Random-primed reverse transcriptase-in vitro transcription method for RNA amplification
US7229765B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Nov 28, 2001 |
| Grant date | Jun 12, 2007 |
| Priority date | — |
| Expiry date | May 6, 2023 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N15/1096
- WIPO fieldBiotechnology
- WIPO sectorChemistry
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.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.