Allele-specific-PCR combined with restriction enzyme digestion to detect mutations in JAK2V617F.docVIP

 PAGE \* MERGEFORMAT 4 Allele-specific-PCR combined with restriction enzyme digestion to detect mutations in JAK2V617F Author: Shen Wei Wu Zhang Mei-xiang Xu Liang Fang-Ping Chen Xiaoqi Shi Zhi Qin Xu Hongliang [Abstract] Objective: To establish a sensitive and specific JAK2V617F point mutation in the clinical detection methods. Methods: Genomic DNA from HEL cells or peripheral blood of normal volunteers extracted. Using allele-specific-PCR (Allele specific-PCR, AS-PCR) and restriction enzyme digestion method to detect genome JAK2V617F mutation. Results: The AS-PCR method can be right JAK2 gene was amplified 364 bp internal reference band, when there JAK2V617F mutation, but also the mutations in 203 bp amplified bands. Only in the light of the wild-type 364 bp band can be cut BsaX Ⅰ digestion. Conclusion: AS-PCR method and restriction enzyme is a sensitive and specific detection of JAK2V617F mutation detection methods can be successfully applied to clinical testing. [Keywords:] allele specific-PCR; JAK2V617F mutation; BsaX Ⅰ restriction endonuclease Abstract Objective: To establish a sensitive and specific test for JAK2V617F point mutation. Methods: Genomic DNA was isolated from HEL cells or Peripheral-blood cells from normal volunteer. Allele-specific polymerase chain reactions (AS-PCR) and restriction enzyme digestion were performed to detect the mutation in genomic DNA.Results: A control band (364 bp) can be obtained by AS-PCR, and a muted band (203 bp) can be obtained only when JAK2V617F was presented.Only wildtype control band (364 bp) can be digested by restriction enzyme BsaX Ⅰ . Conclusion: AS-PCR and restriction enzyme digestion were sensitive and specific tests for JAK2V617F point mutation, and can be successfully used for clinical test. Keywords: Allele specific-PCR; JAK2V617F mutation; Restriction enzyme BsaX Ⅰ JAK2 gene is JAKs (Janus kinases / Just another kinases) family members, is the nature of cytoplasmic non-receptor tyrosine kinase,