Application of Created Restriction Site PCR-RFLP to Identify POT1 Gene Polymorphism

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Tuanwei Wang^1#, Sihua Wang^1,2#, Xiaoran Duan^1#, Xiaolei Feng^1#, Pengpeng Wang¹, Wu Yao¹, Yongjun Wu³, Feifei Feng³, Songcheng Yu⁴, Yiming Wu¹, and Wei Wang^1*

¹Zhengzhou University, College of Public Health, Department of Occupational Health and Occupational Disease, 450001 Zhengzhou, China; fax: +86-371-6778-1466; E-mail: ww375@126.com

²Henan Institute of Occupational Health, Department of Occupational Health, 450052 Zhengzhou, China; E-mail: 13526723488@163.com

³Zhengzhou University, College of Public Health, Department of Hygiene Toxicology, 450001 Zhengzhou, China; E-mail: wuyongjun@zzu.edu.cn

⁴Zhengzhou University, College of Public Health, Department of Sanitary Chemistry, 450001 Zhengzhou, China; E-mail: scyu@zzu.edu.cn

^# These authors contributed equally to this work.

^* To whom correspondence should be addressed.

Received December 17, 2015; Revision received February 22, 2016

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Protection of telomeres protein 1 (POT1) plays pivotal roles in protection of chromosome ends and regulation of telomere length with other telomere binding proteins; its genetic polymorphisms are associated with many diseases. In this study, we explored a novel PCR-RFLP method for typing the single nucleotide polymorphism (SNP) rs1034794 of the human POT1 gene. A new restriction enzyme site was introduced into a POT1 gene amplification product by created restriction site PCR (CRS-PCR). One primer was designed based on changed sequence; after PCR amplification, a new restriction enzyme site for AluI was introduced into the PCR products. One hundred and seventy eight samples from Han Chinese individuals were tested to evaluate this new method. The 3′-end of the forward primer was next to the polymorphic site, and the third base from the 3′-end was the mismatched base A. The final PCR product contained the AGCT sequence (AluI recognition site) when the ancestral POT1 alleles were amplified. The data obtained with the new method perfectly matched those obtained with the sequencing method. Thus, CRS-PCR is a new low-cost and high-efficiency alternative for rs1034794 typing.

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KEY WORDS: created restriction site, single nucleotide polymorphism, protection of telomeres 1

DOI: 10.1134/S0006297916060092

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