US 2003/0211528 A1Published application

Exponential amplification of sub-picogram nucleic acid samples with retention of quantitative representation

OwnerNorman Iscoveinventor

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Abstract

Within the near future it will be possible to survey expression of all genes in a sample by microarray analysis. Current methods for nucleic acid amplification require microgram amounts of complementary DNA or RNA for hybridization to microarrays. Without amplification, such amounts are only obtainable from millions of cells. However, frequently such numbers are not available: aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells are examples where few or even only single cells containing the desired information may be at hand. The current invention reduces the input amount of RNA needed for microarray analysis by a million-fold, and yields reproducible results from the picogram range of total RNA obtainable from a single cell. Of central importance to the present claims, the invention generates an amplified cDNA product in which the abundance relationships of the original RNA are faithfully preserved throughout amplification.

Claims27

Dependent claims

A process for generating nucleic acid targets or probes comprising the steps of:

(A) providing an RNA preparation that comprises polyadenylated mRNA;
(B) providing a first oligonucleotide primer that comprises
(1) a first segment containing a unique sequence;
and
(2) a second segment being substantially complementary to the polyadenylated mRNA and capable of template-dependent first strand synthesis;
(C) contacting the mRNA with the first primer to generate by DNA polymerase or reverse transcriptase reaction from the polyadenylated mRNA, DNA strands that are substantially complementary to the polyadenylated mRNA;
(D) adding a polynucleotide tail to the 3′ end of the DNA strands whereby the DNA strands have a first portion that is complementary to the polyadenylated mRNA and a tail portion;
(E) providing a second oligonucleotide primer that comprises
(1) a first segment containing a unique sequence;
and
(2) a second segment being substantially complementary to the tail portion of the DNA strand and capable of Template-dependent second strand synthesis;
(F) contacting the complementary DNA with the second primer to generate by a DNA polymerase reaction from the tailed DNA, DNA strands that are substantially complementary to the tailed DNA;
and
(G) contacting the DNA strands with the first primer and the second primer to amplify exponentially, by at least 1000-fold, the DNA strands by repetitive cycles of thermal denaturation, annealing and DNA polymerase reaction, to produce the targets or probes.

11 more elements

Dependencies are inferred from the claim text.