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

    Authors

    S. L. Yu; Y. Q. Tang; Y. Li; H. S. Zhang;X. L. Wu

    Comments

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    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

    WOS:000284342700020

    ISSN

    1389-1723

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