Nucleic Acid Amplification

摘要

The present invention provides improved methods for the amplification of nucleic acid molecules. Methods for amplifying target polynucleotides, including mRNA, using oligonucleotides, DNA and RNA polymerases are provided. The invention further provides compositions and kits for practicing the methods, as well as methods which use the amplification products.

主权项

1. A method of producing amplified RNA (aRNA), said method comprising
a) reverse transcribing an RNA template using a promoter-primer complex and an RNA dependent DNA polymerase to produce a first strand cDNA; b) optionally treating the reverse transcription product with RNAse H enzymatic activity; c) producing a second strand cDNA complementary to said first strand cDNA using a DNA dependent polymerase, optionally in the presence of random primers to prime synthesis of said second strand cDNA; and d) producing amplified RNA from the eluted double stranded cDNA by in vitro transcription using a DNA dependent RNA polymerase which initiates transcription from the primer of said promoter-primer complex; wherein after c) and before d) above, after d) above, or both, the product produced by c) or d) is purified by contacting said product with a solid phase which binds nucleic acids followed by eluting bound nucleic acids from said solid phase in an elution volume of less than 50 microliters.