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Published online 20 May 2004

Nucleic Acids Research, 2004, Vol. 32, No. 9 e72
© 2004 Oxford University Press

Real-time measurement of in vitro transcription

Salvatore A. E. Marras*, Benjamin Gold, Fred Russell Kramer, Issar Smith and Sanjay Tyagi

Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA

*To whom correspondence should be addressed. Tel: +1 973 854 3373; Fax: +1 973 854 3374; Email: marras{at}phri.org
Present address:
Benjamin Gold, Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Received March 23, 2004; Revised and Accepted April 16, 2004

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.


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