U.S. patent application number 10/136035 was filed with the patent office on 2003-09-04 for single primer pcr amplification of rna.
Invention is credited to Arnold, Lyle, Bjeldanes, Erik, Daniel, Steve.
Application Number | 20030165885 10/136035 |
Document ID | / |
Family ID | 24474205 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165885 |
Kind Code |
A1 |
Arnold, Lyle ; et
al. |
September 4, 2003 |
Single primer PCR amplification of RNA
Abstract
The invention provides methods and compositions for amplifying
RNA sequences by (a) hybridizing to a target RNA a first primer
comprising a 3' target RNA hybridizing sequence and a first 5'
defined amplifyable sequence; (b) extending the first primer with a
reverse transcriptase to form a first cDNA strand; (c) hybridizing
to the first cDNA strand a second primer comprising a 3' random
cDNA hybridizing sequence and a second 5' defined amplifyable
sequence; (d) extending the second primer with a DNA polymerase to
form a second cDNA strand; and (e) amplifying the second cDNA
strand with a third primer comprising the first 5' defined
amplifyable sequence.
Inventors: |
Arnold, Lyle; (Poway,
CA) ; Bjeldanes, Erik; (Lafayette, CA) ;
Daniel, Steve; (The Woodlands, TX) |
Correspondence
Address: |
RICHARD ARON OSMAN
SCIENCE AND TECHNOLOGY LAW GROUP
75 DENISE DRIVE
HILLSBOROUGH
CA
94010
|
Family ID: |
24474205 |
Appl. No.: |
10/136035 |
Filed: |
April 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10136035 |
Apr 29, 2002 |
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09617578 |
Jul 17, 2000 |
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6379932 |
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Current U.S.
Class: |
435/6.11 ;
435/6.16; 435/91.2 |
Current CPC
Class: |
C12Q 2521/107 20130101;
C12Q 2521/319 20130101; C12Q 2525/179 20130101; C12Q 2525/173
20130101; C12Q 2525/173 20130101; C12Q 2521/107 20130101; C12N
15/1096 20130101; C12Q 1/6844 20130101; C12Q 1/6844 20130101; Y10T
436/143333 20150115; C12Q 1/6844 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. A method for amplifying a RNA sequence, comprising the steps of:
(a) hybridizing to a target RNA a first primer comprising a 3'
target RNA hybridizing sequence and a first 5' defined amplifiable
sequence; (b) extending the first primer with a reverse
transcriptase to form a first cDNA strand; (c) hybridizing to the
first cDNA strand a second primer comprising a 3' random cDNA
hybridizing sequence and a second 5' defined amplifiable sequence;
(d) extending the second primer with a DNA polymerase to form a
second cDNA strand; and (e) amplifying the second cDNA strand by a
polymerase chain reaction with a third primer comprising the first
5' defined amplifiable sequence; wherein step (b) yields a
heteroduplex of the target RNA and the first cDNA and further
comprises the step of digesting the target RNA of the heteroduplex
with a RNAse sufficient to permit hybridization of the first cDNA
strand with the second primer without a melting step.
2. A method according to claim 1, wherein the 3' target RNA
hybridizing sequence is random.
3. A method according to claim 1, wherein the 3' target RNA
hybridizing sequence is nonrandom.
4. A method according to claim 1, wherein the 3' target RNA
hybridizing sequence is nonrandom and is complementary to a
predetermined sequence selected from a coding region, a poly A
junction, or a poly A tail.
5. A method according to claim 1, wherein the first and second 5'
defined amplifiable sequences are the same.
6. A method according to claim 1, wherein: the first and second 5'
defined amplifiable sequences are different, and the method further
comprises the step of functionally depleting the first primer
between steps (b) and (c).
7. A method according to claim 1, wherein: the first and second 5'
defined amplifiable sequences are different, the method further
comprises the step of functionally depleting the first primer
between steps (b) and (c), and step (e) further comprises
amplifying the second cDNA strand with a fourth primer comprising
the second 5' defined amplifiable sequence.
8. A method according to claim 1, wherein: the first and second 5'
defined amplifiable sequences are different, the method further
comprises the step of functionally depleting the first primer
between steps (b) and (c), step (e) further comprises amplifying
the second cDNA strand with a fourth primer comprising the second
5' defined amplifiable sequence, and the method further comprises
step: (f) amplifying the amplified cDNA with an excess of either
the third or fourth primer to form a predominantly single stranded
amplified probe of a predetermined orientation.
9. A method according to claim 1, wherein the method is practiced
in a single tube (homogeneous).
10. A method according to claim 1, wherein the method is practiced
in a single tube and the third primer of step (e) is added in
functional excess of the first primer.
11. A method according to claim 1, wherein the method comprises the
step of functionally depleting remaining first and second primers
between steps (d) and (e).
12. A method according to claim 1, wherein: the first and second 5'
defined amplifiable sequences are the same and the 3' target RNA
hybridizing sequence is random.
13. A method according to claim 1, wherein: the first and second 5'
defined amplifiable sequences are the same and the 3' target RNA
hybridizing sequence is nonrandom.
14. A method according to claim 1, wherein: the first and second 5'
defined amplifiable sequences are the same, the 3' target RNA
hybridizing sequence is nonrandom and the method is practiced in a
single tube.
15. A method according to claim 1, wherein: the 3' target RNA
hybridizing sequence is nonrandom and the first and second 5'
defined amplifiable sequences are different and the method further
comprises the step of functionally depleting the first primer
between steps (b) and (c).
16. A method according to claim 1, wherein: the 3' target RNA
hybridizing sequence is nonrandom, the first and second 5' defined
amplifiable sequences are different and the method further
comprises the step of functionally depleting the first primer
between steps (b) and (c) and the method is practiced in a single
tube.
17. A method for amplifying a RNA sequence, comprising the steps
of: (a) hybridizing to a target RNA a first primer comprising a 3'
target RNA hybridizing sequence and a first 5' defined amplifiable
sequence; (b) extending the first primer with a reverse
transcriptase to form a first cDNA strand; (c) hybridizing to the
first cDNA strand a second primer comprising a 3' random cDNA
hybridizing sequence and a second 5' defined amplifiable sequence;
(d) extending the second primer with a DNA polymerase to form a
second cDNA strand; and (e) amplifying the second cDNA strand with
a third primer comprising the first 5' defined amplifiable
sequence; wherein the 3' target RNA hybridizing sequence is random;
the first and second 5' defined amplifiable sequences are
different; and step (b) comprises the step of functionally
depleting the first primer to prevent it from hybridizing with the
first cDNA strand in subsequent steps.
18. A method according to claim 17, wherein step (e) further
comprises amplifying the second cDNA strand with a fourth primer
comprising the second 5' defined amplifiable sequence.
19. A method according to claim 17, wherein step (e) further
comprises amplifying the second cDNA strand with a fourth primer
comprising the second 5' defined amplifiable sequence and the
method further comprises step: (f) amplifying the amplified cDNA
with an excess of either the third or fourth primer to form a
predominantly single stranded amplified probe of a predetermined
orientation.
20. A method according to claim 17, wherein step (e) further
comprises amplifying the second cDNA strand with a fourth primer
comprising the second 5' defined amplifiable sequence and the
method further comprises step: (f) amplifying the amplified cDNA
with an excess of either the third or fourth primer to form a
predominantly single stranded amplified probe of a predetermined
orientation, and the method is practiced in a single tube.
21. A composition for amplifying a RNA sequence, said composition
comprising instructions reciting the steps of: (a) hybridizing to a
target RNA a first primer comprising a 3' target RNA hybridizing
sequence and a first 5' defined amplifiable sequence; (b) extending
the first primer with a reverse transcriptase to form a first cDNA
strand; (c) hybridizing to the first cDNA strand a second primer
comprising a 3' random cDNA hybridizing sequence and a second 5'
defined amplifiable sequence; (d) extending the second primer with
a DNA polymerase to form a second cDNA strand; and (e) amplifying
the second cDNA strand by a polymerase chain reaction with a third
primer comprising the first 5' defined amplifiable sequence;
wherein step (b) is said to yield a heteroduplex of the target RNA
and the first cDNA and further comprises the step of digesting the
target RNA of the heteroduplex with a RNAse sufficient to permit
hybridization of the first cDNA strand with the second primer
without a melting step.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority under 35USC120 to U.S. Ser.
No. 09/617,578, having the same title and inventors, filed on Jul.
17, 2000, now U.S. Pat. No. 6,379,932, which is incorporated herein
by reference.
INTRODUCTION
[0002] 1. Field of the Invention
[0003] The field of the invention is amplifying RNA.
[0004] 2. Background
[0005] RNA is a frequent starting material for genetic analysis,
such as microarray-based diagnostics and sequencing, and a wide
variety of methods have been devised to amplify RNA, generally by
first copying the RNA to cDNA and then using PCR and/or repeated
rounds of transcription to obtaine an amplified product. For
example, Silver et al. (1992) U.S. Pat. No.5,104,792; Liang et al.
(1997) U.S. Pat. No.5,599,672; and Shuber (1999) U.S. Pat.
No.5,882,856 describe methods for amplifying RNA. The present
invention provides an improved method of amplifying RNA which is
adaptable to total RNA input, low quantity input (100 pg or less
mRNA) and linear or quantitative PCR amplification.
SUMMARY OF THE INVENTION
[0006] The invention provides methods and compositions for
amplifying RNA sequences. In one aspect, the invention comprises
the steps of:
[0007] (a) hybridizing to a target RNA a first primer comprising a
3' target RNA hybridizing sequence and a first 5' defined
amplifiable sequence;
[0008] (b) extending the first primer with a reverse transcriptase
to form a first cDNA strand;
[0009] (c) hybridizing to the first cDNA strand a second primer
comprising a 3' random cDNA hybridizing sequence and a second 5'
defined amplifiable sequence;
[0010] (d) extending the second primer with a DNA polymerase to
form a second cDNA strand; and
[0011] (e) amplifying the second cDNA strand with a third primer
comprising the first 5' defined amplifiable sequence.
[0012] In one principal embodiment, step (b) yields a heteroduplex
of the target RNA and the first cDNA and further comprises the step
of digesting the target RNA of the heteroduplex with an RNase
sufficient to permit hybridization of the first cDNA strand with
the second primer without a melting step. In various applications,
the 3' target RNA hybridizing sequence may be random or nonrandom,
such as complementary to a predetermined sequence (e.g. a coding
region, a polyA junction, or a polyA tail), and the first and
second 5' defined amplifiable sequences may be the same or
different. In particular embodiments wherein the first and second
5' defined amplifiable sequences are different, the method further
comprises the step of functionally depleting the first primer
between steps (b) and (c); step (e) further comprises amplifying
the second cDNA strand with a fourth primer comprising the second
5' defined amplifiable sequence; and/or the method further
comprises step (f) amplifying the amplified cDNA with an excess of
either the third or fourth primer to form a predominantly single
stranded amplified probe of a predetermined orientation. In
particular applications, the method may be practiced in a single
tube (homogeneous assay).
[0013] In another principal embodiment, the 3' target RNA
hybridizing sequence is random; the first and second 5' defined
amplifiable sequences are different; and step (b) comprises the
step of functionally depleting the first primer to prevent it from
hybridizing with the first cDNA strand in subsequent steps.
[0014] In aspects of both principal embodiments, interference by
the first and/or second primers with the amplification step (e) may
be reduced by adding the third and/or fourth primer of step (e) in
functional excess of the first and/or second primer; and/or
functionally depleting remaining first and second primers between
steps (d) and (e).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0015] The following descriptions of particular embodiments and
examples are offered by way of illustration and not by way of
limitation. Unless contraindicated or noted otherwise, in these
descriptions and throughout this specification, the terms "a" and
"an" mean one or more and the term "or" means and/or.
[0016] The first step of the disclosed methods comprises (a)
hybridizing to a target RNA a first primer comprising a 3' target
RNA hybridizing sequence and a first 5' defined amplifiable
sequence. A wide variety of target RNAs may be employed, including
total cellular RNA, amplified RNA, purified RNA species such as
rRNA, tRNA or preferably, mRNA, etc. The first primer comprises a
3' sequence of length and sequence sufficient to hybridize with the
target RNA. Depending on the application, this 3' hybridizing
sequence may be random, specific or a combination of random and
specific sequences. For example, a primer population comprising
random 3' hybridizing sequences provides a "universal" primer set
capable of targeting any RNA species. In other embodiments, primers
comprising polyT 3' hybridizing sequences may be used to target
polyA tails of mRNA; primers comprising predetermined specific
sequences may be used to target particular, predetermined RNA
species comprising complementary sequences; and primers comprising
a random region joined to a wobble nucleotide (A, C or G) joined to
a polyT region may be used to target mRNA polyA junctions. The
first primer also comprises a first 5' defined amplifiable
sequence, which may be any sequences which can be used in the
subsequent specific amplification step and preferably comprises a
PCRable tag. Suitable reaction conditions for effecting
hybridization between the target RNA and first primer are known in
the art, readily ascertained empirically, and/or described and/or
exemplified herein.
[0017] The second step of the methods comprises (b) extending the
first primer with a reverse transcriptase to form a first cDNA
strand. Depending on the application, an RNAse activity may be
present during this step, which can effect the degradation of the
original RNA template subsequent to, or coincident with reverse
transcription, allowing, for example, priming of the new cDNA
strand with the same primer. In a preferred embodiment, the RNAse
activity is provided by the reverse transcriptase, such as Moloney
Murine Leukemia Virus (MMLV) reverse transcriptase, Avian
Myeloblastosis Virus (AMV) reverse transcriptase (both available
from Promega, Madison, Wis.), Rous Associated Virus 2 (RAV2) and
Human Immunodeficiency Virus 1 (HIV1) reverse transcriptases (both
available from Amersham Pharmacia), etc. In applications where the
3' target RNA hybridizing sequence is random and the first and
second 5' defined amplifyable sequences are different, RNAse
activity is preferably avoided so that second strand cDNA synthesis
does not occur in the same reaction mixture. Exemplary suitable
reverse transcriptases without RNase activity include MMLV-RT RNase
H minus (e.g Promega Catalog #M5301 and # M3682), display THERMO-RT
(Display Systems Biotech, Vista Calif.), Strat-Script RT
(Stratagene, San Diego, Calif.), etc.
[0018] The third step of the methods comprises (c) hybridizing to
the first cDNA strand a second primer comprising a 3' random cDNA
hybridizing sequence and a second 5' defined amplifiable sequence.
Note that depending on the application, the second primer may have
the same 3' hybridizing sequence and/or the same 5' defined
amplifiable sequences as does the first primer, or one or both
sequences may differ; see examples, below. For example, where the
first and second 5' defined amplifiable sequences are different,
the method may also comprise the step of functionally depleting the
first primer between steps (b) and (c). Functional depletion
reduces interference of the first primer with the second primer
extension reaction and may be effected by any convenient means such
as removal (e.g. size exclusion or affinity chromatography),
inactivation (e.g. hydrolysis, conjugation, etc.), etc.
[0019] The fourth step of the methods comprises (d) extending the
second primer with a DNA polymerase to form a second cDNA strand.
Suitable DNA polymerases and reaction conditions are known in the
art, readily ascertained empirically, and/or described and/or
exemplified below.
[0020] The fifth step of the method comprises (e) amplifying the
second cDNA strand with a third primer comprising the first 5'
defined amplifiable sequence. To reduce interference from the first
primer, the third primer of step (e) may be added in functional
excess of the first primer, and/or remaining first (and/or second
primer, if present and distinct from the first) may be functionally
depleted between steps (d) and (e). Depending on the particular
application, amplification step (e) may employ additional primers
and reactions. For example, where the first and second 5' defined
amplifiable sequences are different, step (e) may further comprise
amplifying the second cDNA strand with a fourth primer comprising
the second 5' defined amplifiable sequence. In a more particular
embodiment of this application, the method further comprises step
(f) amplifying the amplified cDNA with an excess of either the
third or fourth primer to form a predominantly single stranded
amplified probe of a predetermined orientation.
[0021] Preferred applications of the method reduce handling steps,
such as wash steps, inherent in prior art methods, preferably to
only a single wash step, more preferably to no wash steps wherein
the method is practiced continuously, preferably homogenously, and
in a single tube (i.e. container or reaction vessel).
[0022] The third and fourth primers comprise sequences identical to
those of the defined sequence portions of the first and second
primers and may contain optional detectable labels at positions
other than their 3' termini. The labels may be directly detectable,
as in the case of fluorescent or radio labels, or indirectly
detectable, as in the case of biotin, nitrophenol, or related
labels for which there are high affinity specific binding reagents
which contain directly detectable labels and which are used in
second binding reactions to measure the presence of the indirect
labels.
[0023] In a preferred mode, the sequences of the sequence specific
portions of the first primer and the second primer are identical.
In this mode, the third and fourth primers that are optionally
labeled are also identical, such that only two primers become
necessary for all amplification and labeling steps. In a further
preferred mode, the hybridization temperature of the first primer
portion that hybridizes to the mRNA and the second primer portion
that hybridizes to the cDNA are between 20.degree. C. and
45.degree. C., and the hybridization temperature of the sequence
specific portions of the first and second primers are between
50.degree. C. and 80.degree. C.
[0024] In yet other modes of this invention, labeling does not
occur during the amplification process, but is done after
amplification. In this mode, the amplification products can be
labeled by a variety of methods including the incubation of
reactive label reagents with sites on the DNA strands that include
the terminal hydroxyl group, exocyclic amines of the DNA bases, and
the bridging internucleotide phosphate groups. Alternatively,
labels may be incorporated by the process of nick-translation
employing appropriately labeled nucleotide triphosphates and an
appropriate DNA polymerase such as the Klenow fragment.
[0025] A wide variety of materials and methods are known in the art
for arraying polynucleotides at discrete elements of substrates
such as glass, silicon, plastics, nylon membranes, etc., including
contact deposition, e.g. U.S. Pat. Nos. 5,807,522; 5,770,151, etc.;
photolithography-based methods, e.g. U.S. Pat. Nos. 5,861,242;
5,858,659; 5,856,174; 5,856,101; 5,837,832, etc; flow path-based
methods, e.g. U.S. Pat. No. 5,384,261; dip-pen
nanolithography-based methods, e.g. Piner, et al., Science Jan. 29,
1999: 661-663, etc.; etc. In a preferred embodiment, the capture
polynucleotides are arrayed at corresponding discrete elements in
high density, generally at least 100, preferably at least 1000,
more preferably at least 10,000, most preferably at least 100,000
discrete elements per square centimeter.
[0026] In one principle application of the method, step (b) yields
a heteroduplex of the target RNA and the first cDNA and further
comprises the step of digesting the target RNA of the heteroduplex
with a RNase sufficient to permit hybridization of the first cDNA
strand with the second primer without a melting step. In particular
embodiments of this application, the first and second 5' defined
amplifiable sequences are the same and the 3' target RNA
hybridizing sequence is random; the first and second 5' defined
amplifiable sequences are the same and the 3' target RNA
hybridizing sequence is nonrandom; the first and second 5' defined
amplifiable sequences are the same, the 3' target RNA hybridizing
sequence is nonrandom and the method is practiced in a single tube;
the 3' target RNA hybridizing sequence is nonrandom, the first and
second 5' defined amplifiable sequences are different and the
method further comprises the step of functionally depleting the
first primer between steps (b) and (c); the 3' target RNA
hybridizing sequence is nonrandom, the first and second 5' defined
amplifiable sequences are different, the method further comprises
the step of functionally depleting the first primer between steps
(b) and (c) and the method is practiced in a single tube.
[0027] In a second principle application of the method, the 3'
target RNA hybridizing sequence is random; the first and second 5'
defined amplifiable sequences are different; and step (b) comprises
the step of functionally depleting the first primer to prevent it
from hybridizing with the first cDNA strand in subsequent steps.
Exemplary protocols for representative examples of these principle
applications are provided below.
EXEMPLARY EXPERIMENTAL PROTOCOLS
EXAMPLE 1
[0028] Random Prime from polyA
[0029] The cDNA microarrays were made according to U.S. Pat.
No.5,807,522 by Incyte Genomics, Inc. The oligonucleotides were
synthesized and purified by Operon Technologies, and used without
further purification.
[0030] The following oligonucleotide sequences were used in the
execution of the example:
1 NNNNNNNNNGTTTCCCAGTCACGATC (SEQ ID NO:1) GTTTCCCAGTCACGATC (SEQ
ID NO:2)
[0031] The oligonucleotide of SEQ ID NO:1 was used to randomly
prime a standard 1.sup.st strand cDNA reverse transcription
reaction using complex polyA purified human mRNA as template. M-MLV
(RNase H+) reverse transcriptase was used as the reaction's enzyme.
During the reverse transcription reaction, a 2.sup.nd cDNA strand
was synthesized which was complementary to the 1.sup.st cDNA
strand. This 2.sup.nd cDNA strand had the SEQ ID NO:2 on one end
and its reverse complement on the other end with an mRNA sequence
in the middle.
[0032] The 2nd strand cDNA product was then used as template for a
PCR reaction in which Seq. ID 2 with a 5' Cy3 or 5' Cy5 was used as
the universal primer for exponential amplification.
[0033] This Cy3 or Cy5 labeled, double stranded, PCR product was
then purified and concentrated to 25 uL and applied to the surface
of a cDNA microarray. It was hybridized in a solution of
5.times.SSC and 0.2% SDS at 50.degree. C. for 6 to 12 hours. After
hybridization, the hybridized microarray was then washed in a
solution of 1.times.SSC and 0.1% SDS for 10 minutes at 45.degree.
C. Immediately following the first wash, the microarray was washed
in a second wash of 0.1.times.SSC and 0.2% SDS for 3 minutes at
35.degree. C. The microarray was then scanned using an Axon Genepix
4000A microarray scanner and the hybridization pattern was
detected.
EXAMPLE 2
[0034] Random Prime from polyA With Nick Translation
[0035] The cDNA microarrays were made according to U.S. Pat.
No.5,807,522 by Incyte Genomics, Inc. The oligonucleotides were
synthesized and purified by Operon Technologies, and used without
further purification.
[0036] The following oligonucleotide sequences were used in the
execution of the example:
2 NNNNNNNNNGTTTCCCAGTCACGATC (SEQ ID NO:1) GTTTCCCAGTCACGATC (SEQ
ID NO:2)
[0037] The oligonucleotide of SEQ ID NO:1 was used to randomly
prime a standard 1.sup.st strand cDNA reverse transcription
reaction using complex poly A purified human mRNA as template.
M-MLV (RNase H+) reverse transcriptase was used as the reaction's
enzyme. During the reverse transcription reaction, a 2.sup.nd cDNA
strand was synthesized which was complementary to the 1.sup.st cDNA
strand. This 2.sup.nd cDNA strand had the SEQ ID NO:2 on one end
and its reverse complement on the other end with an mRNA sequence
in the middle.
[0038] The 2.sup.nd strand cDNA product was then used as template
for a PCR reaction in which Seq. ID 2 with a 5' Cy3 or 5' Cy5 was
used as the universal primer for exponential amplification.
[0039] This Cy3 or Cy5 labeled, double stranded, PCR product was
then purified and EtOH precipitated. This probe sample was then
used as the template for nick translation. The Promega kit: Nick
Translation System #U1001 was used, incorporating Cy3 or Cy5
labeled dCTP at a 4:1 cold to hot ratio.
[0040] The nick translated product was then concentrated to 25 uL
and applied to the surface of a cDNA microarray. It was hybridized
in a solution of 5.times.SSC and 0.2% SDS at 60.degree. C. for 6 to
12 hours. After hybridization, the hybridized microarray was then
washed in a solution of 1.times.SSC and 0.1% SDS for 10 minutes at
45.degree. C. Immediately following the first wash, the microarray
was washed in a second wash of 0.1.times.SSC and 0.2% SDS for 3
minutes at 25.degree. C. The microarray was then scanned using an
Axon Genepix 4000A microarray scanner and the hybridization pattern
was detected.
EXAMPLE 3
[0041] Amplify From Total Without Wobble
[0042] The cDNA microarrays were made according to U.S. Pat.
No.5,807,522 by Incyte Genomics, Inc. The oligonucleotides were
synthesized and purified by Operon Technologies, and used without
further purification.
[0043] The following oligonucleotide sequences were used in the
execution of the example:
3 ACTCAGCGTTGTTACCATTTTTTTTTTTTTTTTT (SEQ ID NO:3)
ACTCAGCGTTGTTACCA (SEQ ID NO:4) ACTCAGCGTTGTTACCANNNNNNNN- N (SEQ
ID NO:5)
[0044] The oligonucleotide of SEQ ID NO:3 was used to prime a
standard 1.sup.st strand cDNA reverse transcription reaction using
complex total RNA as template. M-MLV (RNase H+) reverse
transcriptase was used as the reaction's enzyme. After the reverse
transcription reaction, excess reagents were purified away and a
2.sup.nd reverse transcription reaction was performed. Using SEQ ID
NO:5, the second reverse transcription reaction generates cDNA
strands which are complementary to the 1.sup.st cDNA strand. This
2.sup.nd cDNA strand had SEQ ID NO:4 on one end and its reverse
complement on the other end with an mRNA sequence in the
middle.
[0045] The 2.sup.nd strand cDNA product was then used as template
for a PCR reaction in which SEQ ID NO:4 with a 5' Cy3 or 5' Cy5 was
used as the universal primer for exponential amplification. This
Cy3 or Cy5 labeled, double stranded, PCR product was then purified
and concentrated to 25 uL and applied to the surface of a cDNA
microarray. It was hybridized in a solution of 5.times.SSC and 0.2%
SDS at 50.degree. C. for 6 to 12 hours. After hybridization, the
hybridized microarray was then washed in a solution of 1.times.SSC
and 0.1% SDS for 10 minutes at 45.degree. C. Immediately following
the first wash, the microarray was washed in a second wash of
0.1.times.SSC and 0.2% SDS for 3 minutes at 35.degree. C. The
microarray was then scanned using an Axon Genepix 4000A microarray
scanner and the hybridization pattern was detected.
EXAMPLE 4
[0046] Amplify From Total RNA With Wobble
[0047] The cDNA microarrays were made according to U.S. Pat.
No.5,807,522 by Incyte Genomics, Inc. The oligonucleotides were
synthesized and purified by Operon Technologies, and used without
further purification.
[0048] The following oligonucleotide sequences were used in the
execution of the example:
4 ACTCAGCGTTGTTACCATTTTTTTTTTTTTTTTTV (SEQ ID NO:6)
ACTCAGCGTTGTTACCA (SEQ ID NO:4) ACTCAGCGTTGTTACCANNNNNNN- NN (SEQ
ID NO:5)
[0049] The oligonucleotide of SEQ ID NO:3 was used to prime a
standard 1.sup.st strand cDNA reverse transcription reaction using
complex total RNA as template. M-MLV (RNase H+) reverse
transcriptase was used as the reaction's enzyme. After the reverse
transcription reaction, excess reagents were purified away and a
2.sup.nd reverse transcription reaction was performed. Using SEQ ID
NO:5, the second reverse transcription reaction generates cDNA
strands which are complementary to the 1.sup.st cDNA strand. This
2.sup.nd cDNA strand had SEQ ID NO:4 on one end and its reverse
complement on the other end with an mRNA sequence in the
middle.
[0050] The 2.sup.nd strand cDNA product was then used as template
for a PCR reaction in which SEQ ID NO:4 with a 5' Cy3 or 5' Cy5 was
used as the universal primer for exponential amplification. This
Cy3 or Cy5 labeled, double stranded, PCR product was then purified
and concentrated to 25 uL and applied to the surface of a cDNA
microarray. It was hybridized in a solution of 5.times.SSC and 0.2%
SDS at 50.degree. C. for 6 to 12 hours. After hybridization, the
hybridized microarray was then washed in a solution of 1.times.SSC
and 0.1% SDS for 10 minutes at 45.degree. C. Immediately following
the first wash, the microarray was washed in a second wash of
0.1.times.SSC and 0.2% SDS for 3 minutes at 35.degree. C. The
microarray was then scanned using an Axon Genepix 4000A microarray
scanner and the hybridization pattern was detected.
EXAMPLE 5
[0051] Amplify From PolyA Pure Without Wobble
[0052] The cDNA microarrays were made according to U.S. Pat.
No.5,807,522 by Incyte Genomics, Inc. The oligonucleotides were
synthesized and purified by Operon Technologies, and used without
further purification.
[0053] The following oligonucleotide sequences were used in the
execution of the example:
5 ACTCAGCGTTGTTACCATTTTTTTTTTTTTTTTT (SEQ ID NO:3)
ACTCAGCGTTGTTACCA (SEQ ID NO:4) ACTCAGCGTTGTTACCANNNNNNNN- N (SEQ
ID NO:5)
[0054] The oligonucleotide of SEQ ID NO:3 was used to prime a
standard 1.sup.st strand cDNA reverse transcription reaction using
complex polyA purified human mRNA as template. M-MLV (RNase H+)
reverse transcriptase was used as the reaction's enzyme. After the
reverse transcription reaction, excess reagents were purified away
and a 2.sup.nd reverse transcription reaction was performed. Using
SEQ ID NO:5, the second reverse transcription reaction generates
cDNA strands which are complementary to the 1.sup.st cDNA strand.
This 2.sup.nd cDNA strand had SEQ ID NO:4 on one end and its
reverse complement on the other end with an mRNA sequence in the
middle.
[0055] The 2.sup.nd strand cDNA product was then used as template
for a PCR reaction in which SEQ ID NO:4 with a 5' Cy3 or 5' Cy5 was
used as the universal primer for exponential amplification. This
Cy3 or Cy5 labeled, double stranded, PCR product was then purified
and concentrated to 25 uL and applied to the surface of a cDNA
microarray. It was hybridized in a solution of 5.times.SSC and 0.2%
SDS at 50.degree. C. for 6 to 12 hours. After hybridization, the
hybridized microarray was then washed in a solution of 1.times.SSC
and 0.1% SDS for 10 minutes at 45.degree. C. Immediately following
the first wash, the microarray was washed in a second wash of
0.1.times.SSC and 0.2% SDS for 3 minutes at 35.degree. C. The
microarray was then scanned using an Axon Genepix 4000A microarray
scanner and the hybridization pattern was detected.
EXAMPLE 6
[0056] Amplify From PolyA Pure With Wobble
[0057] The cDNA microarrays were made according to U.S. Pat.
No.5,807,522 by Incyte Genomics, Inc. The oligonucleotides were
synthesized and purified by Operon Technologies, and used without
further purification.
[0058] The following oligonucleotide sequences were used in the
execution of the example:
6 ACTCAGCGTTGTTACCATTTTTTTTTTTTTTTTTV (SEQ ID NO:6)
ACTCAGCGTTGTTACCA (SEQ ID NO:4) ACTCAGCGTTGTTACCANNNNNNN- NN (SEQ
ID NO:5)
[0059] The oligonucleotide of SEQ ID NO:3 was used to prime a
standard 1.sup.st strand cDNA reverse transcription reaction using
complex polyA purified human mRNA as template. M-MLV (RNase H+)
reverse transcriptase was used as the reaction's enzyme. After the
reverse transcription reaction, excess reagents were purified away
and a 2.sup.nd reverse transcription reaction was performed. Using
SEQ ID NO:5, the second reverse transcription reaction generates
cDNA strands which are complementary to the 1.sup.st cDNA strand.
This 2.sup.nd cDNA strand had SEQ ID NO:4 on one end and its
reverse complement on the other end with an mRNA sequence in the
middle.
[0060] The 2.sup.nd strand cDNA product was then used as template
for a PCR reaction in which SEQ ID NO:4 with a 5' Cy3 or 5' Cy5 was
used as the universal primer for exponential amplification. This
Cy3 or Cy5 labeled, double stranded, PCR product was then purified
and concentrated to 25 uL and applied to the surface of a cDNA
microarray. It was hybridized in a solution of 5.times.SSC and 0.2%
SDS at 50.degree. C. for 6 to 12 hours. After hybridization, the
hybridized microarray was then washed in a solution of 1.times.SSC
and 0.1% SDS for 10 minutes at 45.degree. C. Immediately following
the first wash, the microarray was washed in a second wash of
0.1.times.SSC and 0.2% SDS for 3 minutes at 35.degree. C. The
microarray was then scanned using an Axon Genepix 4000A microarray
scanner and the hybridization pattern was detected.
EXAMPLE 7
[0061] Amplify PolyA Pure Random Onbead
[0062] The cDNA microarrays were made according to U.S. Pat.
No.5,807,522 by Incyte Genomics, Inc. The oligonucleotides were
synthesized and purified by Operon Technologies, and used without
further purification.
[0063] The following oligonucleotide sequences were used in the
execution of the example:
7 NNNNNNNNNGTTTCCCAGTCACGATC (SEQ ID NO:1) GTTTCCCAGTCACGATC (SEQ
ID NO:2)
[0064] The oligonucleotide of SEQ ID NO: 1 was used to randomly
prime a standard 1.sup.st strand cDNA reverse transcription
reaction using complex polyA mRNA which was captured onto oligo
dT/latex beads provided by the Qiagen mRNA Mini Kit. No elution of
the mRNA off of the latex beads was done. M-MLV (RNase H+) reverse
transcriptase was used as the reaction's enzyme. During the reverse
transcription reaction, a 2.sup.nd cDNA strand was synthesized
which was complementary to the 1.sup.st cDNA strand. This 2.sup.nd
cDNA strand had the SEQ ID NO:2 on one end and its reverse
complement on the other end with an mRNA sequence in the
middle.
[0065] The 2.sup.nd strand cDNA product was then used as template
for a PCR reaction in which Seq. ID 2 with a 5' Cy3 or 5' Cy5 was
used as the universal primer for exponential amplification. This
Cy3 or Cy5 labeled, double stranded, PCR product was then purified
and concentrated to 25 uL and applied to the surface of a cDNA
microarray. It was hybridized in a solution of 5.times.SSC and 0.2%
SDS at 50.degree. C. for 6 to 12 hours. After hybridization, the
hybridized microarray was then washed in a solution of 1.times.SSC
and 0.1% SDS for 10 minutes at 45.degree. C. Immediately following
the first wash, the microarray was washed in a second wash of
0.1.times.SSC and 0.2% SDS for 3 minutes at 35.degree. C. The
microarray was then scanned using an Axon Genepix 4000A microarray
scanner and the hybridization pattern was detected.
[0066] All publications and patent applications cited in this
specification and all references cited therein are herein
incorporated by reference as if each individual publication or
patent application or reference were specifically and individually
indicated to be incorporated by reference. Although the foregoing
invention has been described in some detail by way of illustration
and example for purposes of clarity of understanding, it will be
readily apparent to those of ordinary skill in the art in light of
the teachings of this invention that certain changes and
modifications may be made thereto without departing from the spirit
or scope of the appended claims.
Sequence CWU 1
1
6 1 26 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Sequence 1 nnnnnnnnng tttcccagtc acgatc 26 2 17 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Sequence 2 gtttcccagt cacgatc 17 3 34 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Sequence 3 actcagcgtt
gttaccattt tttttttttt tttt 34 4 17 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Sequence 4 actcagcgtt
gttacca 17 5 26 DNA Artificial Sequence Description of Artificial
Sequence Synthetic Sequence 5 actcagcgtt gttaccannn nnnnnn 26 6 35
DNA Artificial Sequence Description of Artificial Sequence
Synthetic Sequence 6 actcagcgtt gttaccattt tttttttttt ttttv 35
* * * * *