Improving PCR efficiency in the promoter region of serum amyloid A-activating transcription factor

doi:10.3969/j.issn.2095-4344.2013.07.020

Abstract

Abstract:

BACKGROUND: Higher levels of guanine and cytosine in the promoter region of serum amyloid A-activating transcription factor are apt to cause unsuccessful PCR reactions.
OBJECTIVE: To optimize the conditions and amplification system for PCR amplification in the promoter region of serum amyloid A-activating transcription factor with rich guanine and cytosine.
METHODS: THP-1 cell genomic DNA was extracted as a template, to optimize amplification conditions through 97 ℃ denaturation and the best annealing temperature. In addition, different concentrations of dimethyl sulfoxide as an enhancer were added to improve the PCR amplification system. The method of promoting PCR efficiency in the promoter region of serum amyloid A-activating transcription factor with rich guanine and cytosine was established, and meanwhile, the effects of different-concentration dimethyl sulfoxide on PCR results were assessed.
RESULTS AND CONCLUSION: During the PCR cycling, the denaturing temperature of 97℃ at which template DNA unlocked the double-stranded and the annealing temperature increased to 60℃ were critical for PCR amplification specificity and efficiency. The use of 2% dimethyl sulfoxide was shown to improve the yield and specificity of PCR products in the promoter region of serum amyloid A-activating transcription factor. However, templates with different guanine and cytosine content had different dependence on the enhancer. It has been aproved that, during the PCR cycling, high denaturing temperature and high annealing temperature at which the primers bind to their specific template DNA unlocking the double-stranded are critical for specificity and efficiency of the PCR amplification. Templates with different guanine and cytosine contents, however, depend on dimethyl sulfoxide in different manners.

Key words: tissue construction, cytology experiments in tissue construction, basic experiments in tissue engineering, serum amyloid A-activating transcription factor, DNA, promoter region, guanine, cytosine, dimethyl sulfoxide, heat denaturation, annealing temperature, polymerase chain reaction, amplification efficiency, the National Natural Science Foundation of China, tissue construction photographs-containing paper

CLC Number:

R318

Cao Xi-mei, Liang Jun-hong, Zhang Chao, Wan Dong-fang, Meng Xiao-ping, Guo Da-wei. Improving PCR efficiency in the promoter region of serum amyloid A-activating transcription factor[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(7): 1251-1258.

Figures/Tables 6

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