RNA amplification method
First Claim
1. A method for amplifying at least one mRNA in a sample containing a plurality of different mRNAs comprising:
- (a) synthesizing first strand cDNA by contacting under conditions conducive to reverse transcriptase activity at least one mRNA in said sample with (i) reverse transcriptase, and (ii) a first primer that is sufficiently complementary to a sequence in the mRNA so as to prime synthesis in a direction toward the 5′
end of the mRNA;
(b) synthesizing double-stranded cDNA by contacting under conditions conducive to DNA polymerase activity the first strand cDNA with (i) a first DNA polymerase, and (ii) a second primer that is sufficiently complementary to a sequence 5′
to said first primer sequence in said first strand cDNA so as to prime synthesis in a direction toward said first primer sequence;
wherein neither said first primer nor said second primer comprises an RNA polymerase promoter sequence in sense or antisense orientation;
(c) amplifying the double-stranded cDNA by subjecting the double-stranded cDNA to a single round of polymerase chain reaction (hereinafter “
PCR”
) of 20 cycles or less, wherein DNA is synthesized by use of a second DNA polymerase and a primer pair comprising a forward primer and a reverse primer, said forward primer and said reverse primer each being sufficiently complementary to a different strand of said double-stranded cDNA so as to prime synthesis in a template-dependent manner, wherein said forward primer or reverse primer comprises an RNA polymerase promoter sequence in sense or antisense orientation; and
(d) transcribing resultant amplified DNA into cRNA by contacting the amplified DNA with an RNA polymerase specific for said RNA polymerase promoter sequence introduced in step (c) under conditions conducive to RNA polymerase activity, such that cRNA is produced.
4 Assignments
0 Petitions
Accused Products
Abstract
The present invention relates to methods and kits for amplification of mRNA using a primer in PCR that contains an RNA polymerase promoter. The invention provides methods for amplification and detection of RNA derived from a population of cells, preferably eukaryotic cells and most preferably mammalian cells, which methods preserve fidelity with respect to sequence and transcript representation, and additionally enable amplification of extremely small amounts of mRNA, such as might be obtained from 106 mammalian cells. In typical embodiments of the invention, an RNA polymerase promoter (RNAP) is incorporated into ds cDNA by priming cDNA amplification by polymerase chain reaction (PCR) with an RNAP-containing primer. Following less than 20 cycles of PCR, the resultant RNAP-containing ds cDNA is transcribed into RNA using an RNA polymerase capable of binding to the RNAP introduced during cDNA synthesis. This combination of PCR and in vitro transcription (IVT) enables the generation of a relatively large amount of RNA from a small starting number of cells without loss of fidelity. RNAs generated using this method may be labeled and employed to profile gene expression in different populations of cells, e.g., by use of a polynucleotide microarray.
164 Citations
102 Claims
-
1. A method for amplifying at least one mRNA in a sample containing a plurality of different mRNAs comprising:
-
(a) synthesizing first strand cDNA by contacting under conditions conducive to reverse transcriptase activity at least one mRNA in said sample with (i) reverse transcriptase, and (ii) a first primer that is sufficiently complementary to a sequence in the mRNA so as to prime synthesis in a direction toward the 5′
end of the mRNA;
(b) synthesizing double-stranded cDNA by contacting under conditions conducive to DNA polymerase activity the first strand cDNA with (i) a first DNA polymerase, and (ii) a second primer that is sufficiently complementary to a sequence 5′
to said first primer sequence in said first strand cDNA so as to prime synthesis in a direction toward said first primer sequence;
wherein neither said first primer nor said second primer comprises an RNA polymerase promoter sequence in sense or antisense orientation;
(c) amplifying the double-stranded cDNA by subjecting the double-stranded cDNA to a single round of polymerase chain reaction (hereinafter “
PCR”
) of 20 cycles or less, wherein DNA is synthesized by use of a second DNA polymerase and a primer pair comprising a forward primer and a reverse primer, said forward primer and said reverse primer each being sufficiently complementary to a different strand of said double-stranded cDNA so as to prime synthesis in a template-dependent manner, wherein said forward primer or reverse primer comprises an RNA polymerase promoter sequence in sense or antisense orientation; and
(d) transcribing resultant amplified DNA into cRNA by contacting the amplified DNA with an RNA polymerase specific for said RNA polymerase promoter sequence introduced in step (c) under conditions conducive to RNA polymerase activity, such that cRNA is produced. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 75, 77, 78, 79, 80, 81, 82, 100, 101, 102)
-
-
45. A method for comparing the presence or amount of at least one mRNA of interest in a first sample and in a second sample, said first sample and said second sample each containing a plurality of different mRNAs from one or more cells, comprising:
-
(a) synthesizing first strand cDNA by contacting under conditions conducive to reverse transcriptase activity at least one mRNA in said first sample with (i) reverse transcriptase, and (ii) a first primer that is sufficiently complementary to a sequence in the mRNA so as to prime synthesis in a direction toward the 5′
end of the mRNA;
(b) synthesizing double-stranded cDNA by contacting under conditions conducive to DNA polymerase activity the first strand cDNA with (i) a first DNA polymerase, and (ii) a second primer that is sufficiently complementary to a sequence 5′
to said first primer sequence in said first strand cDNA so as to prime synthesis in a direction toward said first primer sequence;
wherein neither said first primer nor said second primer comprises an RNA polymerase promoter sequence in sense or antisense orientation;
(c) amplifying the double-stranded cDNA by subjecting the double-stranded cDNA to a single round of PCR of 20 cycles or less, wherein DNA is synthesized by use of a second DNA polymerase and a primer pair comprising a forward primer and a reverse primer, said forward primer and said reverse primer each being sufficiently complementary to a different strand of said double-stranded cDNA so as to prime synthesis in a templatedependent manner, wherein said forward primer or reverse primer comprises an RNA polymerase promoter sequence in sense or antisense orientation;
(d) transcribing resultant amplified DNA into cRNA by contacting the amplified DNA with an RNA polymerase specific for said RNA polymerase promoter sequence introduced in step (c) under conditions conducive to RNA polymerase activity, such that cRNA is produced;
(e) labeling the cRNA produced in step (d) with a first label;
(f) repeating steps (a)-(d) with said second sample;
(g) labeling the cRNA produced in step (f) with a second label distinguishable from said first label;
(h) detecting or measuring the mRNA of interest in the first sample by contacting the cRNA labeled with said first label with a polynucleotide probe capable of hybridizing to said cRNA of the mRNA of interest under conditions conducive to hybridization; and
detecting any hybridization that occurs between said probe and said cRNA;
(i) detecting or measuring the mRNA of interest in the second sample by contacting the cRNA labeled with said second label with said polynucleotide probe capable of hybridizing to said cRNA of the mRNA of interest under conditions conducive to hybridization; and
detecting any hybridization that occurs between said probe and said cRNA; and
(j) comparing the mRNA of interest detected or measured in said first sample with the mRNA of interest detected or measured in said second sample. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 83, 84, 85, 86)
-
-
87. A kit comprising in one or more containers:
-
(a) a mixture of first primers, each first primer comprising a 3′
end sequence of 1-5 nucleotides, said mixture of first primers comprising primers having an A, a G, or a C nucleotide present in each position of said 3′
end sequence;
wherein each primer in said mixture of first primers further comprises at its 5′
end an identical selected sequence of 5-12 nucleotides, and optionally further comprises an oligo (dT) sequence situated between said 5′
end sequence and said 3′
end sequence;
(b) a mixture of second primers, each second primer comprising at its 5′
end an identical selected sequence of 8-12 nucleotides, and at its 3′
end a sequence of 1-6 nucleotides, said mixture of second primers comprising primers having an A, a G, a C, or a T nucleotide present in each position of said 3′
end sequence;
wherein neither said first primer nor said second primer comprises an RNA polymerase promoter sequence in sense or antisense orientation; and
(c) a primer pair suitable for use in PCR, said primer pair comprising a forward primer and a reverse primer, wherein said forward primer or reverse primer comprises an RNA polymerase promoter sequence in sense or antisense orientation. - View Dependent Claims (88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99)
(h) a set of directions for carrying out PCR; and
(i) a set of directions for carrying out transcribing of amplified double-stranded cDNA into cRNA.
-
-
96. The kit of claim 87 further comprising a control nucleic acid.
-
97. The kit of claim 87 further comprising a microarray.
-
98. The kit of claim 97 further comprising means for stimulating and detecting fluorescent light emissions from fluorescently labeled RNA.
-
99. The kit of claim 98 further comprising expression profile projection and analysis software capable of being loaded into the memory of a computer system.
Specification