14/210,884
Technology Center 1600
Before ULRIKE W. JENKS, TIMOTHY G. MAJORS, and
DAVID COTTA, Administrative Patent Judges.
JENKS, Administrative Patent Judge.
DECISION ON APPEAL
Pursuant to 35 U.S.C. § 134(a), Appellant1 appeals from the
Examiner’s decision to reject claims directed to a method of analyzing DNA
as indefinite and obvious. We have jurisdiction under 35 U.S.C. § 6(b).
We AFFIRM.
STATEMENT OF THE CASE
Claims 1-5 and 8-16 are on appeal, and can be found in the Claims
Appendix of the Appeal Brief. Claim 1 representative of the claims on
appeal, and reads as follows:
1 Appellant is the Applicant, True Health Diagnostics, LLC, which,
according to the Brief, is the real party in interest. Appeal Br. 2.
Appeal 2017-004573
Application 14/210,884
1. A method for analyzing genomic DNA, the method
comprising:
introducing a plurality of samples comprising human
cells into individual vessels in each of a plurality of multi­
vessel well plates, each of said plurality of multi-vessel well
plates comprising 96-sample multi-vessel well plates;
lysing at least a subset of the human cells in the plurality
of samples without a heating step;
isolating DNA in the at least a subset of lysed human
cells by introducing into individual vessels a plurality of
paramagnetic beads;
analyzing the isolated DNA to identify one or more
single nucleotide polymorphisms (SNPs ), wherein the lysing,
isolating, and analyzing steps are performed in parallel for each
of the plurality of samples; and
performing the introducing, lysing, isolating, and
analyzing steps for at least 1000 samples in a 24-hour period,
wherein each of the introducing, lysing, isolating, and
analyzing steps is performed in a single liquid sample handling
instrument.
Appeal Br. 17 (Claims Appendix) (emphasis added).
Appellant requests review of the following grounds of rejection made by
the Examiner:
I. claim 8 under 35 U.S.C. § 112, second paragraph;
II. claims 1-3 and 8-16 under 35 U.S.C. § 103(a) as unpatentable over
DYNAL2 in view of admitted prior art (see Spec. 3-17); and
III. claims 4 and 5 under 35 U.S.C. § 103(a) as unpatentable over
DYNAL in view of admitted prior art (see Spec. 3-17) and further
in view of Weindel.3
2 DYNAL® Bioscience Catalogue 1999, pages 3, 44, and 45 (“DYNAL”).
3 Weindel et al., US 8,129,118 B2, issued Mar. 6, 2012 (“Weindel”).
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Appeal 2017-004573
Application 14/210,884
I Indefiniteness
The Examiner has rejected claim 8 as being incomplete because the
claim depends on a canceled claim. Final Act. 2. Appellant intends to
amend the claim once allowable subject matter is indicated. See Appeal
Br. 16. The Examiner acknowledges this intent (see Ans. 6), but has not
otherwise withdrawn this rejection, therefore, we affirm the rejection for the
reason set out by the Examiner in the Final Office Action. See Final Act. 2.4
I. Obviousness over DYNAL
Appellant contends that the Examiner has not met the burden of
presenting a prima facie case “because the scope and content of the prior art
is not correctly identified, [and] differences between the claims and the prior
art are not identified.” Appeal Br. 10.
We are not persuaded by Appellant’s contention. “[A]ll that is
required of the office to meet its prima facie burden of production is to set
forth the statutory basis of the rejection and the reference or references relied
upon in a sufficiently articulate and informative manner as to meet the notice
requirement of § 132.” In re Jung, 637 F.3d 1356, 1363 (Fed. Cir. 2011).
Section 132, in turn, requires notice to the applicant sufficient to inform him
of the reasons for rejection, “together with such information and references
as may be useful in judging of the propriety of continuing the prosecution of
his application.” 35 U.S.C. § 132.
Here, the Examiner notified Appellant that the claims were being
rejected as unpatentable under 35 U.S.C. § 103(a) and cited three pages from
4 Final Office Action mailed Oct. 8, 2015 (“Final Act.”).
3
Appeal 2017-004573
Application 14/210,884
the DYNAL product catalog that form the basis for the conclusion of
obviousness for the method of lysing cells from a blood sample and isolating
DNA. Final Act. 2. The Examiner’s rejection satisfies the notice
requirement of § 132, and, therefore, meets the burden of establishing a
prima facie case of unpatentability.
DYNAL teaches an automated, high-throughput system for isolation
of PCR-ready genomic DNA from blood. DYNAL 44. A flow diagram of
DYNAL’s method is shown in the figure below:
4
Appeal 2017-004573
Application 14/210,884
Blood
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DYNAL’s figure, reproduced above, shows a flow diagram that takes a 96
well plate, subjects the samples to a lysis step, adsorbs DNA onto the surface
of the Dynabeads, washing, and resuspension of the Dynabead-DNA
complex with or without a further eluting step to arrive at a PCR ready DNA
sample. Id. “This automated 96-well format allows simultaneous isolation
of 96 PCR-ready DNA samples in 80 minutes.” Id.
5
Appeal 2017-004573
Application 14/210,884
Appellant contends that the Examiner has not identified any teaching
or suggestion of motivation to combine the references (Appeal Br. 11), and
there is no “showing that there is some teaching, suggestion, or knowledge
generally available that the [PCR] analysis could be conducted within the
same experiment.” Appeal Br. 12. Appellant further contends that “the
Specification discloses the general understanding in the field to require a
heating step to lyse cells, for example, at paragraph [0014]-[0015].” Id.
at 13. “DYNAL teaches isolation of PCR-ready DNA, but is silent as to
lysing at room temperature without any heating.” Id.
We are not persuaded by Appellant’s contention. “The combination
of familiar elements according to known methods is likely to be obvious
when it does no more than yield predictable results.” KSR Int 7 Co. v.
Teleflex Inc., 550 U.S. 398, 416 (2007). A central question in analyzing
obviousness is “whether the improvement is more than the predictable use of
prior art elements according to their established functions.” KSR, 550 U.S.
at 417. Here, the Specification acknowledges that at the time of the
invention there were multiple known methods for lysing cells to obtain
DNA, and the DNA is then subjected to further analysis according to known
methods. See Spec. 11-14. In making the rejection, the Examiner relies
on the teachings of the various cell lysis protocols set out in the background
section of Specification, these protocols include alkaline lysis carried out at
room temperature as well as proteinase K digestion carried out at elevated
temperatures. See Ans. 2—4; see Spec. 11-14 (citing various references).
The Manual of Patent Examining Procedure (“MPEP”) (9th Ed.,
Rev. 7 (2015)) provides that the background of the invention can include
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Appeal 2017-004573
Application 14/210,884
[a] paragraph(s) describing to the extent practical the state of
the prior art or other information disclosed known to the
applicant, including references to specific prior art or other
information where appropriate. Where applicable, the problems
involved in the prior art or other information disclosed which
are solved by the applicant’s invention should be indicated.
MPEP § 608.01(c)(2). The background knowledge attributable to one of
ordinary skill in the art includes what was admittedly known in the art at the
time of the invention. Constant v. Advanced Micro-Devices Inc., 848 F.2d
1560, 1570 (Fed. Cir. 1988) (“A statement in a patent that something is in
the prior art is binding on the applicant and patentee for determinations of
anticipation and obviousness”); In reNomiya, 509 F.2d 566, 570-71 (CCPA
1975) (using the admitted prior art in applicant’s Specification to determine
the patentability of a claimed invention); In re Font, 675 F.2d 297, 301
(CCPA 1982) (“[i]t is not unfair or contrary to the policy of the patent
system that appellants’ invention be judged on obviousness against their
actual contribution to the art”) (footnote omitted). The above cited cases
explain that the teachings in the Specification that are recognized as
establishing what was known in the art at the time of the invention can
reasonably be relied upon in making patentability assessments.
Here, the Examiner relies on the disclosure in the background of the
Specification to establish what was known in the area of cell lysis and DNA
analysis at the time of the invention and not matters known and identified
only by Appellant. Specifically, the Examiner relies on knowledge in the art
that lysing cells to obtain DNA for further analysis was known. Pessara5
cited in paragraph 11 of the Specification teaches DNA preparation using
5 Pessara et al., US 2004/0265855 Al, published Dec. 30, 2004 (“Pessara”).
7
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Application 14/210,884
alkaline lysis at room temperature. Pessara ^ 124; see Spec. TJ 14 (citing
other alkaline lysis protocols). Thus, Pessara evidences that cell lysis
protocols that occur at room temperature were known. Harvey,6 also cited in
paragraph 11 of the Specification, discloses another cell lysis protocol, this
one relies on a proteinase K at a temperature between 56-65°C. Harvey
9:34-37. Although claim 1 recites that the lysis occurs without a heating
step, the claim does not otherwise limit the type of lysis used. Therefore, we
agree with the Examiner that one of ordinary skill in the art would recognize
that DNA extraction with a lysis buffer is capable of being carried out at a
range of temperatures including room temperature and the choice of which
particular lysis protocol to apply would reasonably be within the grasp of the
ordinary artisan as these various protocols are art recognized known
equivalents. See Ans. 2 (“the inclusion of any or all of the admittedly old
methods and materials being an arbitrary matter of choice”). The flexible
analysis set out by the Supreme Court in KSR recognizes the obviousness of
pursuing known options within the technical grasp of the skilled artisan, e.g.,
known equivalents. See KSR, 550 U.S. at 421. Here, the predictable lysing
methods known in the art and the selection of one that does not require
increased temperature is the ‘“product not of innovation but of ordinary skill
and common sense. See Wm. Wrigley Jr. Co. v. Cadbury Adams USA
LLC, 683 F.3d 1356, 1364-65 (Fed. Cir. 2012) {quotingKSR, 550 U.S. at
421).
Appellant contends that the Examiner’s reliance on DYNAF’s silence
with respect to the lysing temperature as meeting the claimed “no heat
6 Harvey, US 6,423,488 Bl, issued July 23, 2002.
8
Appeal 2017-004573
Application 14/210,884
requirement” is in error; specifically, the explanation of “‘[w]hat is naturally
flowing from the context of the lysis step is that the lysis step is performed at
naturally default or room temperature’ is merely conclusory.” Reply Br. 3
(citation omitted); see also Appeal Br. 12 (“While DYNAL is silent on
temperature of the steps, the fact that a certain result or characteristic may
occur or be present in the prior art is not sufficient to establish the inherency
of that result or characteristic”).
We are not persuaded by Appellant’s contention. We find that the
silence with respect to lysing temperature in DYNAL would reasonably
suggest to the ordinary artisan that any temperature suitable for lysing a
sample to obtain DNA would also be suitable in this method. Therefore, we
agree with the Examiner that based on the disclosure in the background
section of the Specification, specifically, the teaching of Pessara and
paragraph 13 that teaches a room temperature alkaline lysing step to obtain
DNA would reasonably be applicable to the cell lysis step in DYNAL’s
method. Absent evidence to the contrary, we find that room temperature
lysis to obtain DNA is an art recognized equivalent useful for the same
purpose. SeeKSR, 550 U.S. at 421.
Appellant contends that the DYNAL references is insufficient for the
DNA analysis as recited in the claim because “the purpose of the preparation
of the genomic DNA is for use in polymerase chain reactions (PCR).”
Reply Br. 4.
We are not persuaded by Appellant’s contention. Here, the Examiner
relied on DYNAL to teach that the DNA sample obtained with the method
was ready for PCR. PCR allows for the detection of single nucleotide
polymorphism (SNP) in DNA, and detecting any known SNP associated
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Appeal 2017-004573
Application 14/210,884
with disease for diagnostic purposes would have been obvious to one of
ordinary skill at the time the invention was made. See Spec. 5-10. We
agree with the Examiner that DYNAL’s method further instructs the user to
perform additional analysis because it instructs that the sample is now PCR-
ready DNA. See DYNAL 44. Furthermore, the Examiner finds
PCR is routinely used for the analysis of DNA (see also the
instant application at paragraph 0034). Thus, DYNAL®
Bioscience Catalogue clearly discloses an analysis step to be
performed on the DNA isolated. Additionally, the gel
electrophoresis result shown on page 45 of the [DYNAL]
reference can be broadly interpreted as an analytical step on the
isolated DNA.
Ans. 4-5. This known use is further supported in the background section of
the Specification. See Spec. 5-10. On this record, the preponderance of
the evidence supports the Examiner’s conclusion of obviousness with
respect to claim 1. Claims 2, 3, and 8-16 were not separately argued and
fall with claim 1.
Although not argued by Appellant in the Appeal Brief, the Reply brief
asserts that DYNAL does not disclose preforming the analysis on “1000
samples in a 24-hour period . . . and analyzing steps is performed in a single
liquid sample handling instrument.” Reply Br. 4 (emphasis removed).
Appellant presents arguments against the “1000 samples” limitation
and “single liquid sample handling instrument” for the first time in the Reply
Brief. Appellant does not explain what good cause there might be to
consider the new arguments. Appellant’s new arguments are, thus, untimely
and accordingly, have not been considered. See Ex parte Borden, 93
USPQ2d 1473, 1476-77 (BPAI 2010) (informative). Although we are not
considering the merits of this new argument, we find that the Examiner has
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Application 14/210,884
reasonably articulated that “[t]he processing of any number of samples
within a given period of time would also have been obvious as a scaling up
action to achieve an economy of scale.” Final Act. 2; Ans. 2-3.
Error! Reference source not found.. Obviousness over DYNAL and
Weindel
Appellant contends Weindel does not teach that proteinase K can be
used for isolating DNA from cells at room temperature. Reply Br. 5; see
Appeal Br. 14-15.
Weindel teaches that “[sjamples according to the invention include
clinical samples such as blood, serum, oral rinses, urine, cerebral fluid,
sputum, stool, biopsy specimens and bone marrow samples.” Weindel
10:60-64. Weindel teaches that multiple lysing procedures for obtaining
DNA are known.
Procedures for lysing samples are known by the expert and can
be chemical, enzymatic or physical in nature. A combination of
these procedures is applicable as well. For instance, lysis can
be performed using ultrasound, high pressure, by shear forces,
using alkali, detergents or chaotropic saline solutions, or by
means of proteinases or lipases.
Weindel 11:15-21. Weindel teaches that proteinase K functions at room
temperature. See Weindel 23:20^13.
In making the rejection, the Examiner explains that Weindel is used to
establish that proteinase K is active at room temperature, therefore, the
limitation of conducting the lysing step at room temperature is but one art
recognized equivalent use of this enzyme. See Ans. 6. The Examiner
explains:
[Ejven in the event that the plasma in Example 5 of Weindel et
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Application 14/210,884
al[.] does not contain cells, it is not necessary for the rejection
to be sustained since the relevant fact is that that [sic]
Proteinase K may be used at room temperature (as is disclosed
in Weindel et al[.] at column 23, lines 1-23) in the lysis step in
the primary reference (i.e., Proteinase K is active at room
temperature as well as at elevated temperatures (e.g., 37° C)).
Id. In other words, at the time of the invention it was known to one of
ordinary skill in the art that the enzyme activity of proteinase K is not
limited to elevated temperature only, even if it may work better at higher
temperature. One of ordinary skill in the art would appreciate that
incubating a sample at a lower temperature would be but one alternative
option resulting in a reasonable expectation that the enzyme function is
maintained. See Ans. 3 (“All that is achieved is the known and expected
result”). Thus, the application of a room temperature incubation step using
proteinase K is but one art recognized equivalent use of that enzyme. See
KSR, 550 U.S. at 421. On this record, the preponderance of the evidence
supports the Examiner’s conclusion of obviousness with respect to claim 4.
Claim 5 was not separately argued and falls with claim 4.
SUMMARY
We affirm the rejection of claim 8 under 35 U.S.C. § 112, second
paragraph.
We affirm the rejection of claim 1 under 35 U.S.C. § 103(a) over
DYNAL in view of the admitted prior art. Claims 2, 3, and 8-16 were not
argued separately and fall with claim 1.
We affirm the rejection of claim 4 under 35 U.S.C. § 103(a) as
unpatentable over DYNAL in view of the admitted prior and further in view
of Weindel. Claim 5 was not argued separately and falls with claim 4.
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Application 14/210,884
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).
AFFIRMED
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Notice of References Cited
]. 4/210,884
Appeal No.
2017-004573
Examiner Art Unit
1634 Page 1 of 1
U.S. PATENT DOCUMENTS
ie Document Number
Country Code-Number-KinrJ Code
Date
MM-YYYY Name Classification
A US- 2004/0265855 A1 12-2004 Pessara et ai.
B US- 6,423,488 B1 07-2002 Harvey
c US-
D US-
E US-
F US-
G US-
H US-
i US-
J US-
K US-
L US-
M US-
FOREIGN PATENT DOCUMENTS
* Document Number
Country Code-Number-Kind Code
Date
MM-YYYY Country Name Classification
N
O
P
Q
R
s
T
NON-PATENT DOCUMENTS
* include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages)
U
V
w
X
’A copy of this reference is not being furnished with this Office action. (See MPFP § 707.05(a).)
Dates in MM-YYYY format are publication dates. Classifications may be US or foreign.
U.S. Patent and Trademark Office
PTO-892 (Rev. 01-2001) Notice of References Cited Part of Paper No.
US 20040265855A1
(19) United States
(12) Patent Application Publication (io) Pub. No.: US 2004/0265855 Al
Pessara et al. (43) Pub. Date: Dec. 30,2004
(54) HIGH-THROUGHPUT DNA-ISOLATION AND
TRANSFECTION FOR ANALYSING THE
FUNCTION OF GENES OR GENETIC
PRODUCTS
(75) Inventors: Ulrich Pessara, Weilheim (DE);
Michael Kazinski, Munchen (DE);
Johannes Gorl, Munchen (DE)
Correspondence Address:
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON, MA 02110-2624 (US)
(73) Assignee: Xantos Biomedicine AG, Munchen (DE)
(21) Appl. No.: 10/773,100
(22) Filed: Feb. 5, 2004
Related U.S. Application Data
(63) Continuation-in-part of application No. PCT/EP02/
08962, filed on Aug. 9, 2002.
(30) Foreign Application Priority Data
Aug. 10, 2001 (EP).................................... 01119347.1
Publication Classification
(51) Int. Cl.7 .................................................C12Q 1/68
(52) U.S. Cl............................................................... 435/6
(57) ABSTRACT
The present invention relates to a method for screening a
collection of nucleic acid molecules for a desired property of
the nucleic acid or of a (poly)peptide encoded thereof,
comprising the steps (a) automated picking of the cell
collection containing the collection of nucleic acid mol­
ecules with a first robot; (b) automated lysis of the cells with
a second robot; (c) automated separation of the cell DNA
from the cell debris with a second robot; (d) optionally
automated separation of endotoxins from the DNA with the
second robot if the cells are bacteria; (e) automated trans­
fection of the cells with the DNA obtained in step (c) or, if
the cells are bacteria, with the DNA obtained in step (d) with
a third robot; and (f) automated screening for the desired
property with a fourth robot. Moreover, the invention relates
to methods for the enhancement of the binding properties of
the (poly)peptide identified by the of the screening method
of the invention or encoded by the DNA identified and
isolated and a method for the production of a pharmaceutical
composition on the basis of (poly)peptides which can be
obtained with the method of the invention and moreover the
formulation of the substance obtained with a pharmaceuti­
cally acceptable carrier or dilutent.
US 2004/0265855 A1
1
Dec. 30, 2004
HIGH-THROUGHPUT DNA-ISOLATION AND
TRANSFECTION FOR ANALYSING THE
FUNCTION OF GENES OR GENETIC PRODUCTS
[0001] The present invention relates to a method for
screening a collection of nucleic acid molecules for a desired
property of the nucleic acid or a (poly)peptide encoded
thereby, the method comprising the steps (a) automated
picking of a collection of cells containing the collection of
nucleic acid molecules by means of a first robot; (b) auto­
mated lysis of the cells by means of a second robot; (c)
automated separation of the cell DNA from cell debris by
means of the second robot; (d) optionally automated sepa­
ration of endotoxins from the DNA by means of the second
robot if the cells are bacteria; (e) automated transfection of
cells with the DNA obtained in step (c) or, if the cells are
bacteria, with the DNA obtained in step (d) by means of a
third robot; and (f) automated screening for the desired
property by means of a forth robot. Moreover, the invention
relates to methods for improving the binding properties of
the (poly)peptide which is identified by the screening
method of the invention or encoded by the infected or
isolated DNA, as well as to methods for producing a
pharmaceutical composition on the basis of (poly)peptides
obtainable by the method of the invention and, furthermore,
to the formulation of the substance obtained with a phar­
maceutically acceptable carrier or diluent.
[0002] In the specification, a number of prior art docu­
ments is cited. The disclosure content of these documents is
herewith incorporated by reference in its entirety in the
present description.
[0003] For years, high through-put screening has been a
tried and tested instrument for finding potential active agents
in pharmaceutical research. It is, however, relatively new to
use said high through-put technology also for methods such
as the isolation of DNA from bacteria and the transfection of
cellular systems. In particular, the screening of cDNA librar­
ies is of interest in this case. The screening of cDNA or
generic libraries which are usually cloned in bacteria
requires a process that can generally be divided into four
steps and comprises 1) the picking of the bacteria colonies,
2) the preparation of DNA, 3) the transfection of DNA and
4) the reading out of a functional test.
[0004] The DNA is usually isolated from bacteria by
means of two different methods: alkaline lysis of bacteria
with subsequent purification of the DNA recovered over
columns or adhesion of the DNA obtained by the alkaline
lysis to special micro-particles (so-called beads).
[0005] A protocol for alkaline lysis has, for instance, been
described in Sambrook et al., “Molecular Cloning, A Labo­
ratory Handbook”, CSH Press, Cold Spring Harbor 1989; or
Ausubel et al.; Current Protocols, in Molecular Biology
2002; John Wiley & Sons, Inc., N.Y. Methods for purifying
DNA, RNAor their hybrids with magnetic silica beads have
been described for instance in U.S. Pat. No. 6,027,945 or
WO 98/31840. Removing cell debris by using magnetic
micro-particles has been shown in U.S. Pat. No. 5,646,283.
[0006] Said purification is usually based on chemical
purification methods and is therefore suitable to a very
restricted extent for screening complex libraries.
[0007] Corresponding methods are designed to be used for
carrying them out in a laboratory or on pipetting robots for
a small through-put of samples. The daily through-put rate
varies and, depending on the method, is limited to a maxi­
mum of 3000 to 6000 preparations per day. Due to this
limited through-put rate of samples, this method is not
suitable for high through-put.
[0008] For transfecting DNA in eukaryotic cell systems,
chemical methods such as lipofection fulfil the requirements
for a high through-put rate of samples. The DNA can be
introduced into the cell by the preparation of cell membrane-
permeable DNA complexes or by penetration or fusion with
the cell membrane. Physical methods such as magnetofec-
tion or electroporation, too, are suitable methods for high
through-put.
[0009] Single steps of screening processes of complex
libraries can be carried out in an automated manner already.
Corresponding devices can be purchased from Beckman
Coulter or Tecan. The devices Biomek 2000 (Beckman
Coulter; Fullerton, USA) or Genesis (Tecan; Durham, USA)
are semi-automated working platforms for the use of
microtitre plates. These systems are general working plat­
forms which can, for instance be adapted to the use for DNA
preparation. The possibilities of application, however, are
limited as, for example, no centrifuges are integrated. Thus,
advantageous test protocols such as, for instance, preparing
a DNA by alkaline lysis (mini-prep) cannot be carried out.
Moreover, manual steps such as, e.g., for pelleting/precip-
iting the bacteria are not necessary.
[0010] An automated high through-put DNA preparation
system for the use of microtitre plates has been described in
EP 569 115 A2. By integrating modified centrifuges, a DNA
preparation after alkaline lysis is made possible. In so far,
compared to the state-of-the-art processes described above,
this method is already an improvement. However, a degree
of purity of the DNA, which is required for the application
of transfections, is not achieved. This is, amongst others, due
to the fact that the DNA is still contaminated by endotoxins.
It is also disadvantageous that this system, just like the
Genesis (Tecan) and the Biomek 2000 (Beckman) systems
are not outlined as conveyor road system or can be enlarged
as such. It is therefore not possible to interconnect the
individual process steps. The sample through-put rate of the
aforementioned systems is thus limited to about 3000 to
6000 preparations/day at maximum.
[0011] PCT/EP00/00683 describes a method for the iden­
tification of nucleic acid sequences that do not have a
selectable activity. The method comprises the steps of pro­
viding the DNA library, cultivating the host cells, preparing
the DNA, transfecting the target cells with the target DNA
and functional determination of the activity of the DNA in
the target cell. This application is a method which has a
certain degree of automation of the DNA preparation.
Accordingly, embodiments of two robots which can each
perform the DNA preparation and the DNA transfection are
presented. With these methods, too, sample through-put
rates in the range of more than 103 preparations per day can
be achieved.
[0012] PCT/EP00/13132 describes a screening method for
nucleic acids which also includes nucleic acids with select­
able activity. Apart from the screening method, also the
automation of the method and a preferred embodiment for
carrying out the DNA preparation and DNA transfection
US 2004/0265855 A1
2
Dec. 30, 2004
using single robots are recorded. With these methods, too,
sample through-put rates in the range mentioned above can
be achieved.
[0013] All aforementioned methods have the disadvantage
that they are not suitable for screening complete gene
libraries for molecules having the desired properties in a
shorter period of time. For screening gene libraries that
have, for instance, a complexity of up to or even more than
106 cDNAs requires a high sample through-put rate per day
in order to be easy to handle and to lead to the desired
properties in a clear time frame. Such a sample through-put
rate is not only made possible by optimising the individual
processes described in the state of the art. It is rather
necessary to try new ways, i.e. new combinations of pro­
cesses have to be found, to subject gene libraries having a
high degree of complexity to functional studies in an accept­
able time frame that is appropriate for therapeutic develop­
ments. The technical problem underlying the present inven­
tion was to provide a method that meets these requirements.
[0014] This technical problem is solved by the embodi­
ments characterised in the claims.
[0015] Accordingly, the invention relates to methods for
screening a collection of nucleic acid molecules for a desired
property of the nucleic acid or of a (poly)peptide encoded
thereby, comprising the steps of (a) automated picking of a
collection of cells containing the collection of nucleic acid
molecules by means of a first robot; (b) automated lysis of
the cells by means of a second robot; (c) automated sepa­
ration of the cellular DNA from the cell debris by means of
the second robot; (d) optionally automated separation of
endotoxins from the DNA by means of the second robot if
the cells are bacteria; (e) automated transfection of cells with
the DNA obtained in step (c) or, if the cells are bacteria,
obtained in step (d) by means of a third robot; and (f)
automated screening for the desired property by means of a
fourth robot.
[0016] Step (d) of the method of the invention is an
optional step. Especially if the sensitivity of the preferably
eukaryotic cells to be transfected to endotoxin is very low,
this step is preferred, it can, however, also be left out.
[0017] Accordingly, the method of the invention either
comprises steps (a), (b), (c), (d), (e) and (f) or the steps (a),
(b), (c), (e) and (f).
[0018] According to the invention, the latter order of steps
can also be defined as (a), (b), (c), (d1) and (e1), with step (d1)
corresponding to step (e) and step (e1) corresponding to step
(0-
[0019] Within the meaning of the invention, the term
“collection” relates to a number of nucleic acid molecules
which is more than 103 different molecules, preferably at
least more than 104 different molecules, more preferably at
least more than 105 different molecules and most preferably
106 different molecules such as 2xl06 or 3xl06 different
molecules.
[0020] The “nucleic acid molecules” are preferably coding
regions together with homologous or heterologous expres­
sion control sequences. It is particularly preferred that they
represent or substantially represent the genome of an organ­
ism.
[0021] Said organism can be a prokaryote, e.g. a bacte­
rium, or a eukaryote, e.g. a yeast. If the organism is a
eukaryote, it is, in a preferred embodiment, a mammal, e.g.
a human.
[0022] The term “(poly)peptide” describes both peptides
and polypeptides (proteins).
[0023] According to the convention, a chain of up to 30
amino acids is called a peptide and a chain of more than 30
amino acids is called a polypeptide.
[0024] Within the meaning of the invention, the term
“automated” means that the step in question is not per­
formed by humans but is only carried by a machine. How­
ever, this definition of said terms, of course, also includes
manipulations and adjustments of the machine (the robot) by
humans.
[0025] Within the meaning of this invention, the term “cell
debris” means the mass of cell components obtained after
lysis of a cell and that can be separated from the aqueous,
DNA-containing supernatant by centrifugation, e.g. at
3000xg. Cell debris usually contains proteins and, in the
case of bacteria, cell membrane components.
[0026] The expression “robot” refers to an automated
working station with grip arms and specific product pro­
cessing stations such as, e.g. centrifuges, incubation places,
etc.
[0027] With the method of the invention, a screening
method is provided in which the four process steps of
picking of the colonies, preparing of the DNA, transfecting
of the DNA and reading out a functional screening assay are
carried out in an automated manner by a robot. In this way,
an automated overall process is made possible which is
suitable for high through-put screening. The automated
removal of endotoxins, preferably using magnetic micro­
particles, can be considered an essential component of this
method in one embodiment (i.e. an embodiment including
step (d)). Only in this way, in combination with further
automated steps, is an acceptable time frame for high
through-put screening of libraries having a high degree of
complexity achieved. For the purification of the DNA from
endotoxins in this embodiment is an essential prerequisite
for being able to use the DNA directly for the transfection.
Only in this way can thus the DNA obtained from the DNA
preparation be directly used for analyses and transfections.
It is of particular advantage that the time-consuming cen­
trifugation steps are considerably reduced. Methods for
removing endotoxins from DNA, RNA or their hybrids
using magnetic silica particles are described in U.S. Pat. No.
6,194,562 or WO 99/54340.
[0028] In another embodiment of the method of the inven­
tion (i.e. the embodiment without step (d)), the removal of
the endotoxins is not essential. This is particularly the case
if the cells to be transfected have a low sensitivity to
endotoxins and are thus not essentially interfered with or
killed by endotoxin contamination in common DNA purifi­
cation processes.
[0029] The combination of the automated individual pro­
cesses which are carried out by interconnected robots makes
it, for the first time, possible to achieve a sample through-put
rate of up to 30,000/40,000 samples per day. In other words,
the combination of a serial production technique using the
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components described (according to the two above-de­
scribed embodiments) makes it possible to achieve a
through-put rate in the preparation of DNA capable for
transfection which has never been achieved before. Using
the same high through-put method, this DNA can be analy­
sed for its biological function after transfection, preferable in
eurkaryotic cells, which makes it possible to screen a
complete cDNA gene library within one month.
[0030] In a preferred embodiment of the method of the
invention, the collection of nucleic acid molecules is a gene
library.
[0031] The term “gene library” is known in the state of the
art and defined as a “Collection of cloned DNA fragments
representing an entire genome” in Winnacker, “Gene und
Klone”, VCH Weinheim 1985 (p. 403). The invention also
includes gene libraries with gaps, i.e. which do not represent
the entire gene or which represent an expression stage, e.g.
of a certain tissue, a stage of a disease or a development.
[0032] In another preferred embodiment of the method of
the invention, the nucleic acid molecules are genomic DNA
or cDNA molecules or RNAi oligonucleotides. Correspond­
ing RNAi oligonucleotides are synthesised for instance by
Dharamcon (LaFayette, USA), Xeragon (Germantown,
USA) or Ambion (Austin, USA).
[0033] In a particularly preferred embodiment of the
method of the invention, the gene library is an expression
cDNA gene library, preferably a eukaryotic gene library, a
human gene library is particularly preferred.
[0034] The term “expression cDNA gene library”, too, is
well-known in the state of the art. In an expression cDNA
gene library, the cDNA molecules are cloned into an expres­
sion vector which allows their expression in a suitable host;
cf. Winnacker, loc. cit. or Sambrook et al., “Molecular
Cloning, A Laboratory Manual”; CSF1 Press, Cold Spring
Flarbour 1989.
[0035] The gene library is preferred to be normalised (i.e.
the number of the genes contained in the gene library is
virtually the same) and/or enriched for “full length cDNA”.
[0036] In another preferred embodiment, the collection of
nucleic acids is a collection of clones. A collection of clones
is a collection of selected cDNA clones which preferably has
“full length cDNA”.
[0037] In a preferred embodiment of the method of the
invention, the cells in step (a) and/or step (e) are mammalian
cells, insect cells, yeast cells or bacteria.
[0038] Examples of mammalian cells are COS cells,
F1UVEC cells, Asperg///«s(niger/nidulans etc.) cells or CFIO
cells. Examples of insect cells are Spodoptera frugiperda
cells. Suitable yeast cells include cells of the species S.
cerevisiae or P. pastoris. Suitable bacteria can be both
Gram-negative and Gram-positive bacteria.
[0039] In a particularly preferred embodiment of the
method of the invention, the bacteria are Gram-negative
bacteria.
[0040] The particularly preferred properties of the method
of the invention, are in particular of importance if the
bacteria are Gram-negative bacteria as they, in particular,
have endotoxins as cell wall or cell membrane components.
With the Gram-negative bacteria, in particular bacteria of
the species E. coli are used for cloning purposes in the state
of the art.
[0041] In a most preferred embodiment of the method of
the invention, the Gram-negative bacteria thus belong to the
species E. coli.
[0042] It is particularly preferred that they are E. coli
DH5a, E. coli Shure and E. coli JM 109.
[0043] In a preferred embodiment of the invention, at least
one of the steps (a) to (f) (with or without step (d)) is carried
out in microtitre plates.
[0044] Conventional microtitre plates have the advantage
that, independent from the number of wells, they have a
standardised size which renders them particularly suitable
for an automated use by the robots. Microtitre plates (e.g.
obtainable from Nunc), are usually made of PVC or poly­
styrene. They can have 6, 24, 96, 384 or 1536 wells. The
microtitre plates that are preferably used in the method of the
invention have 96 or 384 wells.
[0045] In a particularly preferred embodiment of the
method of the invention, all steps (a) to (f) (with our without
step (d)) are carried out in microtitre plates.
[0046] In an additional preferred embodiment of the
method of the invention, the microtitre plates are marked
with bar codes.
[0047] Therefore, this embodiment is particularly advan­
tageous as it allows a complete tracking of all plates, also
after changing from one robot to another. Thus, an assign­
ment starting from plating the cells for processing by the first
robot to functional screening and reading-out by the forth
robot is particularly easy and can be done in a time-saving
manner. In this was, it is easily possible to go back to the
initial clones on the screening plate after the functional
screening.
[0048] The bar code technique on the robots 2 and 3 makes
it moreover possible that the individual processes are inter­
laced within the conveyor road system.
[0049] In another preferred embodiment of the method of
the invention, the first robot is characterised by at least one
and preferably all of the following features: (a) a digital
image processing system for collecting the plated bacteria,
(b) a working station with a grip arm for microtitre plates for
transferring the microtitre plates between the processing
stations, (c) a separation module having one or more heads
with needles for picking the plated single colonies and for
placing them into the microtitre plates, (d) integrated prod­
uct processing stations for cleaning the needles between the
working steps and replicating the placed single colonies in
the microtitre plates and (e) a computer-based bar code
identification and tracking system.
[0050] The microtitre plates are preferably plates with 96
or 384 wells. The integrated product processing stations
include a sterilisation system. Moreover, it is preferred that
the grip arm is a robot arm which has at least two heads with
needles, wherein the heads are used for cross-picking and
are cleaned on the sterilisation station. In addition, a modu­
lar set-up of the robot arm is preferred which allows an
exchange of grip arm modules for separation head modules.
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[0051] In a preferred embodiment of the method of the
invention, the lysis is an alkaline lysis.
[0052] The conduction of the alkaline lysis is described,
amongst others, in Sambrook, loc. cit., and in another
passage of this description.
[0053] In an additional preferred embodiment of the
method of the invention, the second robot is characterised by
at least one and preferably all of the following features: (a)
a conveyor road transport system combined with grip arms
for the microtitre plates for reloading the products and for
transferring the microtitre plates between the product pro­
cessing stations, (b) product processing stations integrated
into the transport system, particularly centrifuges, pipetting
automats, shakers and incubation places for incubation at
different temperatures, (c) a sensor technology for the detec­
tion of product positions as well as for the detection of
errors, (d) a software for the interlaced handling of several
processes which are in the machine for a continual produc­
tion process and (e) a computer-based bar code identification
and tracking system, preferably with an internal product
tracking containing a time stamp function for the interlacing
of time-critical sub-processes.
[0054] In this case, too, the microtitre plates are preferred
to have 96, 384 or 1536 wells. In another preferred embodi­
ment of the method of the invention, the cellular DNA in
step (c) is separated by silica particles.
[0055] Within the meaning of this invention, the term
“separation of the cellular DNAby means of silica particles”
means that the cellular DNA (i.e. the plasmid DNA or the
chromosomal DNA in another embodiment) is bound to
these particles and separated from the cell debris. In prin­
ciple, this separation step therefore is a purification step. The
silica particles can be removed easily by centrifugation from
cell debris.
[0056] In a particularly preferred embodiment of the
method of the invention, the silica particles are magnetic
silica particles.
[0057] Thus, the embodiment is particularly preferred as
the magnetic particles can easily be removed from the cell
debris and other supernatant by using a magnet. Correspond­
ing methods are described, for example, in U.S. Pat. No.
6,027,945 and WO 98/31840.
[0058] In a preferred embodiment of the method of the
invention, the separation of the endotoxins in step (d) is
carried out by precipitation with SDS/isopropanol.
[0059] A suitable composition is 2.5% SDS in isopro­
panol.
[0060] In a particularly preferred embodiment of the
method of the invention, the DNA bound to silica particles
is further purified by washing with acetone.
[0061] In another preferred embodiment of the method of
the invention, the endotoxins in step (d) are separated by
means of endotoxin-binding particles which are preferred to
be magnetic endotoxin-binding particles.
[0062] The endotoxin-binding particles can preferably be
provided as magnetic particles.
[0063] In another preferred embodiment of the method of
the invention, the transfection of cells in step (e) is mediated
by calcium phosphate, electroporation or lipofection.
[0064] In another preferred embodiment of the method of
the invention, the transfection of cells in step (s) is mediated
by calcium phosphate or lipofection. Mediation of the lipo­
fection can be effected by lipids, liposomes or lipid combi­
nations. Examples thereof are Effectene (Qiagen; Hilden),
Fugene (Roche; Basle), Metafectene (Biontex), lipo-
fectamins or Lipfectamine 2000, Lipofectin, Oligofectamine
(Invitrogen; Karlsruhe).
[0065] Metafectene, Oligofectamine or calcium phosphate
are particularly suitable for the transfection of RNAi oligo­
nucleotides.
[0066] Corresponding methods are known in the state of
the art and are described, for instance, in “Transfection
Technologies” (Methods Mol. Biol. 2000; 130: 91-102) or
Current Protocols (Ausubel et al., 2002; 9.1).
[0067] In an additional preferred embodiment of the
method of the invention, the transfection is carried out using
DNA-binding magnetic biocompatible micro-particles.
[0068] The term “biocompatible micro-particle” means
micro-particles that are biologically inert or that can be
metabolised in a cell.
[0069] In this preferred embodiment, modified micro­
particles can already be used in the step of DNApreparation,
wherein said micro-particles can then be used directly for
transfection. The method, which is hereinafter called mag­
neto-transfection, is based on the following parameters:
[0070] The DNA suitable for transfection is bound to
biocompatible magnetic micro-particles. The micro-par­
ticles with the DNA bound thereto are applied to the cell
cultures. By application of a magnetic field, the DNA
micro-particle complexes are concentrated on the cell sur­
face and taken up into the cell by endocytotoxic processes.
Alternatively, the DNA micro-particles can be inserted into
the cell/nucleus by increase of the magnetic field. Such a
method of magneto-transfection is known in the state of the
art and described, for instance, in PCT/EP01/07261. The
effectiveness of said method can still be improved by using
lipophilic substances that enhance the uptake, e.g. by lipo-
fectamin.
[0071] The magnetic concentration of the complexes or
the insertion of the DNA micro-particles in the cell/nucleus
on the cell surface leads to an increased transfection effi­
ciency. In this way, the amount of sample DNA can be
reduced and, with regard to the amount of samples used in
a high through-put system, the costs can be reduced signifi­
cantly. Furthermore, by using said micro-particles, the trans­
fection process can be carried out on a robot system which
has similar specifications as the robot system used for DNA
preparation. In addition, the process steps can be reduced
further and the overall process can be sped up.
[0072] This preferred embodiment provides a high
through-put transfection system with which a daily through­
put rate of up to 40,000 samples can be achieved in a
particularly cost- and money-saving manner.
[0073] In another preferred embodiment of the method of
the invention, the third robot is characterised by at least one
and preferably all of the following features: (a) a conveyor
road transport system combined with grip arms for microti­
tre plates for reloading the products and for transferring the
microtitre plates between the product processing stations,
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(b) product processing stations integrated into the transport
system, particularly pipetting stations, shakers and incuba­
tion places and an incubator for culturing the transfectants,
(c) a sensor technology for the detection of product positions
as well as for the detection of errors, (d) sterile overpressure
ventilation to prevent contaminations of the cell cultures, (e)
a software for the interlaced handling of several processes
which are in the machine for a continual production process
and (f) a computer-based bar code identification and track­
ing system, preferably with an internal product tracking
containing a time stamp function for the interlacing of
time-critical sub-processes.
[0074] In another preferred embodiment of the method of
the invention, the forth robot is characterised by at least one
and preferably all of the following features: (a) a system for
determining the fluorescence, luminescence or colour reac­
tions from cell culture assays, (b) a pipetting station with a
grip arm for microtitre plates for transferring the microtitre
plates from the incubator to and between the product pro­
cessing stations, (c) processing places for adding and with­
drawing cell culture media or reagents and incubation in the
incubator and (d) computer-based bar code identification
and tracking system.
[0075] The system is preferably an ELISA reader or a
microtitre plate imaging system. It is moreover preferred
that the system is suitable for determining the cell morphol­
ogy. As is the case with the other robots, it is preferred that
the microtitre plate has 96 or 384 wells. Apart from pro­
cessing places for adding and withdrawing cell culture
media, etc., the robot may have two other product processing
stations such as, e.g. shakers, incubation places.
[0076] In an additional preferred embodiment of the
method of the invention, the forth robot is characterised by
at least one and preferably all of the following features: (a)
a digital image processing system and image acquisition
system for determining the cell morphology, luminescence
and/or fluorescence, (b) a pipetting station with grip arm for
microtitre plates for transferring the microtitre plates from
the incubator to and between the product processing sta­
tions, (c) processing places for adding and withdrawing cell
culture media or reagents and incubation in the incubator
and (d) a computer-based bar code identification and track­
ing system.
[0077] The term “image processing system” means a
system that can detect and analyse automatically differences
in the luminescence or fluorescence properties and the
morphology of the cells to be examined. Preferably, the data
processing of such a system is based on neuronal networks
or other corresponding digital image-analytical algorithms
of the state of the art.
[0078] The term “image acquisition system” is an auto­
mated microscoping station which can generate images of
the cells to be examined using camera or scanning systems.
[0079] In this case, both the image processing and the
acquisition system are suitable for a high through-put pro­
cess.
[0080] In still another preferred embodiment of the
method of the invention, the automated screening is a
functional screening.
[0081] Within the meaning of this invention, the term
“functional screening” means that the nucleic acid such as
DNA or the (poly)peptide encoded thereby is tested for a
function. An RNA can be tested for a ribosyme property, an
anti-sense property or the binding property within the mean­
ing of an aptamer. Mostly however, the (poly)peptide
encoded is tested for a desired property.
[0082] An RNAi oligonucleotide (double-stranded RNA)
(Elbashir et al., 2002) can be tested for its property to reduce
or block the expression of genes.
[0083] In a particularly preferred embodiment of the
method of the invention, the functional screening is a
screening for an enzymatic, pharmacological or therapeutic
property.
[0084] Said property is usually tested with the (polypep­
tide. The property, for instance, to induce apoptosis in the
cell can be determined by means of the cell morphology or
cell assays such as the CDD+ assay (Roche Diagnostics;
Basle/Switzerland) or by caspase activation.
[0085] In another preferred embodiment, the functional
screening is a screening for the function of secreted proteins.
In this case, the proteins encoded by the transfected cDNA
are secreted into the cell supernatant. Said supernatant is
transferred to target cells and the function of the protein
secreted is determined by its effect on the target cell.
Alternatively, the cell transfected with the cDNA can be
contacted with the target cell and the function of the protein
expressed (e.g. on the cell surface) can be determined by its
effect on the target cell.
[0086] In another particularly preferred embodiment of
the method of the invention, the functional screening is a
screening for activation or suppression of a reporter system.
[0087] Suitable reporter systems are known in the state of
the art and comprise reporter gene assays (e.g. for transcrip­
tional activation of indicator proteins, enzymatic activation/
deactivation of indicator proteins). Examples thereof are the
green fluorescent protein (GFP), luciferase (Firefly) from the
field of fluorescence-based reporter systems.
[0088] In other preferred embodiments, the screening is a
screening for modified cell morphology, cell death or pro­
liferation.
[0089] In a preferred embodiment of the method of the
invention, 2, 3 or all 4 robots are arranged in a conveyor
road.
[0090] In this preferred embodiment, at least 2, i.e. 3 or all
4, individual processing stations/robots for colony picking,
DNA preparation, DNA transfection and reading-out of the
functional screening assay are additionally connected or
combined by conveyor road systems. In this way, interme­
diary steps between the individual processes, which have so
far been necessary, are avoided and the sample through-put
rate is increased further.
[0091] By using conveyer belt transport systems in com­
bination with overhead manipulators by a corresponding
interlacing of the process steps, a serial production process
is arrived at which, in contrast to classical pipetting stations,
has no limitation with respect to the production volume. If
alternatively 96-well or 384-well plates are used, flexibility
is even more increased.
[0092] In a further preferred embodiment of the process
according to the invention, a DNA, (poly)peptide or a
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transfectant containing these which has been identified in a
screening process, is purified or isolated.
[0093] For the further processing of the DNA/RNAi oli-
gonucleotides/(poly)peptides which were tested positively
in the screening process, it is desirable that the substances or
the corresponding tranfectant is purified to a no longer
contaminated and thus pure form. This is particularly easy
with the process of the invention, as e.g. the positively tested
substance is directly available by referring back to the
master plate. The further purification steps for the substances
or the corresponding transfectants can be carried out accord­
ing to the conventional processes.
[0094] In another preferred embodiment, the present
invention also relates to a process for improvement of the
binding properties of the (poly)peptide encoded by the DNA
identified or isolated in the screening process of the inven­
tion, comprising the steps of (a) identification of the binding
sites of the (poly)peptide or its binding partner by site
specific mutagenesis or chimeric protein studies; (b)
molecular modelling of the binding site of both the
(poly)peptide and the binding partner; and (c) modification
of the (poly)peptide in order to improve the binding speci­
ficity or the affinity of the binding.
[0095] The (poly)peptide can be modified so as to increase
the binding affinity or effectiveness and specificity. If e.g.
electrostatic interactions between a certain residue of the
(poly)peptide in question and a region of the (poly)peptide
exists, the total charge of this region can be changed in order
to increase the existing interation in this manner.
[0096] Computer programs can be useful for identifying
binding sites. Thus, suitable computer programs can be used
for identifying interactive sites of an alleged inhibitor and
the polypeptide by computer-based screening for comple­
mentary structural motifs (Fassina, Immunomethods 5
(1994), 114-120). Further suitable computer systems for the
computer-based design of proteins and peptides are
described in the state of the art, e.g. in Berry, Biochem. Soc.
Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci.
501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-
5991. Modifications of the (poly)peptide can be achieved by
e.g. peptidomimetics. Other inhibitors can also be identified
by means of synthesis of combinatorial peptidomimetic
libraries by successive chemical modification and testing of
the compositions which have been obtained. Processes for
the production and use of combined peptidomimetic librar­
ies are described in the state of the art, e.g. in Ostresh,
Methods in Enzymology 267 (1996), 220-234 and Dorner,
Bioorg. Med. Chem. 4 (1996), 709-715. Moreover, the
three-dimensional and/or crystallographic structure of the
activators of the expression of the (poly)peptide of the
invention can be used for the design of peptidomimetic
activators, e.g. in connection with the (poly)peptide identi­
fied according to the invention (Rose, Biochemistry 35
(1996), 12933-12944, Rutenber, Bioorg. Med. Chem. 4
(1996), 1545-1558).
[0097] In a particularly preferred embodiment of the pro­
cess of the invention, the modification in step (c) is a
reproduction of the (poly)peptide by petidomimetics.
[0098] In an additional preferred embodiment of the pro­
cess of the invention, the (poly)peptide as leading structure
is further modified in order to obtain (i) a modified site of
action, a modified spectrum of activity, a modified organ
specificity and/or (ii) an improved activity and/or (iii) a
reduced toxicity (an improved therapeutic index) and/or (iv)
reduced side effect and/or (v) a delayed on-set of the
therapeutic action, of the duration of the therapeutic effect
and/or (vi) modified pharmacokinetic parameters (resorp­
tion, distribution, metabolism or excretion) and/or (vii)
modified physicochemical parameters (solubility, hygro­
scopic properties, colour, taste, odour, stability, state) and/or
(viii), improved general specificity, organ/tissue specificity
and/or (ix) optimised application form and route by (i)
esterification of carboxylic groups or (ii) esterification of
hydroxyl groups with carboxylic acids or (iii) esterification
of hydroxyl groups to form e.g. phosphates, pyrophosphates
or sulfates or amber acid semi-esters or (iv) formation of
pharmaceutically acceptable salts or (v) the formation of
pharmaceutically acceptable complexes or (vi) the synthesis
of pharmaceutically active polymers or (vii) the introduction
of hydrophilic moieties or (viii) the introduction/exchange
of substituents in aromates or side chains, change of the
substituent pattern or (ix) modification by introduction of
isosteric or bioisosteric moieties or (x) the synthesis of
homologous compounds or (xi) introduction of branched
side chains or (xii) conversion of alkyl substituents to form
cyclic analogues or (xiii) derivatisation of hydroxyl groups
to form ketals or acetals or (xiv) N-acetylation to form
amides, phenylic carbamates or (xv) synthesis of Mannich
bases, imines or (xvi) transformation of ketones or alde­
hydes to Schiff’s bases, oximes, acetals, ketals, enolic esters,
oxazolidines, thiozolidines or combinations thereof.
[0099] The different above-mentioned steps are generally
known in the art. They comprise or are based on quantitative
structure-effect-relationships (QSAR) analyses (Kubinyi,
“Flausch-Analysis and Related Approaches”, VCF1 Verlag,
Weinheim, 1992), combined biochemistry, classical chem­
istry and others (cf. e.g. Flolzgrabe and Bechtold, Deutsche
Apotheker Zeitung 140(8), 813-823, 2000).
[0100] Moreover, the present invention relates to a process
for the production of a pharmaceutical composition com­
prising the steps of the process of the invention and further­
more the formulation of the substance obtained with a
pharmaceutically acceptable carrier or diluent.
[0101] The pharmaceutical composition can be produced
in a conventional manner.
[0102] Examples of suitable pharmaceutically acceptable
carriers and/or diluents are known to the person skilled in the
art and comprise e.g. phosphate buffered physiological
salines, water, emulsions, such as e.g. oil/water emulsions,
different kinds of wetting agents or detergents, sterile solu­
tions, etc. Pharmaceutical compositions comprising such
carriers can be formulated by means of known conventional
processes. These pharmaceutical compositions can be
administered to an individual in a suitable dose. The admin­
istration can be effected orally or parenteraly, e.g. intrave­
nously, intraperitoneally, subcutaneously, intramuscularly,
locally, intranasally, intrabronchially or intradermally or by
means of a catheter somewhere in an artery. The dosage
form is chosen by the physician in charge according to the
clinical factors. It is known to the person skilled in the art
that the dosage form depends on several factors such as e.g.
the body size or the weight, the body surface, the age, the sex
or the general health of the patient but also on the substance
US 2004/0265855 A1
7
Dec. 30, 2004
to be administered in particular, the duration and form of the
administration and on other pharmaceutical preparations
which are possibly administered at the same time. Atypical
dose can e.g. be in a range from 0.001 to 1,000 fig, with
doses below or above this exemplary range being possible,
in particular when considering the above-identified factors.
In general, the dose should range from 1 fig and 10 mg units
per day if the composition of the invention is administered
regularly. If the composition is administered intravenously,
which is not recommended as being preferred in order to
minimize the danger of anaphylactic reactions, the dose
should range from 1 fig and 10 mg units per kilogram body
weight per minute.
[0103] The composition of the invention can be adminis­
tered locally or systemically. Preparations for a parenteral
administration comprise sterile aqueous or non-aqueous
solutions, suspensions and emulsions. Examples of non-
aqueous solvents are propylene glycol, polyethylene glycol,
plant oils such as e.g. olive oil and organic ester composi­
tions such as e.g. ethyloleate which are suitable for injec­
tions. Aqueous carriers comprise water, alcoholic-aqueous
solutions, emulsions, suspensions, saline solutions and buff­
ered media. Parenteral carriers comprise sodium chloride
solutions, Ringer’s dextrose, dextrose and sodium chloride,
Ringer’s lactate and bound oils. Intravenous carrier com­
prise e.g. fluid, nutrient and electrolyte supplements (such as
e.g. those based on Ringer’s dextrose). The composition
according to the invention can moreover comprise preserv­
ing agents and other additives such as e.g. antimicrobial
compounds, antioxidants, complex former and inert gasses.
Moreover, dependent on the intended use, compounds such
as e.g. interleukins, growth factors, differentiation factors,
interferons, chemotactic proteins or an unspecific immuno­
modulatory agent can be contained.
[0104] In general, the complete process on which the
invention is based can e.g. be presented as follows:
[0105] 1. Picking the Bacterial Colonies and Replication
(Robot 1)
[0106] cDNA banks are plated on agar plates, the indi­
vidual colonies are picked and transferred to microtitre
plates where the bacteria are cultivated for propagation. In
a second step, several growth plates are inoculated from
these master plates and are cultivated for propagation to
generate sufficient bacteria for the isolation of the DNA
(replication).
[0107] 2. DNA Preparation (Robot 2)
[0108] The growth plates with the bacterial suspension are
centrifuged and the supernatant is sucked off. Subsequently,
the pellets are resuspended in a buffer containing RNAse
(PI), an alkaline lysis buffer (P2) is added and is then
neutralised (P3).
[0109] These steps are carried out on an orbital shaker to
which a multi-channel dispenser is fixed.
[0110] After a short incubation, the plates are centrifuged
and the supernatant is transferred to a support plate. Subse­
quently, P4 is dispended in order to bind bacterial endotox­
ins, is recentrifuged after an incubation and the supernatant
is transferred to a second support plate. Silica is dispensed
to this supernatant in order to bind the DNA. A centrifuga­
tion is carried out, the supernatant is removed and the pellet
is washed with acetone. After having carried out another
centrifugation, the acetone supernatant is sucked off, the
silica pellet is resuspended with hot water with a tempera­
ture of 60° C. (removal of the DNA), centrifuged and the
DNA-solution is transferred to the final plates. (Buffer 1:
Tris EDTA with RNAse, P2: NaOH/SDS, P3: potassium
acetate buffer, P4: SDS in isopropanol).
[0111] 3. DNA Transfection (robot 3) A defined amount of
the DNA solution from the DNA plates produced by robot 2
is pipetted in support plates and a control plasmid ((3-Gal),
calcium chloride, HBS are added. After an incubation for
complex formation chloroquine is dispensed to the prepa­
ration and after mixing, a defined amount of the preparation
is pipetted onto the cell culture. After 4 to 5 hours, the
medium is changed.
[0112] 4. Functional Screening Assay (Robot 4)
[0113] After 24 to 48 hours, a substrate is added to the cell
culture plates which causes a change in colour in apoptotic
cells. This change in colour is evaluated in the ELISA reader
and the cells are discarded.
[0114] 2) DNA Preparation and Transfection Method by
Using Magnetic Micro-Particles:
[0115] After their growth, bacteria are centrifuged in
growth plates and are treated with an RNAse buffer. The
bacteria are resuspended on an orbital shaker. Subsequently,
a lysis and a neutralising buffer are added. By adding a first
kind of magnetic micro-particle, cell debris and proteins are
bound. The magnetic micro-particles are separated on a
magnetic plate and the supernatant is transferred to a support
plate. Afterwards, optionally, a second kind of magnetic
micro-particle is added which bind to bacterial endotoxins.
These are also separated magnetically and the supernatant is
transferred into a second support plate. These steps can be
combined by adding a mixture of both kinds of micro­
particles.
[0116] Alternatively, endotoxin precipitation reagents can
be used which are removed after the precipitation of the
endotoxins in the first micro-particle separation step.
[0117] In a last step, magnetic micro-particles are added
which bind to the DNA. The DNA can either be eluted from
these magnetic micro-particles and used for transfection or,
if the micro-particles are formulated accordingly, it can be
used directly for transfections.
[0118] In a preferred embodiment, the DNAmicro-particle
complexes produced during the DNA isolation can be used
directly for transfections.
[0119] The example illustrates the invention.
EXAMPLE 1
Carrying Out the Screening Method for the
Determination of the Function of Genes or Gene
Products
[0120] 1. Colony Picking and Replication
[0121] The bacteria containing DNA were plated in such
a way with a selection antibiotic on agar plates that as high
an amount as possible of single clones was evenly distrib­
uted on the plates. After an overnight incubation at 37° C.,
the colonies were picked by a robot and were transferred into
US 2004/0265855 A1
8
Dec. 30, 2004
microtitre plates with 384 wells (MTP), in which 60 fA LB
medium with a selection antibiotic was present. These plates
were incubated overnight at 37° C. and, on the following
day, were coated with a mixture of LB medium and glyc­
erine so that the final concentration of glycerine amounted to
15%. Subsequently, the plates (hereinafter referred to as
master MTP) were stored at -80° C.
[0122] For further use, the master MTPs were thawed and
replicated with a replication tool on a first robot in 4x“Deep-
well” MTP with 96 wells. 1.5 ml LB medium with a
selection antibiotic was plated into each of these 96-well
MTPs. After inoculation, the plates were incubated over­
night in a shaking container, the shaking speed amounting to
280 rpm.
[0123] 2. DNA Preparation
[0124] The MTPs with 96 wells were centrifuged at 3,000
g for 5 minutes and the supernatant was removed. 170 fA PI
(50 mM Tris pH 8.0; 10 mM EDTA pH 8.0; 100 fig/ml
RNAse A (Qiagen) were added on a shaking station with
dispenser, shaken at 1,000 rpm for 5 minutes, 170 fA P2 (200
mM NaOH, 1% SDS) were added, shaken for 10 s at 300
rpm and incubated at room temperature for 5 minutes.
Subsequently, 170 fA P3 (3 M KAc, pH 5.5) were added and
shaken for 30 seconds at 1,000 rpm. After 5 minutes of
incubation at 4° C., the MTPs were centrifuged for 5 minutes
at 3,500 g. The supernatant was removed and was trans­
ferred to a support MTP. 120 fA P4 (2.5% SDS (Roth) in
isopropanol) were added to the supernatant and were incu­
bated for 20 minutes at 4° C. Subsequently, a centrifugation
was carried out for 10 minutes at 3,500 g and the supernatant
was transferred onto a support plate. 120 fA silica (50 mg/ml
Si02 (12.5 g per 250 ml water)) were added and incubated
for 5 minutes at room temperature. In this case, the silica
suspension was prepared as follows: 12.5 g silica per 250 ml
water was stirred for 30 minutes, the supernatant (contains
silica powder) was sedimented; removed; 150 fA concen­
trated HC1 was added, filled up with H20 to 250 ml
(graduated cylinder) and autoclaved. Subsequently, a cen­
trifugation was carried out for 5 minutes at 2,000 g and the
supernatant was discarded. 400 fA acetone were added,
shaken for 1 minute at 1,000 rpm and subsequently centri­
fuged for 5 minutes at 2,000 g. Then, the supernatant was
sucked off and the plates with the silica pellets were dries for
20 minutes on a heating plate at 70° C. Subsequently, 140 fA
bidistilled water was added at a temperature of 65° C., was
shaken for 5 minutes at 800 rpm, centrifuged for 5 minutes
at 3,000 g and the supernatant was stored with the DNA in
a 96-well polystyrene MTR
[0125] 3. Transfection
[0126] On the day prior to the transfection, the cells to be
transfected were plated with a cell density of approximately
8,000 cells/well in a 96 well cell culture plate. 5 (3-Gal
plasmid (c=100 ng/1) were dispensed in a support MTP and
subsequently 20 fA of the DNA solution (c=100 ng/1) were
added. Subsequently 20 fA (0.25 M CaCl2) were added,
briefly shaken and subsequently 25 fA L2 (2xHBS) were
added. After an incubation for 20 minutes at room tempera­
ture, 15 fAJi (2 mM chlorochin-solution) were added and
briefly shaken. 91 fA of this mixture were placed on the cells
and incubated 5 to 6 at 37° C. Subsequently, a medium
change (DMEM/10% FCS) was carried out. After an incu­
bation overnight the medium (DMEM/10% FCS) was
changed again.
[0127] 4. Functional Reading Out
[0128] 30 /A CPRG solution 2.31 ml, 0.1 M sodium
phosphate solution, 30 fA 100xMgCl2, 660 fA CPRG solu­
tion were added to the transfected cell to each well of the cell
culture plate and incubated for 1 to 3 hours. Subsequently,
the plates were measured in an ELISA reader (absorption
measurement at 570 nm).
EXAMPLE 2
Functional Screening for Secreted Proteins
[0129] COS-7 cells are seeded with a cell density of
approximately 5,000 cells/well in 10% DMEM and incu­
bated for 24 hours at 37° C. in an incubator. The cDNA is
introduced into the cells by lipofection with Metafectene
(Biontex, Munich) and incubated for 3 hours at 37° C. in the
incubator. After complete removal of the medium, the endot­
helial cell growth medium (PromoCell, Heidelberg) is added
and the cells are incubated in an incubator for 48 hours at
37° C. Subsequently, the supernatant is removed and is
transferred to the endothelial cells (human umbelical vein
endothelial cells, HUVECs or microvascular endothelial
cells, HMVECs). Beforehand, these HUVEC cells are
seeded with a cell density of 2,000 cells/well in endothelial
cell growth medium (PromoCell, Heidelberg). After com­
plete removal of the medium, the supernatant of the COS-7
cells is transferred to the endothelial cells. The cells are
incubated for 6 days at 37° C. in an incubator and the
activities of the secreted proteins are determined by the
cytosolic reduction of Alamar Blue (BioSource, Solingen).
[0130] If no other indications are given, the individual
assay steps are carried out with protocols according to
Current Protocols (Ausubel et. al, 2002).
1. A method for screening a collection of nucleic acid
molecules for a desired property of the nucleic acid or of a
(poly)peptide encoded thereby, comprising the steps of
(a) automated picking of a collection of cells containing
the collection of nucleic acid molecules by means of a
first robot;
(b) automated lysis of the cells by means of a second
robot;
(c) automated separation of the cellular DNAfrom the cell
debris by means of a second robot;
(d) optionally automated separation of endotoxins from
the DNA by means of the second robot if the cells are
bacteria;
(e) automated transfection of cells with the DNA obtained
in step (c) or, if the cells are bacteria, obtained in step
(d) by means of a third robot; and
(f) automated screening for the desired property by means
of a fourth robot.
2. The method according to claim 1 wherein the collection
of nucleic acid molecules is a gene library or a collection of
clones.
3. The method according to claim 1 or 2 wherein the
nucleic acid molecules are genomic DNA or cDNA mol­
ecules or RNAi oligonucleotides.
US 2004/0265855 A1
9
Dec. 30, 2004
4. The method according to claim 2 or 3 wherein the gene
library is an expression cDNA gene library, preferably a
eukaryotic gene library, a human gene library is particularly
preferred.
5. The method according to any one of claims 1 to 4
wherein the cells in step (a) and/or step (e) are mammalian
cells, insect cells, yeast cells or bacteria.
6. The method of claim 5 wherein the bacteria are
Gram-negative bacteria.
7. The method of claim 6 wherein the Gram-negative
bacteria belong to the species E. coli.
8. The method of any one of claims 1 to 7 wherein at least
one of the steps (a) to (f) is carried out in microtitre plates.
9. The method according to claim 8 wherein all steps (a)
to (f) are carried out in microtitre plates.
10. The method according to claim 8 or 9 wherein the
microtitre plates have bar codes.
11. The method according to any one of claims 1 to 10
wherein the first robot is characterised by
(a) a digital image processing system for collecting the
plated bacteria,
(b) a working station with a grip arm for microtitre plates
for transferring the microtitre plates between the pro­
cessing stations,
(c) a separation module having one or more heads with
needles for picking the plated single colonies and for
placing them into the microtitre plates,
(d) integrated product processing stations for cleaning the
needles between the working steps and replicating the
placed single colonies in the microtitre plates and
(e) a computer-based bar code identification and tracking
system.
12. The method of any one of claims 1 to 4 wherein the
lysis is an alkaline lysis.
13. The method of any one of claims 1 to 12 wherein the
second robot is characterised by
(a) a conveyor road transport system combined with grip
arms for the microtitre plates for reloading the products
and for transferring the microtitre plates between the
product processing stations,
(b) product processing stations integrated into the trans­
port system, particularly centrifuges, pipetting
automats, shakers and incubation places for incubation
at different temperatures,
(c) a sensor technology for the detection of product
positions as well as for the detection of errors,
(d) a software for the interlaced handling of several
processes which are in the machine for a continual
production process and
(e) a computer-based bar code identification and tracking
system, preferably with an internal product tracking
containing a time stamp function for the interlacing of
time-critical sub-processes.
14. The method according to any one of claims 1 to 13
wherein the separation of the cellular DNA in step (c) is
carried out with silica particles.
15. The method according to claim 14 wherein the silica
particles are magnetic silica particles.
16. The method according to any one of claims 1 to 15
wherein the separation of the endotoxins in step (d) is carried
out with endotoxin-binding particles, which are preferably
magnetic endotoxin-binding particles.
17. The method according to any one of claims 1 to 15
wherein the separation of the endotoxins in step (d) is carried
out by precipitation with SDS/isopropanol.
18. The method according to any one of claims 14 to 16
wherein the DNA bound to silica particles is further purified
by washing with acetone.
19. The method according to any one of claims 1 to 18
wherein the transfection of cells in step (e) is mediated by
calcium phosphate, electroporation or by lipofactors.
20. The method according to any one of claims 1 to 18
wherein the transfection is carried out by means of DNA-
binding magnetic biocompatible micro-particles.
21. The method of any one of claims 1 to 20 wherein the
third robot is characterised by
(a) a conveyor road transport system combined with grip
arms for microtitre plates for reloading the products
and for transferring the microtitre plates between the
product processing stations,
(b) product processing stations integrated into the trans­
port system, particularly pipetting stations, shakers and
incubation places and an incubator for culturing the
transfectants,
(c) a sensor technology for the detection of product
positions as well as for the detection of errors,
(d) sterile overpressure ventilation to prevent contamina­
tions of the cell cultures,
(e) a software for the interlaced handling of several
processes which are in the machine for a continual
production process and
(f) a computer-based bar code identification and tracking
system, preferably with an internal product tracking
containing a time stamp function for the interlacing of
time-critical sub-processes.
22. The method of any one of claims 1 to 21 wherein the
fourth robot is characterised by
(a) a system for determining the fluorescence, lumines­
cence or colour reactions from cell culture assays,
(b) a pipetting station with a grip arm for microtitre plates
for transferring the microtitre plates from the incubator
to and between the product processing stations,
(c) processing places for adding and withdrawing cell
culture media or reagents and incubation in the incu­
bator and
(d) computer-based bar code identification and tracking
system.
23. The method according to any one of claims 1 to 21
wherein the fourth robot is characterised by
(a) a digital image processing system and image acquisi­
tion system for determining the cell morphology, fluo­
rescence and/or luminescence
(b) a pipetting station with grip arm for microtitre plates
for transferring the microtitre plates from the incubator
to and between the product processing stations,
US 2004/0265855 A1
10
Dec. 30, 2004
(c) processing places for adding and withdrawing cell
culture media or reagents and incubation in the incu­
bator and
(d) a computer-based bar code identification and tracking
system.
24. The method according to any one of claims 1 to 23
wherein the automated screening is a functional screening.
25. The method according to claim 24 wherein the func­
tional screening is a screening for an enzymatic, pharmaco­
logical or therapeutic property.
26. The method according to claim 14 or 25 wherein the
functional screening is a screening for activation or suppres­
sion of a reporter system or wherein the screening is a
screening for the function of a secreted protein.
27. The method according to any one of claims 1 to 26
wherein 2, 3 or 4 robots are arranged in a conveyor road.
28. The method according to any one of claims 1 to 27
wherein a DNA, (poly)peptide or a transfectant containing
the same, which has been identified in the screening process
is purified or isolated.
29. The method according to any one of claims 1 to 28
which moreover comprises the improvement of the binding
properties of the (poly)peptide encoded by the DNA iden­
tified or isolated in the screening process according to any
one of claims 1 to 28, comprising the steps of
(a) identification of the binding sites of the (poly)peptide
or its binding partner by site-specific mutagenesis or
chimeric protein studies;
(b) molecular modelling of the binding site of the
(poly)peptide and of the binding partner; and
(c) modification of the (poly)peptide in order to improve
the binding specificity or the affinity of the binding.
30. The method according to claim 29 wherein the modi­
fication in step (c) is a reproduction of the (poly)peptide by
peptidomimetics.
31. The method according to any one of claims 1 to 28
wherein the (poly)peptide as a leading structure is further
modified in order to obtain
(i) a modified site of action, a modified spectrum of
activity, a modified organ specificity, and/or
(ii) an improved activity, and/or
(iii) a decreased toxicity (an improved therapeutic index),
and/or
(iv) decreased side effects, and/or
(v) a delayed onset of the therapeutic action, of the
duration of the therapeutic effect and/or
(vi) modified pharmacokinetic parameters (resorption,
distribution, metabolism or exretion), and/or
(vii) modified physico-chemical parameters (solubility,
hygroscopic properties, colour, taste, odour, stability,
state), and/or
(viii) improved general specificity, organ/tissue specific­
ity, and/or
(ix) optimised application form and route by
(i) esterification of carboxyl groups, or
(ii) esterification of hydroxyl groups with carboxylic
acids, or
(iii) esterification of hydroxyl groups to e.g. phosphates,
pyrophosphates or sulfates or succinic acid semiesters,
or
(iv) formation of pharmaceutically acceptable salts, or
(v) formation of pharmaceutically acceptable complexes,
or
(vi) synthesis of pharmacologically active polymers, or
(vii) introduction of hydrophilic moieties, or
(viii) introduction/exchange of substituents in aromates or
side chains, change of the substituent pattern, or
(ix) modification by introduction of isosteric or bioisos-
teric moieties, or
(x) synthesis of homologous compounds, or
(xi) introduction of branched side chains, or
(xii) conversion of alkyl substituents to cyclic analogues,
or
(xiii) derivatisation of hydroxyl groups to ketals or
acetals, or
(xiv) N-acetylation to amides, phenylcarbamates, or
(xv) synthesis of Mannich bases, imines, or
(xvi) transformation of ketones or aldehydes to Schiff’s
bases, oximes, acetals, ketals, enolic esters, oxazo-
lidines, thiozolidines or combinations thereof.
32. The method for the manufacture of a pharmaceutical
composition comprising the steps of the method according
to any one of claims 23 to 31 and, moreover, formulating of
the substance obtained with a pharmaceutical acceptable
carrier or diluent.
(12) United States Patent
Harvey
US006423488B1
(io) Patent No.: US 6,423,488 B1
(45) Date of Patent: Jul. 23,2002
(54) HIGH THROUGHPUT SCREENING ASSAY
FOR DETECTING A DNA SEQUENCE
(75) Inventor: Alex J. Harvey, Athens, GA (US)
(73) Assignee: AviGenics, Inc, Athens, GA (US)
( * ) Notice: Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 0 days.
(21) Appl. No.: 09/760,048
(22) Filed: Jan. 13, 2001
Related U.S. Application Data
(60) Provisional application No. 60/176,255, filed on Jan. 15,
2000.
(51) Int. Cl.7......................... C07H 21/02; C12Q 1/68;
C12Q 1/70; A21C 11/16
(52) U.S. Cl................................. 435/5; 435/6; 536/23.1;
425/288.1
(58) Field of Search ....................... 435/5, 6; 536/23.1;
425/288.1
(56) References Cited
PUBLICATIONS
J. Sambrook et al., Molecular Cloning.*
“DNA Extraction from Nucleated Red Blood Cells”; J.F.
Medrano, E. Aasen, L. Sharrow, BioTechnques 8 (1), 43,
1990.
“Detection of specific polymerase chain reaction product by
ulilizing the 5'—»3'exonuclease activity of Thermits acquati-
cus DNA polymerase”; P.M. Holland, R.D. Abramson, R.
Watson, D.H. Gelfand, Proc. Natl. Acad. Sci. USA 88,
7260-7280, 1991.
“Genomic DNA Microextraction: A Method to Screen
Numerous Samples”; R. Ramirez-Solis, J. Rvera-Perez J.D.
Wallace, M. Wims, H. Zheng, A. Bradlely, Analytical Bio­
chemistry 201, 331-335, 1992.
“Isolation of Genomic DNA from Avian Whole Blood”; J.N.
Petitte, A.E. Kegelmeyer, M.J. Kulik, Bio Techniques 17 (4),
664, Jul. 14, 1994.
“Microplate DNA Preparation , PCR Screening and Cell
Freezing for Gene Targeting in Embryonic Stem Cells”;
G.B. Udy, M.J. Evans, BioTechniques 17 (5), 887-894, Aug.
16, 1994.
“Germline transmission of exogenous genes in chickens
using helper-free ecotropic avian leukosis virus-based vec­
tors”, P. Thoraval, M. Afanassieff, F.L. Cosset, F. Lasserre,
G. Verdier, F. Coudert, G. Dambrine, Transgenic Research 4,
369-376, 1995.
“Continuous Fluorescence Monitoring of Rapid Cycle DNA
Amplification”; C.T. Wittwer, M.G. Herrmann, A.A. Moss,
R.P. Rasmussen, BioTechniques 22(1), 130-138, Jan. 1997.
“Improved Protocol for Using Avian, Red Blood Cells as
Substrates for the Polymerase Chain Reaction”D. Bercov-
ich, Y. Plotsky, Y. Gruenbaum, BioTechniques 26 (6),
1080-1082, Jun. 1999.
* cited by examiner
Primary Examiner—Andrew Wang
Assistant Examiner—Karen A. Lacourciere
(74) Attorney, Agent, or Firm—Judy Jarecki-Black
(57) ABSTRACT
Genetic modification or selection of avians requires that
large numbers of birds be genetically analyzed for sequences
of interest. Typically, DNA is extracted on an individual
basis from samples taken from the birds. Current methods of
DNA extraction extract the DNA from blood or other tissues
using tedious and time-consuming procedures. The present
invention provides a high throughput screening assay for
detecting a genetic sequence in multiple samples. The assay
further provides a DNA extraction method that allows DNA
to be extracted rapidly from multiple avian samples, such as
red blood cells. Hie extraction method is extremely reliable
and does not require that each sample be quantitated post­
extraction. The extracted DNA can be used for a variety of
genetic assays, including a high throughput screening assay
to identify insertion of a transgene. The present invention is
particularly useful for extracting DNA from nucleated
RBCs. Therefore, the method can be applied towards genetic
analysis of avians, fish, reptiles and amphibians.
16 Claims, 8 Drawing Sheets
High tbroughpu t extrsrcfclmj of avian fetooti DNA.
J. Add bls'id to
lysis buffer 1.
bxv&ivx-?:-!r l|!|||| i _
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buffet 1 lysis
buffet' 2.
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c-thanol. dry, and ethaaoi. DNA t:> .Nuclei lyse,
resiisiwocl i:i. v.qste'. insttuoi of iveiS.
U.S. Patent Jul. 23,2002 Sheet 1 of 8 US 6,423,488 B1
Figure 1. High throughput extraction of avian blood BN A.
1. Add blood to
lysis buffer L
at} 0 ®
\
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2, Pia«a membranes
lyse* releasing
■cytoplasm into
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3, Pellet ttttctei. 4, Replace lysis
buffer I with lysis
buffer 2,
7. Wash with 70%
ethanol, dry, and
resuspend in water.
6, Precipitate with 5. Incubate at
ethanol DNA attaches to 65°C. Nuclei lyse,
bottom of well.
U.S. Patent Jul. 23,2002 Sheet 2 of 8 US 6,423,488 B1
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Neo for- 1
TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT
ACCTAACGTG CGTCCAAGAG GCCGGCGAAC CCACCTCTCC GATAAGCCGA
Neo probe Neo rev-1
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US 6,423,488 B1
1
HIGH THROUGHPUT SCREENING ASSAY
FOR DETECTING A DNA SEQUENCE
The present application claims the benefit of priority
from a provisional application filed Jan. 15,2000 and having
U.S. Ser. No. 60/176,255.
FIELD OF THE INVENTION
The present invention relates generally to a screening
assay and, more specifically, to a high-throughput screening
assay useful for detecting the presence of a foreign DNA
sequence in a sample. The present invention further includes
a high throughput extraction method for extracting DNA
from nucleated cells, particularly red blood cells.
BACKGROUND OF THE INVENTION
The present invention provides a high throughput screen­
ing assay useful for detecting the presence of an exogenous
DNA sequence in a sample. The method of the present
invention further includes a high throughput DNA extraction
method useful for extracting DNA from avian blood for
subsequent use in a screening assay as, for example, an assay
to detect the insertion of foreign DNA in the genome of a
recipient.
The publications cited herein to clarify the background of
the invention and in particular, materials cited to provide
additional details regarding the practice of the invention, are
incorporated herein by reference, and for convenience are
cited in the following text.
Transgenesis is the ability to introduce foreign or exog­
enous DNA into the genome of a recipient, as for example,
into a sheep, a cow or even a chicken. The ability to alter the
genome of an animal immediately suggests a number of
commercial applications, including the production of an
animal able to express an exogenous protein in a form that
is harvested easily.
The main obstacle to avian transgenesis is the low effi­
ciency of introduction of foreign DNA into the chicken
genome. The insertion of foreign DNA into the chicken
genome using procedures that have worked for other ani­
mals is a difficult task and attempts at such have been mostly
unsuccessful, partly due to the unique physiology of the
chicken (Love et al., Transgenic birds by DNA
microinjection, Biotechnology 12: 60-63,1994; Naito et al.,
Introduction of exogenous DNA into somatic and germ cells
of chickens by microinjection into the germinal disc of
fertilized ova, Mol Reprod Dev 37: 167-171, 1994).
Through the use of retroviruses, a number of research
groups have successfully introduced foreign DNA into the
chicken genome at acceptable but low efficiencies
(Bosselman et al., Germline transmission of exogenous
genes in the chicken, Science 243: 533-5, 1989;
Petropoulos, et al., Appropriate in vivo expression of a
muscle-specific promoter by using avian retroviral vectors
for gene transfer [corrected] [published erratum appears in J
Virol 66: 5175,1992]/ Virol 66: 3391-7, 1992; Thoraval. et
al., Germline transmission of exogenous genes in chickens
using helper-free ecotropic avian leukosis virus-based
vectors, Transgenic Res 4: 369-377, 1995). The retroviral
vectors used have been engineered such that they will not
result in the replication and spread of any new retroviruses.
This allows production of transgenic chickens that are free
of any retrovirus. However, because the retroviral vectors
cannot propagate in the chicken, the transgene is not trans­
mitted from cell to cell. Retroviral vectors are typically
injected into the embryo of a freshly laid egg through a small
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window in the egg shell. Approximately 1% of the embry­
onic cells are transduced, such that one copy of the transgene
is inserted into the cell’s DNA. After sexual maturity and
meiosis, 0.5% of sperm or oocytes carry the transgene. In
order to obtain one transgenic bird, at least 200 chicks have
to be screened. It is often desirable to obtain several trans­
genic chicks because different chromosomal insertions can
lead to different levels of transgene expression. Thus, it is
necessary to breed and screen hundreds to thousands of
chicks, necessitating a method for high throughput genetic
screening for detecting the desired genetic sequence.
Random chromosomal insertion of transgenes via non-
retroviral methods has become the mainstay of transgenics
in some domesticated animals including pigs, sheep, goats
and cows. The primary method to introduce the transgene is
the injection of linearized DNA containing the desired
transgene into the pro nucleus of a zygote. Up to 20% of Ga
offspring contain the transgene. The relative high efficiency
of transgenesis offsets the high technical costs incurred
during the procedure. Transgenes have been inserted into
goats, for instance, that direct the expression of pharmaceu­
ticals in mammary glands for subsequent secretion into milk
(Ebert, et al., Transgenic production of a variant of human
tissue-type plasminogen activator in goat milk: generation of
transgenic goats and analysis of expression, Biotechnology
9: 835-8, 1991).
In chickens, injection of the zygote germinal disk has
been accomplished but with limited success, in part due to
additional complications associated with unique aspects of
chicken physiology and embryogenesis (Love et al., 1994;
Naito et al., 1994). One lab has successfully produced
several transgenic chickens, which have incorporated the
injected DNA into their chromosomes and passed the trans­
gene on to offspring. Another lab attempted to reproduce the
technique but failed. Zygote injections in chickens are
difficult because the nucleus is very small and is about 50
microns below the yolk membrane. Thus, the DNA must be
injected into the cytoplasm. As in mice, cytoplasmic injec­
tion of DNA results in inefficient incorporation of the
transgene into the chromosomes. Chickens must be sacri­
ficed in order to remove the zygote and each chicken yields
only one zygote.
An important technical breakthrough was pioneered by
Gibbins, Etches, and their colleagues at the University of
Guelph by using blastodermal cells (BDCs) collected from
embryonic stage X embryos at oviposition, e.g., the time
when the egg is laid (Brazolot et al., Efficient transfection of
chicken cells by lipofection, and introduction of transfected
blastodermal cells into the embryo, Mol Reprod Dev 30:
304-12. 1991; Fraser, et al., Efficient incorporation of trans­
fected blastodermal cells into chimeric chicken embryos, Int
J Dev Biol 37: 381-5, 1993). Coupled with recent progress
in the culturing of BDCs, which can still reconstitute the
germline, the method theoretically enables random trans­
gene addition via nonhomologous recombination as well as
targeted gene engineering via homologous recombination.
At stage X, the embryonic blastoderm consists of 40,000
to 60,000 cells organized as a sheet (area pellucida) sur­
rounded by the area opaca; it harbors presumptive primor­
dial germ cells (PGCs) that have not yet differentiated into
migrating PGCs. Dispersed BDCs can be transfected with an
appropriate transgene and introduced into the subgerminal
cavity of y-irradiated, recipient stage X embryos. Irradiation
may selectively destroy presumptive PGCs and retard recipi­
ent embryo growth allowing injected cells additional time to
populate the recipient blastoderm. Using genetic markers for
feather color (black for Barred Rock and white for White
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Leghorn), Etches, Gibbins and their colleagues were able to
show that, of injected embryos surviving to hatch, 50% or
greater of these were somatic chimeras of which nearly half
were also germline mosaics (Petitte, et al., Production of
somatic and germine chimeras in the chicken by transfer of
early blastodermal cells, Development 108: 185-9, 1990).
Gibbins and her colleagues have determined that random
gene addition occurs in in vitro cultured BDCs in 1 out of
every 300 transfected cells (Gibbins and Leu, personal
communication). They did not determine whether BDCs
with random gene additions can be re-introduced into stage
X embryos to produce germline Ga chimeras. Therefore, the
actual efficiency of transgenesis has not yet been deter­
mined.
Gene targeting, the ability to specifically modify a specific
gene, is a much sought-after technology in a variety of
species, including chickens, because such modifications will
result in very predictable transgene expression and function.
Gene targeting has been successfully accomplished in mice
because mouse embryonic stem (ES) cells can be cultured in
vitro for long periods of time and still contribute to the
germline (Mountford et al., Dicistronic targeting constructs:
reporters and modifiers of mammalian gene expression,
Proc Natl Acad Sci USA 91: 4303-7, 1994). The long-term
culture of mouse ES cells allows the researcher to select for
and expand colonies of cells transfected with the targeting
vector that have the transgene inserted into the proper site.
Similar to the use of the feather color alleles in chimeric
birds, coat color of different breeds of mice are used to track
the donor cells in offspring. The difficulty in applying the
mouse ES cell technology to other species is that it has been
impossible to isolate ES cells of other species. While cells
resembling ES cells have been isolated from goats and pigs
and cultured in vitro, these cells are not able to contribute to
recipient embryos after long-term culture. Nuclear transfer
technology offers an alternative to the use of ES cells and it
is probable that gene targeting in animals will, in the future,
be implemented via nuclear transfer. Presently, however,
nuclear transfer is very inefficient and expensive, making its
implementation a slow process.
Recent advances in the in vitro short-term culture of
chicken blastodermal cells, combined with the unique physi­
ology of avian reproduction, indicate that gene targeting is
possible in chickens. The division rate of stage X BDCs can
be maintained in vitro at one division every 8-10 hours for
4-8 days using culture conditions developed by the Ivarie
laboratory (University of Georgia, Athens, Ga.) and
AviGenics, Inc. (Athens, Ga.) (Speksnijder and Baugh,
unpublished data). The ability to propogate BDCs in vitro at
this rate, while maintaining totipotency, will allow for the
rapid expansion of cell colonies containing the desired
genetic modification. This, combined with the fact that large
numbers of BDCs (40,000 to 60,000 cells/egg) can easily be
isolated from freshly laid chicken eggs, makes it feasible to
screen large numbers of transfected BDC colonies for those
having a desired gene of interest.
Currently, BDCs can only be cultured for 4 to 8 days
before they lose the ability to contribute to germ tissues in
the recipient embryo (Speksnijder and Baugh, unpublished
data). Therefore, it is likely that BDCs carrying the desired
genetic modification can only be enriched to perhaps 0.1 to
10% of the total number of donor cells. While sufficient to
enable gene targeting, the rate of transmission of the desired
genetic modification from chimeric founder animals (those
that were directly derived from injection of donor BDCs into
recipient embryos) to their offspring will be low. Elundreds
to thousands of offspring will have to be screened, again
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necessitating a method for high throughput genetic screen­
ing for detecting a desired sequence.
The enrichment of BDCs for desired genetic modifica­
tions can be applied to transgenesis projects involving
random insertion of a gene into the avian genome, as well as
modification of a specific gene. Therefore, a method for high
throughput genetic screening will have broad applications in
the fast-growing field of avian transgenesis.
To determine if an organism contains a novel or new gene,
DNA is extracted from a tissue sample (blood, skin, sperm)
and is subjected to an assay that will detect the gene. The
method of choice was the Southern assay, which is
extremely sensitive and reliable (Southern, E. M., Detection
of specific sequences among DNA fragments separated by
gel electrophoresis,/Mol Biol 98, 503-17,1975). Elowever,
the Southern assay is very labor intensive and time consum­
ing.
The Southern assay was replaced by the polymerase chain
reaction (PCR) method (Mullis et al., Specific enzymatic
amplification of DNA in vitro: the polymerase chain reac­
tion. Cold Spring Harbor Symp Quant Biol 51 (Pt 1):
263-73, 1986), which is a more sensitive and rapid assay.
Recently developed techniques, such as the TAQMAN
sequence detection system (Applied Biosystems, Foster
City, Calif.) allow hundreds of samples to be analyzed in
hours without requiring a time-consuming gel electrophore­
sis step (Heid et al. Real time quantitative PCR, Genome Res
6: 986-94, 1996). During a TAQMAN reaction run, which
is setup like a PCR reaction, a fluorogenic probe consisting
of an oligonucleotide with both a reporter and a quencher
fluorescent dye attached, anneals specifically between the
forward and reverse primers. The probe and primers are
complementary to the sequence of the desired transgene.
When the probe is cleaved by the 5' nuclease activity of Taq
DNA polymerase, the reporter dye is separated from the
quencher dye and a sequence-specific signal is generated.
With each cycle, additional reporter dye molecules are
cleaved from their respective probes, and the fluorescence
intensity is monitored during the PCR. Samples are analyzed
in 96-well plates and, at the end of a run, it is obvious which
samples contain the desired sequence.
While high throughput methods for sequence detection
are available, no comparable methods exist for the extraction
of DNA useful in a high throughput assay for sequence
detection. Rather, existing DNA extraction methods are still
labor intensive and time consuming. The majority of extrac­
tion methods require the DNA samples to be treated in
individual tubes. Samples are subjected to a number of steps,
including proteinase digestion, extraction with organic
solvents, and precipitation. The extraction step is particu­
larly problematic because of the awkwardness of manipu­
lation of the solution phases. Salting out has been used as an
alternative for extraction of unwanted proteins, but this
method requires multiple centrifugations and tube transfers.
Kits are available which avoid the extraction steps by using
DNA binding resins and allow for the processing of 96
samples at a time. However, the resins are not reusable, and
their use can result in poor yield and inconsistent DNA
quality. In addition, these kits are not cost-effective, costing
up to $3.00 per sample processed for extraction.
Existing methods for extracting DNA extraction from
multiple samples of avian tissue are labor intensive and
tedious. Avian blood, like all non-mammalian vertebrates,
has a special quality in that the erythrocytes are nucleated
(Rowley and Ratcliffe, Vertebrate blood cells, Cambridge
University Press, Cambridge, N.Y., 1988). The presence of
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nucleated cells allows one to extract a large amount of DNA
from a very small amount of blood. But existing DNA
extraction techniques have not taken advantage of this
aspect of avian blood. Grimberq. et al. developed a method
in which the plasma membrane, but not the nuclear
membrane, of red blood cells (RBCs) was lysed (Grimberg
et al., A simple and efficient non-organic procedure for the
isolation of genomic DNA from blood, Nucleic Acids Res
17: 8390, 1989). Subsequently, Petitte et al. augmented
Grimberg’s method by optimizing the initial lysis and spool­
ing ethanol-precipitated DNA out on a glass rod, resulting in
a more pure DNA preparation but requiring a more labor-
intensive protocol (Petitte, et al., Isolation of genomic DNA
from avian whole blood, Biotechniques 17: 664-6, 1994).
Thoraval, et al. used a similar procedure, however, each
sample was required to be treated individually (Thoraval et
al., Germlne transmission of exogenous genes in chickens
using helper-free ecotropic avian leukosis virus-based
vectors, Transgenic Res 4: 369-377, 1995).
All of the aforementioned procedures possess similar
disadvantages in that the each sample must be treated
individually and the DNA extracted must be transferred
between multiple tubes. In addition to being labor-intensive,
these DNA extraction procedures increase both the possi­
bility of contamination among samples and loss of DNA.
In order to target genes in mice, hundreds of mouse
embryonic stem (ES) cell colonies have to be individually
analyzed for the presence of the desired genetic modifica­
tion. In order to facilitate DNA extraction from a large
number of colonies, Ramirez-Solis et al. devised an inge­
nious method in which ES cells are lysed in 96-well plates
(Ramirez-Solis et al., Genomic DNA microextraction: a
method to screen numerous samples. Anal Biochem 201:
331-5, 1992). Using the method of Ramirez-Solis, et al.,
DNA is precipitated such that it sticks to the bottom of the
microtiter well without centrifugation. This is due in part to
the affinity of DNA for polystyrene, the major component of
96-well tissue culture plates. While the DNA is stuck to the
plates, all the unwanted protein and salts can be removed by
washing the wells multiple times with 70% ethanol. In this
way, 96 samples can be processed simultaneously. Because
the DNA is not transferred among tubes, the possibility of
both sample loss and contamination is minimized.
Ramirez-Solis et al. attempted to isolate DNA from
human blood samples using the above-described method,
however the inefficiency of the procedure required process­
ing a large volume of blood to obtain enough cells for
efficient extraction. At least 0.3 ml, and most probably about
1.0 ml, of human blood is required per well to obtain enough
DNA for efficient extraction, however the maximum capac­
ity of each microtiter well is only about 0.25 ml. Thus, the
method of Ramirez-Solis, et al. is not useful for the high
throughput extraction of DNA from genomic blood.
Udy and Evans developed a 96-well plate method for
DNA extraction from embryonic stem (ES) cells, similar to
the method of Ramirez-Solis et al., but never applied their
method to the extraction of DNA from blood (Udy and
Evans, Microplate DNA preparation, PCR screening and cell
freezing for gene targeting in embryonic stem cells, Bio­
techniques 17: 887-94,1994).
In view of the aforementioned deficiencies of the prior art,
there is a need for a method providing for the rapid and easy
extraction of DNA from a large number of blood samples
without necessitating large sample volumes or labor inten­
sive steps, or requiring the transfer of DNA between mul­
tiple tubes. Further, there is a need for a DNA extraction
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method that can be used in an high throughput assay to
rapidly screen a large number of samples to detect a desired
DNA sequence or transgene. Finally, there is a need for a
high-throughput assay useful for detecting the presence of a
desired genetic sequence in a large number of samples when
the copy number is low, i.e., between about 5 to about 50
copies.
SUMMARY OF THE INVENTION
The present invention recognizes and addresses the above
noted deficiencies and drawbacks of the prior art. The
present invention provides a rapid method for extracting and
preparing DNA for use in a subsequent high-throughput
screening assay. The method of the present invention is
especially useful for extracting DNA from avian blood for
use in a high throughput screening assay as, for example, an
assay to detect the insertion of foreign DNA in the genome
of a recipient.
The present invention is also directed to an assay useful
for rapidly screening a large number of nucleated blood
samples to detect a desired genetic sequence. In one embodi­
ment of the present invention, the nucleated blood samples
may be avian blood such as, for example, from a chicken or
turkey. The genetic sequence may be an endogenous DNA
gene or a foreign sequence such as, for example, a transgene,
or alternately, the genetic sequence may be a plasmid.
In one embodiment of the present invention, a nucleic
acid is isolated from a nucleated blood sample, particularly
an avian blood sample, by placing the sample in a microtiter
well, lysing the cells to release the DNA, precipitating the
nucleic acid within the well of the microtiter plate such that
the nucleic acid is attached to the well, removing any
extraneous material from the well by washing, and subject­
ing the isolated nucleic acid to a screening assay to detect a
desired genetic sequence.
The present invention also provides a high throughput
assay for detecting a desired sequence; the assay further
comprising a sequence tag that permits a desired genetic
sequence to be detected at low copy numbers even in the
presence of interfering genomic DNA. In one embodiment,
the high-throughput assay provides a sequence tag which
permits a target plasmid to be detected in the presence of
chicken genomic DNA at a level of from about 5 to about 50
copies.
BRIEF DESCRIPTION OF THE FIGURES
A full and enabling disclosure of the present invention,
including the best mode thereof, to one of ordinary skill in
the art, is set forth more particularly in the remainder of the
specification, including reference to the accompanying
Figures, in which:
FIG. 1 is a schematic illustrating the method of the present
invention;
FIG. 2 is a photograph of an agarose gel. DNA was
extracted from blood obtained from White Leghorn chickens
using either a conventional phenol-based method or the
method of the present invention. After extraction, DNA
samples were quantitated by absorbance at 260 nanometers
and 1, 2 and 5 fig of each sample was separated on a 0.8%
agarose gel. Samples extracted using the phenol-based
method are shown in lanes marked as L, while lanes marked
as H contain DNA samples extracted according to the
method of the present invention. Lane M contains a DNA
standard with molecular sizes indicated as kilobase pairs;
FIG. 3 is a graph illustrating results of an experiment
performed as described in Example 3, using the high
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throughput DNA extraction method of the present invention
with an assay to detect the insertion of the chicken
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
gene. Primers and a FAM/TAMRA-modified oligonucle­
otide probe complementary to the chicken GAPDF1 gene
was used in a TAQMAN reaction to confirm the reliability
of the high throughput DNA extraction method;
FIG. 4 is a graph illustrating results of an experiment
conducting high throughput screening of transgenic off­
spring according to the present invention. Using the DNA
extraction method of the present invention, DNA was
extracted from 82 chicks that were bred from a male that was
partially transgenic. A TAQMAN reaction with primers and
a TET/TAMRA-modified probe complementary to the bac­
terial neomycin resistance gene was used to detect the
presence of the transgene. Curves that did not demonstrate
an increase in ARn until after cycle 33 indicate that the
respective chicks were not transgenic. DNA extracted from
a transgenic chick gave rise to amplification at cycle 18. The
second curve that began to amplify at cycle 18 was gener­
ated by DNA extracted from the same chick on a different
96-well plate;
FIG. 5 is a schematic of a targeted gene showing the
sequence tag. The targeting vector is modified such that the
sequence tag (Tag) is inserted at the 3' end of the polyade-
nylation signal sequence (pA). Upon introduction of the
vector into the desired cells, the vector recombines with the
target gene. DNA is extracted from the cells and screened for
those with a targeted gene using a PCR assay with primers
NeoRev-1 and primer 2. A TAQMAN probe (Neoprobe) can
be added to the reaction if a realtime PCR reaction is to be
run;
FIG. 6 shows the nucleotide sequence of the sequence tag;
FIG. 7 is an agarose gel showing the results of an
experiment using the high throughput assay and sequence
tag of the present invention. Results showed detection of the
targeted gene at copy number, even in the presence of 150
ng of chicken genomic DNA. Each sample, comprising 10
microliters of a TAQMAN reaction, was run on a 1%
agarose gel and stained with ethidium bromide. Lane one is
one microgram of one kB DNA Ladder from Gibco-BRL.
Plasmid DNA is TTV-TTrev. The number of copies of
plasmid in each reaction is indicated above each lane. The
desired 941 bp product is indicated.
FIG. 8 is a graph depicting real-time PCR detection of a
targeted gene using the high throughput assay of the present
invention. The reactions were conducted as specified in FIG.
7 above.
DETAILED DESCRIPTION OF THE
PREFERRED EMBODIMENTS
Reference now will be made in detail to the presently
preferred embodiments of the invention, one or more
examples of which are illustrated in the accompanying
drawings. Each example is provided by way of explanation
of the invention, not limitation of the invention. In fact, it
will be apparent to those skilled in the art that various
modifications and variations can be made in the present
invention without departing from the scope or spirit of the
invention. For instance, features illustrated or described as
part of one embodiment, can be used in another embodiment
to yield a still further embodiment. It is intended that the
present invention covers such modifications and variations
as come within the scope of the appended claims and their
equivalents.
The present invention is directed to a high-throughput
assay useful for rapidly screening a large number of samples
8
to detect a desired genetic sequence as, for example, a
transgene or plasmid.
In one aspect of the present invention, a rapid method for
extracting and preparing DNA for use in the high-throughput
screening assay is provided. The method of the present
invention is especially useful for extracting DNA from
nucleated blood for use in a high throughput screening assay
including, but not limited to, a polymerase chain reaction
(PCR), ligase chain reaction (LCR), or other conventional
DNA detection assay for the detection of genetic markers or
foreign DNA in the genome of a recipient.
In one embodiment of the present invention, a high
throughput method for extracting DNA from multiple
samples of chicken blood is disclosed, as for example, from
White Leghorn and Barred Rock chicks and fully mature
birds. However, the method of the present invention can be
used for the high throughput extraction of a DNA from any
nucleated blood cell including, but not limited to, avian, fish,
reptile and amphibian nucleated blood cells.
The present invention provides a high throughput method
for extracting DNA from multiple blood samples containing
nucleated blood cells without requiring the repositioning of
the DNA into separate tubes or vessels during the extraction
procedure. In one embodiment, a nucleic acid such as DNA
is extracted from a nucleated blood sample, particularly an
avian blood sample, by placing the sample in a microtiter
well, lysing the cells to release the DNA, precipitating the
nucleic acid within the well of the microtiter plate such that
the nucleic acid is attached to the well, removing any
extraneous material from the well by washing, and subject­
ing the isolated nucleic acid to an assay.
The present invention further provides a sequence tag for
use in a high throughput assay to permit detection of the
desired genetic sequence at low copy numbers. For example,
in one embodiment, the sequence tag is used in the high
throughput assay of the present invention to allow a plasmid
to be detected at a level of from about 5 to about 50 copies
in the presence of chicken genomic DNA.
Also contemplated within the scope of the present inven­
tion is a high throughput DNA extraction method adapted
for use with blood from species other than avian. For
example, an alternate embodiment of the present invention
provides a DNA extraction method that uses mammalian red
blood cells in the high throughput assay of the present
invention. In one aspect of the present invention, mamma­
lian blood is enriched for those RBCs that are nucleated, as
with cell sorting, centrifugation, or the administration of a
hemopoietic agent, such that a sufficient amount of nucle­
ated cells can be transferred to each well of a microtiter plate
in a volume of 250 fil or less.
In yet another embodiment of the present invention, DNA
or other nucleic acid is extracted from nucleated cells other
than red blood cells. For example, white blood cells, includ­
ing granulocytes, neutrophils and mast cells, can be used in
the high throughput assay of the present invention.
The present invention may be better understood with
reference to the accompanying Examples, which Examples
are provided for the purpose of illustration and should not be
construed to limit the scope of the invention, which is
defined in the claims appended hereto.
EXAMPLE 1
DNA Extraction Method
Briefly, the protocol for DNA extraction from avian blood
according to the present invention is as follows:
A. To pre-chilled 96 well-flat bottom polystyrene tissue
culture plates, 0.2 ml (can go as high as 0.25 ml) of
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lysis buffer LB1 (containing 0.32 M sucrose, 10 mM
Tris-Cl, 5 mM MgC12, and 1% Triton X-100, at pH
7.5) was added to each well. Duplicate plates were set
up for each set of 96 chicks. The 96-well plates were
kept on ice until step C below.
B. One to 10 day old White leghorn chicks were heated
under a heat lamp to facilitate bleeding, and a heparin­
ized 0.05 ml capillary tube (Fisher, Pittsburgh, Pa.) was
filled half-full by pricking a leg vein. Over-filling the
capillary tube will allow too much blood to go into the
first 96-well plate. Upon filling the capillary tube, one
drop (about 8 microliter or VAh of the capillary) of
blood was transferred into one well and its duplicate,
each containing LB1. Following transfer, the blood and
LB1 were mixed in each well using the capillary tube.
If chicks older than 10 days are used as blood donors, a
25 G needle and 1 cc syringe primed with 0.05 ml of heparin
can be used to collect blood. Transfer one drop (about 8 fA)
into each well.
Note that the lysis solution can hold only so much blood,
otherwise the quality of the DNA will significantly decrease.
Add enough blood such that the lysis solution is light to
medium red. If significant clotting occurs, the cell pellet is
lost during subsequent steps, or the DNA appears yellow or
brown after resuspension, it is likely that too much blood
was added to LB1.
C. Each microtiter plate was centrifuged at about 960 g
(about 2000 rpm in a tabletop centrifuge) for 7 minutes
to pellet nuclei.
D. The supernatent was carefully aspirated from each
well, leaving a layer of nuclei remaining at the bottom
of each microtiter well. Most of the red color was gone.
E. 0.05 ml of lysis buffer 2 (LB2 containing 10 mM
Tris-Cl, 10 mM NaCl, 10 mM EDTA, and 1 mg/ml
proteinase K at pH 8.0) was added to each well, and the
plates incubated for 2 hours at 56-65° C.
F. To each well, 1.5 /A M NaCl and 0.01 ml cold ethanol
(premixed) was added, without mixing, and the plates
were left overnight at 4° C.
G. The supernatent was then removed by carefully invert­
ing the plate and pouring the supernatent into a large
beaker.
H. The pellet was washed 3-4 times with 70% ethanol,
using about 0.2 ml per well. The supernatent was
removed by carefully inverting the plate and, following
the last wash, the plate was blotted onto a paper towel.
I. Wells which lost their DNA were marked by holding up
the plates against a black background and marking
wells which had no dense white mat on the bottom of
the well.
J. The DNA samples were air-dried completely (as indi­
cated by complete transparency of the DNA) by incu­
bating the plates at 65° C. for one hour.
K. 0.2 ml PCR or DNA grade water was added to each
well, a sheet of Parafilm was placed over the wells, and
a lid tightly placed on top of the parafilm. The DNA
samples were allowed to resuspend overnight at 4° C.
The next day each plate was gently shaken at the lowest
speed on a vortexer with a microplate holder at room
temperature for 6-8 hours or overnight. The resulting
DNA solution appeared completely clear.
Referring now to FIG. 1, a schematic is provided to
illustrate the steps of the DNA extraction method according
to the present invention. As illustrated in the schematic, 8 to
12 fA of avian blood is added to lysis buffer 1 (LB1) in each
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well of a 96-well plate. After lysis of the red blood cell
plasma membrane occurs, the nuclei are spun down and the
supernatents containing cytoplasmic proteins are removed.
A proteinase K solution is added such that the bed of nuclei
is not disturbed. After lysis of the nuclei, a solution of
ethanol and NaCl is gently added. The chromosomal DNA
precipitates and forms a dense white mat which adheres
tightly to the bottom of the well. The DNA mat can be easily
washed with 70% ethanol several times without centrifuga­
tion. The solutions are simply poured off by hand between
each wash. After the last wash, the plate is inverted onto
some paper towels, dried and water is added to each well to
resuspend the DNA.
If the DNA extracted according to the present invention is
to be used in a qualitative assay, the amount of DNA present
in each well does not need to be quantitated. Rather, after the
last 70% ethanol wash and before drying, a visual inspection
of the plate will indicate which wells do not have an
adequate amount of DNA. A well containing an adequate
amount of DNA will have a dense white mat of DNA at its
bottom, which is easily visualized if the plate is held up
against a black background.
EXAMPLE 2
Average DNA Yield Using High Throughput DNA Extrac­
tion
Three separate DNA extraction experiments were con­
ducted using blood samples obtained from White Leghorn
chickens as described in Example 1 above. To quantify yield
following high throughput extraction, 2 ul of DNA was
added to 5 ul of Picogreen (Molecular Probes, Eugene,
Oreg.) in 1.0 ml of TE buffer (containing 0.1 M Tris-base,
and 0.005 M EDTA at pH 7.5). Samples were read on a
Turner Designs TD-700 Fluorometer using CsCl-banded
plasmid DNAquanitated by absorbance at A260 as a standard
Results of these experiments showed that 1 fA of DNA
extracted and resuspended according to the high throughput
method of the present invention typically contained 100 to
600 ng of genomic DNA. The average DNA yield was
approximately 340 ng/wl±120 ng/wl, as summarized in the
following table:
Yield using High Througput DNA Extraction
from Chicken Red Blood Cells
Experiment Average (ng//d) Standard deviation Number of samples
1 362.5 116.0 23
2 357.8 149.1 8
3 313.9 120.7 8
Referring now to FIG. 2, a photograph of an agarose gel
is presented which compares DNA extracted according to
the method of the present invention with that obtained using
a conventional phenol-based method (see, for example, the
standard phenol extraction protocol provided in “Molecular
Cloning: A Laboratory Manual, ” 2nd ed., J. Sambrook et
al., eds., Cold Spring Harbor Press, 1989 and Methods in
Plant Molecular Biology: A Laboratory Course Manual, P.
Maliga et al., eds., Cold Spring Harbor Press, 1994).
Blood obtained from White Leghorn chickens was
extracted according to either the high throughput method of
the present invention, as described in Example 1, or a
conventional phenol based method. After extraction, DNA
samples were quantitated by absorbance at 260 nanometers,
loaded onto an 0.8% agarose gel (at 1, 2 and 5 fig concen­
trations of DNA) and subjected to electrophoresis using a
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conventional protocol. The gel was visualized using an
ethidium bromide stain to compare the quality of the DNA
extracted according to the present invention (lanes marked
as H) with that extracted using a conventional phenol-based
technique (lanes marked as L). Lane M contains a DNA
standard with molecular sizes indicated. As can be seen in
FIG. 2, the quality of the DNA extracted using the high
throughput method of the present invention is comparable to
that extracted with the conventional technique.
EXAMPLE 3
Identification of a GPDF1 Transgene in the Chicken Genome
Using the Fligh Throughput Assay
To demonstrate the compatibility of DNA extracted
according to the present invention, two different TAQMAN
assays were performed. First, a primer/probe set comple­
mentary to the chicken glyceraldehyde-3-phosphate dehy­
drogenase (GAPDF1) was designed and made commercially.
The primers were made at Gibco BRL (Gaithersburg, Md.)
and the probe was synthesized by Operon Technologies
(Alameda, Calif.). The primers used were designed as fol­
lows:
chGAPDH-l: 5'-TCCCAGATTTGGCCGTA
TTG-3' (SEQ ID NO: 1)
and
chGAPDH-2: 5'-CCACrTGGACTTTGCC
AG AG A-3' (SEQ ID NO: 2).
The sequence of the chGAPDFl probe was
5'-CCGCCTGGTCACCAGGGCTG-3' (SEQ ID NO: 3).
The chGAPDFl probe was labeled with FAM
(6-carboxyfluorescin) at the 5' end and TAMRA (N,N,N',
N'-tetramethyl-6-carboxyrhodamine) at the 3' end. The
TAQMAN assay measures the increase of relative fluores­
cence due to hybridization of the chGPDFl probe to the PCR
product and the resulting endonucleolytic cleavage of the
probe. The cleavage releases the FAM molecule from the
probe so that its fluorescence is no longer quenched by
TAMRA.
TAQMAN reactions were carried out in 50 ul volumes by
adding 100 to 300 ng of DNA, extracted from blood
obtained from randomly-selected White Leghorn chicks
according to the method of the present invention described
in Example 1 above. To each reaction tube, 0.75xPCR
Buffer (Perkin-Elmer, Foster City, Calif.), 0.25xTAQMAN
buffer (Perkin-Elmer), 2.5 mM MgC12,5% DMSO, 125 fiM
dATP, 125 fiM dCTP, 125 fiM dGTP, 250 fiM UTP, 0.9 fiM
forward primer, 0.9 fiM reverse primer, 40 nM chGAPDFl
probe, 0.05 U/wl AmpliTaq Gold DNA Polymerase (Perkin-
Elmer), 0.004 U/fi\ and AmpErase UNG (Perkin-Elmer) was
added according to the manufacturer’s recommendations.
Reactions were analyzed on a Perkin-Elmer Applied Bio­
systems Sequence Detector Model 7700 using the following
conditions: 50° C. for 2 minutes, 950° C. for 10 minutes,
followed by 40 or 50 cycles of 95° C. for 15 seconds and 60°
C. for 1 minute.
Results of the TAQMAN reaction were visualized as an
increase in the fluorescence (eRn) during each cycle of the
PCR reaction. An increase in eRn at an earlier cycle indi­
cates the presence of more copies of that particular
sequence, whereas an increase in eRn at a later cycle
indicates that fewer copies of the sequence are present. Thus,
TAQMAN data can determine the presence of a specific
sequence and the relative quantity of that sequence.
FIG. 3 depicts the results of the TAQMAN amplification
assay measuring fluorescence at each cycle of the PCR
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reaction. The cycle number is shown on the x-axis (only
cycles 18-50 are shown). ARn is the increase of relative
fluorescence due to hybridization of the chGAPDH probe to
the PCR product and the resulting endonucleolytic cleavage
of the probe. As shown in FIG. 3, the three control samples
(blanks) produced overlapping curves that show no increase
in ARn, while the DNA samples obtained from all 21 White
Leghorn chicks gave rise to very similar amplification plots
showing hybridization of the probe to the chGAPDH gene.
These results indicate that the high throughput DNA extrac­
tion method of the present invention used with TAQMAN
amplification assay provides an accurate and consistent
method to detect the presence of a specific gene sequence in
genomic DNA.
EXAMPLE 4
Construction of a Sequence Tag for Use in a High Through­
put Genetic Sequence Method
A significant hurdle in the design of targeting vectors is
the inability to detect plasmids that mimic a targeted gene
using PCR. We found that, under a variety of conditions, the
limit of detection of test plasmids was 5000 copies or
greater. The main obstacle is that, when nanogram amounts
of chicken genomic DNA was added to a PCR reaction, the
reactions were significantly inhibited although, in the
absence of chicken DNA, detection limits of our assay were
10 to 50 copies. Chicken genomic DNA prepared by several
different methods and derived from different breeds of
chicken was tried, but all resulted in unacceptable detection
limits in the PCR assay, making it impossible to correctly
identify targeted cells.
Different primers can be tried to overcome this problem,
but this can be a costly and time-consuming process. In
certain cases, such as designing primers to detect integration
of a targeting vector into its target gene, the sequences from
which to choose the primers is limited to specific areas of the
targeting vector and the target gene. For instance, the primer
specific for the targeting vector should reside within the 3'
untranslated region (UTR) of the selection cassette.
However, most 3' UTRs are very short, limiting the choice
of potential primer binding sites. In addition, the primer
binding site should reside relatively close to the 3' end of the
3' UTR to keep the length of the PCR product as short as
possible. The longer the PCR product, the more inefficient
the PCR reaction.
In an attempt to improve the limits of detection in the
presence of chicken genomic DNA, a sequence tag, NeoR-
evl (SEQ ID NO.: 6) was constructed as is described in more
detail below. Results using the sequence tag with a template
in the high throughput assay of the present invention show
that the template can be detected in extremely low copy
numbers (5-20 copies) even in the presence of genomic
DNA (100 ng of chicken DNA). The NeoRevl sequence tag
can be used in combination with almost any primer that
anneals to a site downstream of NeoRevl and primes DNA
synthesis in the opposite direction.
A 62 bp sequence from the Neomycin resistance gene,
having the sequence GTG CCC AGT CAT AGC CGAATA
GCC TCT CCA CCC AAG CGG CCG GAG AAC CTG
CGT GCA ATC CA (SEQ ID NO.: 5), was cloned into the
bovine growth hormone 3' untranslated region (UTR) or
polyadenylation sequence such that the new sequence
resides just downstream of the UTR (see FIG. 6). This
positions a binding site for the primer NeoRevl (SEQ ID
NO.: 6) that will prime DNA synthesis away from the UTR
using a PCR reaction. The PCR reactions are relatively
insensitive to the type of polymerase used or the magnesium
concentration, an indication of the robustness of the reac­
tion.
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The inserted sequence contains a binding site for a
neomycin probe (Neoprobe; SEQ ID NO.: 7) that can be
used in a variety of real-time PCR reactions, including
TAQMAN (Perkin Elmer), allowing high throughput detec­
tion of a gene targeting event. The inserted sequence con­
tains a second primer binding site (NeoForl; SEQ ID NO.:
4) which primes synthesis in the direction opposite to that of
NeoRevl. The combination of these two primers and the
probe enables detection of this sequence, regardless of the
sequence context, in an efficient and high throughput man­
ner. Because the amplicon is short (62 bp), amplification is
highly efficient. This primer set can be used in a quantitative
PCR reaction (realtime or gel-based) to accurately determine
the copy number of the transgene. This would be useful, for
example, if a transgene has integrated randomly because, in
many cases of random insertion, multiple copies of the
transgene inserts. Thus, one is required to determine the
copy number of the transgene. A second example in which
copy number must be determined occurs when the animals
are bred to be homozygous for the transgene. In this case,
desired animals have twice as many copies of the transgene
as their parents or hemizygous (single copy) siblings.
Referring now to FIG. 5, a targeting vector was con­
structed by subcloning of the 62 bp sequence (SEQ ID NO.:
5) shown in FIG. 6 into a restriction site at the 3' end of the
polyadenylation signal. In this particular case, a 62 bp
product was produced by PCR by using the neomycin
resistance gene (E.coli Transposon Tn5) as the template and
using the following primers:
NeoForl: 5'-TGGATTGCACGCAGGTTCT-3' (SEQ ID NO.: 4),
and
NeoRevl: 5'-GTGCCCAGTCATAGCCGAAT-3' (SEQ ID NO.: 6).
The primers were kinased with T4 DNA Kinase and ATP
prior to PCR. The vector was cut with a restriction site that
produced a blunt end and ligated to the PCR product. A
subclone was selected in which the PCR product had
inserted in the reverse orientation such that the NeoRevl
primer primed DNA synthesis away from the polyadenyla­
tion signal, as shown in FIG. 5. For the purposes of this
application, this vector is referred to as Targeting Vector-
Transgene Tag-rev or TV-TTrev.
To mimic a targeted gene, the 3' flank of the targeting
vector, which is homologous to a region of the chicken
ovalbumin gene, was replaced by a longer segment of the
same region of the gene. This vector is referred to as
Targeting Test Vector-Transgene Tag-rev or TTV-TTrev.
A clone in which the PCR product was in the forward
orientation was also selected. In this case the NeoForl
sequence tag primes DNA synthesis away from the poly­
adenylation signal. The analogous test vector is referred to
as Targeting Test Vector-Transgene Tag-for or TTV-TTfor.
When this vector is used, the NeoForl sequence tag would
be used to prime DNA synthesis. Results comparing a high
throughput detection assay for TTV-TTfor using the Neo­
Forl sequence tag and OV18rev primer (SEQ ID NO.: 8;
5'-CAA TAG AAG ATT TAT ACT TGT TCT GTC TGT TT)
with an assay detecting TTV-TTrev with NeoRevl and
OV18rev show the NeoForl sequence tag and OV18rev
assay has a much lower sensitivity (10-100 fold) than that
of the TTV-TTrev and primers NeoRevl and OV18rev.
The sensitivity of detection using the NeoRevl sequence
tag was tested as follows: TAQMAN reactions were carried
out in 20 ul volumes and all reactions had 150 ng of White
Feghorn DNA, extracted from blood obtained from
14
randomly-selected chicks according to the method of the
present invention described in Example 1 above. To each
reaction tube, 0.75xPCR Buffer (Perkin-Elmer, Foster City,
Calif.), 0.25xTAQMAN buffer (Perkin-Elmer), 2.5 mM
MgC12, 5% DMSO, 125 dATP, 125 /rM dCTP, 125 /rM
dGTP, 250 tM UTP (dNTPS and UTP were from Perkin-
Elmer), 0.9 fjM Neofor-1, 0.9 fjM OV18rev, 40 nM
Neoprobe, 0.05 U/^mI AmpliTaq Gold DNA Polymerase
(Applied Biosystems, Foster City, Calif.) and 0.004 U/wl
AmpErase UNG (Perkin-Elmer) was added according to the
manufacturer’s recommendations. In some cases AmpliTaq
Gold DNA Polymerase was replaced with Promega Taq
DNA polymerase (Promega, Madison, Wis.). Additionally
the Perkin-Elmer dNTPs/UTP mixture can be substituted
with dNTPs from Roche (catalog number 1969064,
Indianapolis, Ind.). Reactions containing AmpliTaq Gold
DNA Polymerase were analyzed on a Perkin-Elmer Applied
Biosystems Sequence Detector Model 7700 using the fol­
lowing conditions: 50° C. for 2 minutes, 95° C. for 10
minutes, followed by 40 or 50 cycles of 95° C. for 20
seconds and 62.8° C. for 2 minutes, 30 seconds. The
following conditions were used when Promega Taq DNA
polymerase was in the reaction mixture: 94° C. for 2
minutes, followed by 40 or 50 cycles of 94° C. for 20
seconds and 62.8° C. for 2 minutes, 30 seconds.
FIGS. 7 and 8 show the results of PCR experiments using
the NeoRev-1 sequence tag (SEQ ID NO.: 6) as the forward
primer and OV18rev (SEQ ID NO.: 8) as .the reverse primer.
As can be seen from the agarose gel shown in FIG. 7, the
expected 941 bp band is detectable in as low as 5 copies of
plasmid DNA. FIG. 8 shows the results from a real-time
PCR detection experiment using the sequence tag in the
presence of 150 ng of chicken DNA. Results confirm the
detection of the desired gene sequence at a 5 copy level.
EXAMPLE 5
Detection of a Neomycin Resistance Gene in the Chicken
Genome Using the High Throughput DNA Extraction
Method
White Leghorn embryos were transduced with a retroviral
vector containing the bacterial neomycin resistance gene
(NeoR). Because of the inefficiency of transduction, even in
the best cases less than 1% of the embryonic cells, including
those that give rise to germ tissues, carry a copy of the
transgene. Males that arose from the transductions were bred
to non-transgenic White Leghorn hens. The resulting chicks
were hatched and DNA was extracted in duplicate via the
high throughput DNA extraction method described in
Example 1 above.
Detection of the neomycin resistance gene was performed
using the TAQMAN assay described in Example 3 above,
except that the sequence of the primers used was as follows:
NeoForl: 5'-TGGATTGCACGCAGGTTCT-3' (SEQ ID NO.: 4)
and
NeoRevl: 5'-GTGCCCAGTCATAGCCGMT-3' (SEQ ID NO.: 6).
The sequence of the TAQMAN probe (Neoprobe), designed
to be complementary to the bacterial neomycin resistance
gene, was 5'-CCTCTCCACCCAAGCGGCCG-3' (SEQ ID
NO.: 7). The Neoprobe was labeled with TET (tetrachloro-
6-carboxy-fluorescein) or FAM (6-carboxyfluorescin) at the
5' end and TAMRA (N,N,N',N'-tetramethyl-6-
carboxyrhodamine) at the 3' end. Reactions were carried out
as described in Example 3 above.
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FIG. 4 shows the results of the neomycin detection assay.
As can be seen in FIG. 4, only duplicate DNA samples from
a fully transgenic chick demonstrated an increase in eRn at
a sufficiently early cycle. The other samples began to
amplify after cycle 34 due to destabilization of the probe and
not due to detection of a specific sequence.
These results demonstrate the feasibility of using the
DNA high throughput extraction method of the present
invention with a TAQMAN assay designed to detect the
presence of a bacterial neomycin resistance transgene. The
results also demonstrate the feasibility of using the high
throughput DNA extraction method in conjunction with the
TAQMAN sequence detection system to screen large num­
bers of chicks for a desired transgene.
The method of the present invention has widespread
implications for the production of transgenic chickens. Not
only can this DNA extraction method be used to facilitate the
isolation of founder transgenic chicks, but also the method
can be used to facilitate the propogation of those chicks into
production flocks. Unless birds that are both homozygous
for the desired transgene are mated to each other, only a
percentage (50-75%) of offspring from a transgenic founder
will carry the transgene, necessitating the screening of
thousands of chicks for the desired transgene.
The method of the present invention also provides a
significant impact for the screening of genetic markers that
are associated with wanted or unwanted traits. Once
identified, these traits can be enriched or selected against to
produce genetically superior offspring using DNA extracted
according to the present invention coupled with a screening
assay.
Although preferred embodiments of the invention have
been described using specific terms, devices, and methods,
such description is for illustrative purposes only. The words
used are words of description rather than of limitation. It is
to be understood that changes and variations may be made
by those of ordinary skill in the art without departing from
the spirit or the scope of the present invention, which is set
forth in the following claims. In addition, it should be
understood that aspects of the various embodiments may be
interchanged both in whole or in part.
What is claimed is:
1. A method for the isolation of nucleic acid from nucle­
ated red blood cells, comprising the steps of:
a) obtaining a biological sample containing said nucleated
red blood cells;
b) adding the biological sample to a first lysis buffer to
lyse plasma membranes, wherein the first lysis buffer is
confined in a container that binds a precipitated nucleic
acid;
c) centrifuging the container to yield a supernatant and a
pellet;
d) removing the supernatant from the pellet in the con­
tainer;
e) adding a second lysis buffer to the pellet in the
container, after which the pellet is incubated in the
second lysis buffer for two hours to release a nucleic
acid;
f) precipitating the nucleic acid in the container with a
nucleic acid precipitating solution;
g) washing the nucleic acid in the container;
h) drying the nucleic acid in the container; and
i) dissolving the nucleic acid in the container in a solvent.
2. The method of claim 1, wherein the biological sample
is obtained from a mammal, a bird, a reptile, a fish or an
amphibian.
16
3. The method of claim 1, wherein the biological sample
is from a bird, a reptile, a fish or an amphibian.
4. The method of claim 1, wherein the biological sample
is from a bird.
5. The method of claim 1, wherein the container is
polystyrene.
6. The method of claim 1, wherein the biological sample
is blood.
7. The method of claim 1, wherein the first lysis buffer
comprises between about 0.05M and about 1.0M sucrose,
between about 5 mM and about 500 mM Tris-HCl, between
about 1 mM and about 50 mM MgCl2 and between about
0.1% w/vol and, about 10% w/vol Triton X-100, and a pH
between about 5.0 and about 9.0.
8. The method of claim 1, wherein the first lysis buffer
comprises between about 0.2M and about 0.5M sucrose,
between about 5 mM and about 50 mM Tris-HCl, between
about 1 mM and about 10 mM MgCl2 and between about
0.1% w/vol and about 5% w/vol Triton X-100, and a pH
between about 6.0 and about 8.0.
9. The method of claim 1, wherein the first lysis buffer
comprises about 0.32M sucrose, about 10 mM Tris-HCl,
about 5 mM MgCl2 and about 1% w/vol Triton X-100 and
a pH about 7.5.
10. The method of claim 1, wherein the second lysis
buffer comprises between about 5 mM and about 100 mM
Tris-HCl, between about 1 mM and about 100 mM NaCl,
between about 1 mM and about 100 mM EDTA, and a
protease, and a pH between about 5.0 and about 9.0.
11. The method of claim 1, wherein the second lysis buffer
comprises between about 5 mM and about 50 mM Tris-HCl,
between about 1 mM and about 50 mM NaCl, between about
1 mM and about 50 mM EDTA, and a protease, and a pH
between about 7.0 and about 9.0.
12. The method of claim 1, wherein the second lysis
buffer comprises about 10 mM Tris-HCl, about 10 mM
NaCl, about 10 mM EDTA, about 1 mg/ml proteinase K, and
a pH about 8.0.
13. The method of claim 1, wherein the container com­
prises a compartmentalized container.
14. The method of claim 1, wherein the container com­
prises a multi-we 11 plate.
15. The method of claim 1, wherein the nucleic acid
precipitating solution comprises about 0.05 M NaCl and
ethanol.
16. A method for the high-throughput isolation of nucleic
acid from a bird, comprising the steps of:
a) obtaining a plurality of blood samples containing
nucleated blood cells from a bird;
b) adding the blood samples of step a) to a first lysis
buffer, wherein the first lysis buffer comprises about
0.3M sucrose, about 10 mM Tris-HCl, about 5 mM
MgCl2 and about 1% wt/vol Triton X-100, at about pH
7.5, and wherein the first lysis buffer is confined in a
compartmentalized polystyrene container, and wherein
a blood sample of the plurality of blood samples is
isolated in a single compartment of the compartmen­
talized polystyrene container;
c) centrifuging the compartmentalized polystyrene con­
tainer to yield a plurality of supernatants and a plurality
of pellets;
d) removing the supernatants from the pellets in the
compartmentalized polystyrene container;
e) adding a second lysis buffer to the pellets in the
compartmentalized polystyrene container, wherein the
second lysis buffer comprises about 10 mM Tris-HCl,
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about 10 mM NaCl, about 10 mM EDTA, about 1
mg/ml proteinase K, at about pH 8.0, after which the
pellets are incubated in the second lysis buffer for two
hours thereby releasing nucleic acid samples from the
pellets;
f) precipitating the nucleic acid samples in the compart­
mentalized polystyrene container with nucleic acid
precipitating solution comprising about 0.05 M NaCl
and ethanol;
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g) washing the nucleic acid samples in the compartmen­
talized polystyrene container;
h) drying the nucleic acid samples in the compartmental­
ized polystyrene container; and
i) dissolving the nucleic acid samples in the compartmen­
talized polystyrene container in a solvent.