中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2010年
6期
634-638
,共5页
王攀%叶璐夷%郭忠慧%朱自严
王攀%葉璐夷%郭忠慧%硃自嚴
왕반%협로이%곽충혜%주자엄
多重聚合酶链反应%定点诱变%等位基因%单核苷酸多态性
多重聚閤酶鏈反應%定點誘變%等位基因%單覈苷痠多態性
다중취합매련반응%정점유변%등위기인%단핵감산다태성
multiplex polymerase chain reaction%site-directed mutagenesis%allele%single nucleotide polymorphism
目的 应用基于聚合酶链反应(polymerase chain reaction,PCR)的基因定点诱变技术(sitedirected mutagenesis,SDM)制备s、Oka血型等位基因检测对照品.用多重聚合酶链反应(multiplex PCR)技术建立3种血型Fy2、s和Ok2的基因分型方法,以了解这3种血型在中国随机献血者中的多态性分布状况.方法 采用基于PCR的基因定点诱变技术对s和Oka血型等位基因的单核苷酸多态性(single nucleotide polymorphism,SNP)位点构建了含有突变SNP位点(GYPB基因cDNA 153位C/T突变和BSG基因cDNA 274位G/A突变)的标准质粒,作为s和Oka血型等位基因检测的对照品.同时针对血型抗原Fya、s和Oka等位基因的SNP位点设计序列特异性(sequence specific primer,SSP)引物,构建多重PCR体系,对438份随机献血者样本进行Fya、s和Oka血型抗原的基因筛选.结果 成功应用定点诱变技术完成了s和Oka基因检测中等位基因对照品的制备,并建立了可同时检测Fya、s和Oka血型的多重PCR体系,438份随机献血者样本中共检出2例Fy(a-)样本,未检出s-和Ok(a-)样本.结论 基于PCR的基因定点诱变技术能够得到难以获得的血型基因检测等位基因对照品,用于验证基因分型方法.本实验建立的多重PCR体系是一种有效的进行Fya、s和Oka血型基因检测的方法.
目的 應用基于聚閤酶鏈反應(polymerase chain reaction,PCR)的基因定點誘變技術(sitedirected mutagenesis,SDM)製備s、Oka血型等位基因檢測對照品.用多重聚閤酶鏈反應(multiplex PCR)技術建立3種血型Fy2、s和Ok2的基因分型方法,以瞭解這3種血型在中國隨機獻血者中的多態性分佈狀況.方法 採用基于PCR的基因定點誘變技術對s和Oka血型等位基因的單覈苷痠多態性(single nucleotide polymorphism,SNP)位點構建瞭含有突變SNP位點(GYPB基因cDNA 153位C/T突變和BSG基因cDNA 274位G/A突變)的標準質粒,作為s和Oka血型等位基因檢測的對照品.同時針對血型抗原Fya、s和Oka等位基因的SNP位點設計序列特異性(sequence specific primer,SSP)引物,構建多重PCR體繫,對438份隨機獻血者樣本進行Fya、s和Oka血型抗原的基因篩選.結果 成功應用定點誘變技術完成瞭s和Oka基因檢測中等位基因對照品的製備,併建立瞭可同時檢測Fya、s和Oka血型的多重PCR體繫,438份隨機獻血者樣本中共檢齣2例Fy(a-)樣本,未檢齣s-和Ok(a-)樣本.結論 基于PCR的基因定點誘變技術能夠得到難以穫得的血型基因檢測等位基因對照品,用于驗證基因分型方法.本實驗建立的多重PCR體繫是一種有效的進行Fya、s和Oka血型基因檢測的方法.
목적 응용기우취합매련반응(polymerase chain reaction,PCR)적기인정점유변기술(sitedirected mutagenesis,SDM)제비s、Oka혈형등위기인검측대조품.용다중취합매련반응(multiplex PCR)기술건립3충혈형Fy2、s화Ok2적기인분형방법,이료해저3충혈형재중국수궤헌혈자중적다태성분포상황.방법 채용기우PCR적기인정점유변기술대s화Oka혈형등위기인적단핵감산다태성(single nucleotide polymorphism,SNP)위점구건료함유돌변SNP위점(GYPB기인cDNA 153위C/T돌변화BSG기인cDNA 274위G/A돌변)적표준질립,작위s화Oka혈형등위기인검측적대조품.동시침대혈형항원Fya、s화Oka등위기인적SNP위점설계서렬특이성(sequence specific primer,SSP)인물,구건다중PCR체계,대438빈수궤헌혈자양본진행Fya、s화Oka혈형항원적기인사선.결과 성공응용정점유변기술완성료s화Oka기인검측중등위기인대조품적제비,병건립료가동시검측Fya、s화Oka혈형적다중PCR체계,438빈수궤헌혈자양본중공검출2례Fy(a-)양본,미검출s-화Ok(a-)양본.결론 기우PCR적기인정점유변기술능구득도난이획득적혈형기인검측등위기인대조품,용우험증기인분형방법.본실험건립적다중PCR체계시일충유효적진행Fya、s화Oka혈형기인검측적방법.
Objective To establish the controls for allele detection of blood groups s and Oka. A multiplex PCR method for the detection of three blood group antigens Fya, s and Oka was developed and used to investigate the distribution of these blood groups in Chinese random blood donors. Methods Polymerase chain reaction (PCR)-based, gene site-directed mutagenesis (SDM) technique were used to make site-directed mutagenesis for the single nucleotide polymorphism (SNP) sites of the blood group alleles (the 153 C/T point mutation of the GYPB gene, and the 274 G/A point mutation of the BSG gene) as controls for allele detection. Sequence specific primers were designed according to the SNP sites of alleles of blood group antigens Fya, s and Oka. A multiplex PCR system was developed and 438 random donor samples were screened for the blood group antigens Fya, s and Oka. Results The controls for alleles in blood groups s and Oka were successfully made with the SDM technique, a multiplex PCR system was set up and successfully used to analyze the genotypes of three blood group antigens Fya, s and Oka. Two Fy(a-)samples were detected in the 438 samples, no s- and Ok(a-) sample was found. Conclusion The PCR-based SDM technique can be used to obtain the unavailable controls in blood group genotyping. The multiplex PCR technique established in this study is an efficient genotyping method for blood groups Fya, s and Oka.
