METHODS OF PREPARING AMPLIFIED NUCLEIC ACID MOLECULES

摘要

New and improved methods are provided for generating amplified nucleic acid molecules from cellular mRNA. The methods are robust and reliable, and can be used to provide gene fragments for use in methods of analyzing gene expression patterns. The methods comprise : (a) preparing double-stranded cDNA by : (i) hybridizing at least one primer comprising an RNA polymerase promoter to said population of RNA molecules and extending said primer by reverse transcription to generate single-stranded cDNA, and (ii) synthesizing double-stranded cDNA from said single-stranded cDNA by priming with an oligonucleotide mixture having a random sequence selected from the group consisting of a tetramer oligonucleotide mixture, a pentamer oligonucleotide mixture, a hexamer oligonucleotide mixture, a heptamer oligonucleotide mixture, an octamer oligonucleotide mixture, a nonamer oligonucleotide mixture, a decamer oligonucleotide mixture and mixtures thereof; and (b) transcribing amplified copies of anti-sense RNA from said double-stranded cDNA.