RNA amplification method

Details RNA amplification method RNA amplification method

1999-10-04

2001-08-07

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S004000, C435S005000, C435S006120, C435S091100, C435S091200, C435S007100, C435S288200, C436S501000, C536S024300, C536S023400

Reexamination Certificate

active

06271002

ABSTRACT:

1. INTRODUCTION
The present invention relates to oligonucleotide primers for amplification of nucleic acids that comprise a synthetic oligonucleotide containing an RNA promoter (an “RNAP-containing” primer). The present invention also relates to methods for amplification and detection of nucleic acids using RNAP-containing primers, and, more specifically, to 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 10
6
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 multiple 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.
2. BACKGROUND OF THE INVENTION
Gene expression is important for understanding a wide range of biological phenomena, including development, differentiation, senescence, oncogenesis, and many other medically important processes. Recently, changes in gene expression have also been used to assess the activity of new drug candidates and to identify new targets for drug development. The latter objective is accomplished by correlating the expression of a gene or genes known to be affected by a particular drug with the expression profile of other genes of unknown function when exposed to that same drug; genes of unknown function that exhibit the same pattern of regulation, or signature, in response to the drug are likely to represent novel targets for pharmaceutical development.
Generally, the level of expression of the protein product of a gene and its messenger RNA (mRNA) transcript are correlated, so that measuring one provides you with reliable information about the other. Since in most instances it is technically easier to measure RNA than to measure protein, variations in mRNA levels are commonly employed to assess gene expression in different cells and tissues or in the same cells and tissues at different stages of disease or development or exposed to different stimuli. One particularly useful method of assaying gene expression at the level of transcription employs DNA microarrays (Ramsay, Nature Biotechnol. 16: 40-44, 1998; Marshall and Hodgson, Nature Biotechnol. 16: 27-31, 1998; Lashkari et al., Proc. Natl. Acad. Sci. (USA) 94: 130-157, 1997; DeRisi et al., Science 278: 680-6, 1997).
Mammalian cells contain as many as 1×10
5
to 3×10
5
different mRNA molecules, each of which varies in abundance (or frequency) within a given cell. The most abundant mRNAs are typically present at many thousands of copies per cell, while others may be present in as few as one copy or less per cell. Techniques for analyzing gene expression at the level of transcription typically require tens to hundreds of micrograms of mRNA, or as much as might be found in 10
7
-10
9
mammalian cells. Oftentimes, it is impractical to obtain this number of cells from a tissue of interest. For example, a blood sample typically contains
10
6
nucleated cells/ml; hence, to obtain 10
9
cells for analysis would necessitate taking a 1000 ml blood sample, which is clearly impractical in most instances.
Various methods have been described in the literature for amplifying the amount of a nucleic acid, such as deoxyribonucleic acid (DNA) and RNA, present in a sample. Among these, the most widely practiced is the polymerase chain reaction (PCR), described in U.S. Pat. No. 4,683,195 (Mullis et al., entitled “Process for amplifying, detecting, and/or-cloning nucleic acid sequences,” issued Jul. 28, 1987) and U.S. Pat. No. 4,683,202 (Mullis, entitled “Process for amplifying nucleic acid sequences,” issued Jul. 28, 1987), and herein incorporated by reference. Briefly, PCR consists of amplifying denatured, complementary strands of target nucleic acid by annealing each strand to a short oligonucleotide primer, wherein the primers are chosen so as to flank the sequence of interest. The primers are then extended by a polymerase enzyme to yield extension products that are themselves complementary to the primers and hence serve as templates for synthesis of additional copies of the target sequence. Each successive cycle of denaturation, primer annealing, and primer extension essentially doubles the amount of target synthesized in the previous cycle, resulting in exponential accumulation of the target.
When PCR is used to amplify mRNA into double-stranded (ds) DNA, the polyadenylated (poly(A)+) fraction is first selected, then a complementary DNA (cDNA) copy of the mRNA is made using reverse transcriptase and an oligo(dT) or random primer. The products of this reaction can be amplified directly using random priming (Ausubel et al., eds., 1994,
Current Protocols in Molecular Biology,
vol. 2, Current Protocols Publishing, New York).
PCR methodologies in general suffer from several limitations that are well-known in the art; see U.S. Pat. No. 5,716,785 (Van Gelder et al., entitled “Processes for genetic manipulations using promoters,” issued Feb. 10, 1998) and Innis et al., eds. (1990,
PCR Protocols: A Guide to Methods and Applications,
Academic Press Inc., San Diego, Calif.) for review and discussion of limitations. One such limitation is due to the poor fidelity of commonly used, thermostable polymerase enzymes, such as Taq. This results in nucleotide base misincorporations that are propagated from one cycle to the next. It is estimated that such misincorporations may occur as often as once per one thousand bases of incorporation. A second limitation is that different cDNAs are amplified with different efficiencies, resulting in underrepresentation of some cDNA sequences and overrepresentation of others in the amplified product. Even a small difference in efficiency may result in a several-thousand fold differential in the representation of these cDNAs in the product after only 30 cycles of amplification.
An alternative method of mRNA amplification is known as in vitro transcription (IVT) and is described in U.S. Pat. No. 5,716,785 (Van Gelder et al., entitled “Processes for genetic manipulations using promoters,” issued Feb. 10, 1998), which is herein incorporated by reference. Unlike PCR, IVT does not result in geometric amplification, but rather in linear amplification. In IVT, an oligo(dT) primer that is extended at the 5′-end with a bacteriophage T7 RNA polymerase promoter (RNAP) is used to prime the poly-A+ mRNA population for cDNA synthesis. After synthesis of the first-strand cDNA, the second-strand cDNA is made using the method of Gubler and Hoffman (Gene 25:263-69, 1983). Addition of RNA polymerase results in in vitro transcription and linear amplification of mRNA that is anti-sense to the poly-A+ RNA. While this method does not suffer from the same limitations in fidelity of amplification as PCR, it is also not as sensitive as PCR and requires a much larger sample of mRNA to generate the same amount of material as PCR. Because of its low efficiency, IVT is thus sometimes not a realistic alternative.
Two other types of nucleotide amplification are described in Stoflet et al. (Science 239:491-494, 1988) and in Sarkar & Sommer (Science 244: 331-334, 1989). Stoflet et al. (Science 239:491-494, 1988) describes a variation of PCR known as genomic amplification with transcript sequencing (GAWTS). In GAWTS, a phage promoter is attached to at least one of the two PCR primers. The DNA segments amplified by

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