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Real-time measurement of in vitro transcription.

Abstract：

:We have developed a simple method to measure RNA synthesis in real time. In this technique, transcription reactions are performed in the presence of molecular beacons that possess a 2'-O-methylribonucleotide backbone. These probes become fluorescent as they hybridize to nascent RNA during the course of synthesis. We found that molecular beacons synthesized from natural deoxyribonucleotides were not suitable, because they are copied by RNA polymerases, generating complementary product strands that bind to the molecular beacons, causing a conformational change that results in unwanted fluorescence. However, when the molecular beacons are synthesized from 2'-O-methylribonucleotides, they are not copied and fluorescence is strictly dependent upon transcription of the added template. Utilizing these modified molecular beacons, quantitative comparisons were made of the activity of a variety of RNA polymerases and the effect of an inhibitor of transcription was determined.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Marras SA,Gold B,Kramer FR,Smith I,Tyagi S

doi

10.1093/nar/gnh068

keywords:

subject

Has Abstract

pub_date

2004-05-20 00:00:00

pages

e72

issue

9

eissn

0305-1048

issn

1362-4962

pii

32/9/e72

journal_volume

32

pub_type

杂志文章

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