发明名称 |
RANDOM-PRIMED REVERSE TRANSCRIPTASE-<i>IN VITRO</i> TRANSCRIPTION METHOD FOR RNA AMPLIFICATION |
摘要 |
A random-primed reverse transcriptase-in vitro 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. |
申请公布号 |
WO0244399(A2) |
申请公布日期 |
2002.06.06 |
申请号 |
WO2001US44821 |
申请日期 |
2001.11.28 |
申请人 |
ROSETTA INPHARMATICS, INC.;ZIMAN, MICHAEL;DAVIS, COLLEEN, P. |
发明人 |
ZIMAN, MICHAEL;DAVIS, COLLEEN, P. |
分类号 |
C12N15/10;C12Q1/68 |
主分类号 |
C12N15/10 |
代理机构 |
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地址 |
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