Establishment of an optimized PCR method using sequence-specific primers for screening multiple gene polymorphisms simultaneously

Yang,Rui; Zhang,Jun-He; Yuan,Guo-Yan

doi:10.3892/mmr.2012.1122

Establishment of an optimized PCR method using sequence-specific primers for screening multiple gene polymorphisms simultaneously

Authors:
- Rui Yang
- Jun-He Zhang
- Guo-Yan Yuan
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Affiliations: Department of Life Science and Technology, Xinxiang Medical University, Xinxiang, Henan 453003, P.R. China, Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang, Henan 453003, P.R. China, Department of Neurosurgery, The First Affiliated Hospital of Xinxiang Medical University, Weihui, Henan 453100, P.R. China
Published online on: October 9, 2012 https://doi.org/10.3892/mmr.2012.1122
Pages: 201-204

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Abstract

The present study aimed to explore the factors that affect polymerase chain reaction using sequence-specific primers (PCR-SSP) and to establish an optimized PCR-SSP method for detecting multiple gene polymorphisms simultaneously. The amplification system parameters, including the concentrations of Mg2+, dNTPs, pfu Taq, primers and control primers, were optimized using the designed PCR-SSP reactions. The resulting optimized reaction system was used to determine the melting temperature of the genomic DNA and the cycling parameters. The optimized PCR-SSP method was used to analyze the polymorphisms of the following genes: mutations -308A/G and -238G/A in TNF-α, -174G/C in IL-6 and C/T mutation at exon 188 of CYP2D6 *10B. The PCR-SSP amplification system was optimized; in a 20 µl reaction system, the quantities of Mg2+, dNTPs, pfu Taq, primers, control primers and genomic DNA were 3.25 µM, 0.5 mM, 2.5 units, 0.5 µM, 0.2 µM and 0.15 µg, respectively. The cycling system comprised 5 start cycles and took 15 min to melt a genomic DNA sample using a touchdown protocol. The optimized PCR-SSP is suitable for polymorphism analysis of polygenic SNPs in large genomic DNA samples and a number of different genes.

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