Journal
ANALYTICAL BIOCHEMISTRY
Volume 439, Issue 2, Pages 201-203Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2013.04.024
Keywords
HIV-1; Digital PCR; Total HIV DNA; Touchdown PCR
Funding
- Research Foundation-Flanders (FWO) [1.8.020.09.N.00]
- Agency for Innovation by Science and Technology in Flanders (IWT) [111286, 111393]
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Digital polymerase chain reaction (PCR) is an emerging absolute quantification method based on the limiting dilution principle and end-point PCR. This methodology provides high flexibility in assay design without influencing quantitative accuracy. This article describes an assay to quantify HIV DNA that targets a highly conserved region of the HIV-1 genome that hampers optimal probe design. To maintain high specificity and allow probe binding and hydrolysis of a probe with low melting temperature, a two-stage touchdown PCR was designed with a first round of amplification at high temperature and a subsequent round at low temperature to allow accumulation of fluorescence. (C) 2013 Elsevier Inc. All rights reserved.
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