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Gradient decrement of annealing time can improve PCR with fluorescent-labeled primers

Paper ID Volume ID Publish Year Pages File Format Full-Text
21409 43220 2010 5 PDF Available
Title
Gradient decrement of annealing time can improve PCR with fluorescent-labeled primers
Abstract

ABSTRACTThe influences of fluorescence labeling on PCR amplification and T-RFLP analysis were examined by the analyses of a soil bacterial and archaeal community using both clone library and T-RFLP methods. The PCR amplification and microbial community structure patterns were compared among the primers labeled with and without fluorescent groups. PCR amplification was negatively affected by the labeling groups of the primers, which may be caused by the increment of primer molecular weight. It is known that thermodynamic movement of molecules will be slowed as molecular weight increased. Therefore it is understandable that the reaction of primer–DNA template hybridization will be inhibited with the fluorescent groups added to the primer(s). An effective “Gradient-Decreasing Annealing Time Program,” in which the annealing time was initially set long and reduced cycle by cycle, can improve PCR efficiency under comparable amplification specificity with the fluorescent-labeled primers. No significant negative impact was observed in the altered conditions.

Keywords
Terminal restriction fragment length polymorphism (T-RFLP); PCR amplification efficiency; Fluorescence labeling; Gradient-decreasing annealing time PCR program; Microbial community analysis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 110, Issue 4, October 2010, Pages 500–504
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us