应用MALDI-TOFMS检测5种白血病细胞株DCK和CDA基因部分单核苷酸多态性

Detection of SNPs of DCK and CDA genes in five leukemia cell lines with MALDI-TOF MS

目的:检测5种白血病细胞株中脱氧胞苷激酶(deoxycytidine kinase,DCK)和胞苷脱氨酶(cytidine deaminase,CDA)基因的单核苷酸多态性。方法:培养人红白血病细胞株K562,人慢性粒细胞白血病细胞株Ka,人急性髓系白血病细胞株HL-60、U937,人Burkkit淋巴瘤细胞株Raji。采用QIAamp DNA Blood Mini Kit提取基因组DNA,设计引物,PCR扩增相应目的片段,采用基质辅助激光解吸电离飞行时间质谱技术(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOFMS)检测DCK基因A674G(rs111454937)、C1644T(rs72552079),CDA基因A79C(rs2072671)、G208A(rs60369023)的单核苷酸多态性。结果:5种细胞株DCK基因A674G(rs111454937)均为A/A纯合子型,C1644T(rs72552079)均为C/C纯合子型。HL-60、U937、Raji细胞株CDA基因A79C(rs2072671)为A/A纯合子,K562、Ka细胞株为C/A杂合子;5种细胞株CDA基因G208A(rs60369023)均为G/G纯合子型。结论:CDA基因的A79C(rs2072671)SNPs位点基因型在5株血液系统肿瘤细胞株中不尽相同,余所测3个SNPs位点基因型在各细胞株中均相同。

OBJECTIVE： This study was aimed to detect single nucleotide polymorphisms （SNPs） of deoxycytidine kinase（DCK） gene,cytidine deaminase（CDA） gene in five different leukemia cell lines. METHODS：Cell line K562 Ka HL-60 U937 Raji were cultured. The genomic DNA was isolated by QIAamp DNA Blood Mini Kit. Designed primers were amplified by PCR,using the related DNA fragments. DCK gene A674G（rs111454937） C1644T （rs72552079）,CDA gene A79C（rs2072671） G208A（rs60369023） was genotyped by means of matrix assisted laser desorption ionisation-time of flight mass spectrometry method （MALDI-TOF MS）. RESULTS：The genotype of locus A674G （rs111454937） on DCK gene in all five leukemia cell lines was A/A. The genotype of locus C1644T （rs72552079） was C/C. The genotype of locus G208A（rs60369023） on CDA gene in all five hematology system cell lines was G/G. For HL-60,U937,Raji cell lines,the genotype of locus A79C（rs2072671） on CDA gene was A/A,while for K562,Ka cell lines it was C/A. CONCLUSION：The genotype of locus A79C（rs2072671） on CDA gene was different in the 5 tested cell lines of hematological malignancy. The genotypes of other 3 loci were the same in all 5 cell lines.