Abstract: AIM: To establish a pyrosequencing based method for detection LAMA5 rs944895 polymorphism and to determine the frequency of this polymorphism in healthy Chinese. METHODS: After preparation of gDNA from blood of 201 subjects, LAMA5 rs944895 target fragments were amplified by PCR, LAMA5 rs944895 polymorphism was detected on PyroMark ID by pyrosequencing technology. The reliability of pyrosequencing method was validated by repeat tests and Sanger sequencing. RESULTS: We established a new pyrosequencing method to detect the LAMA5 rs944895 polymorphism in healthy Chinese. The detection rate and repetition rate were both 100%. The frequencies of A allele and G allele were 72.4% and 27.6%, respectively. And the frequencies of AA, AG and GG genotype were 53.2%, 38.3% and 8.5%, respectively. The genotype frequencies match the Hardy-Weinberg equilibrium (P=0.55). CONCLUSION: This pyrosequencing assay to detect the LAMA5 rs944895 polymorphism is proved to be a rapid, accurate and high-throughput alternative to conventional methods, and it can be a preferred option in research and clinical application.

Key words: Pyrosequencing, Polymorphism, LAMA5, Sequencing