Title
Gradient decrement of annealing time can improve PCR with fluorescent-labeled primers
Abbreviated Journal Title
J. Biosci. Bioeng.
Keywords
Terminal restriction fragment length polymorphism (T-RFLP); PCR; amplification efficiency; Fluorescence labeling; Gradient-decreasing; annealing time PCR program; Microbial community analysis; 16S RIBOSOMAL-RNA; FRAGMENT-LENGTH-POLYMORPHISM; BACTERIAL COMMUNITY; STRUCTURE; RICE FIELD SOIL; MICROBIAL COMMUNITY; T-RFLP; CLONE; LIBRARIES; GENES; DIVERSITY; AMPLIFICATION; Biotechnology & Applied Microbiology; Food Science & Technology
Abstract
The 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. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.
Journal Title
Journal of Bioscience and Bioengineering
Volume
110
Issue/Number
4
Publication Date
1-1-2010
Document Type
Article
Language
English
First Page
500
Last Page
504
WOS Identifier
ISSN
1389-1723
Recommended Citation
"Gradient decrement of annealing time can improve PCR with fluorescent-labeled primers" (2010). Faculty Bibliography 2010s. 1000.
https://stars.library.ucf.edu/facultybib2010/1000
Comments
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