HLA组特异性PCR及DNA测序确认新基因HLA-A*110202

目的:用HLA组特异性PCR扩增,DNA测序方法确认新等位基因。方法:用SSO(序列特异性寡核苷酸探针)反向流式荧光微珠法对标本62011550进行常规HL-A、B、DRB1分型检测,A位点反应格局清晰而无法给出确切分型结果,对该基因扩增2、3外显子,以此为模板采用组特异性引物区分杂合子,分别对杂合子2、3外显子测序,将测序结果与已知的基因序列进行比较。结果:测序得到A*1102v(1102的变异体)序列与已知序列比对发现,在第2对外显子215出现C>A,导致第75密码子GGA>AGA,未引起氨基酸改变(R>R),该序列为新的HLA序列,经向WHO HLA因子命名委员会申报确定为HLA-A 110202,注册号HWS.10003507-DQ3544411(www.ebi.ac.uk/imgt/hla)结论:DNA测序是HLA分型的最直接准确方法,常用于新基因的发现,用组特异性引物区分杂合子进而进行测序简便易行,避免了基因克隆操作。

Identification a new HLA allele A * 110202 using group specific PCR and DNA based sequencing

Objective:Conform a novel HLA-A*110202 by group specific PCR and DNA based sequencing.Methods: low resolution HLA-A,B,DRB1 typing was taken with PCRSSO LABMAS method(Lambda Array Beads Multi-Analyte System、),a sample with ID 62011550 have an ambiguous result A* 1102V、31 if beads 64、66 were FN(false negative) but the back ground of reaction for the whole beads is clear.Amplify the exon 2 and 3 of locus A,then distinguish A*11 and 31 with groups specific PCR before sequencing separately to determine the new HLA*A sequence.Results: Compare the new sequence with A*1102 a single nucleotide C>A can be find at position 215 of exon 2 region.a Change of GGA>AGA found which have the same amino acid codon,This new sequence was registered and named A*110202 by the WHO Nomenclature Committee for Factors of HLA,The registered number is HWS l0003507-DQ354441.Conclusion: DNA based sequencing is a golden stander HLA typing method with high resolution.The group specific primer PCR is an very convenient method to distinguish a heterozygote which can be difficult for direct sequencing.