Colleen M Davis

US Patent:

20130344491, Dec 26, 2013

Filed:

May 17, 2013

Appl. No.:

13/896872

Inventors:

COLLEEN DAVIS - SEATTLE WA, US

Assignee:

LIFE TECHNOLOGIES CORPORATION - Carlsbad CA

International Classification:

C12N 15/10

US Classification:

435 612, 435 9121

Abstract:

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.