Hejtmancik J F, Holcomb J D, Howard J, Vanderford M
Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas.
Prenat Diagn. 1989 Mar;9(3):177-86. doi: 10.1002/pd.1970090306.
Discrimination of the M, Z, and S alleles of alpha 1-antritrypsin (AAT) has been carried out using in vitro gene amplification with the polymerase chain reaction (PCR). Amplification of 90 nucleotides surrounding the Z mutation site and 120 nucleotides surrounding the S mutation site dramatically improves the sensitivity and reliability of allele-specific oligonucleotide (ASO) hybridization for direct detection of these alleles. Analysis is performed using Southern blots or dot blots hybridized with 19 base oligonucleotides and differentially washed for allele specificity. Amplification of the Z and S mutation sites can be combined in one PCR to allow detection of both mutations when analysed by gel electrophoresis and Southern transfer. This technique can be performed reliably using less than 0.1 micrograms of genomic DNA or less than 100 amniocytes or white blood cells. This technique has been used to perform prenatal diagnosis on a chorionic villus sample (CVS) in a fetus at risk for the ZZ Pi type form of AAT deficiency.
利用聚合酶链反应（PCR）进行体外基因扩增，已实现对α1-抗胰蛋白酶（AAT）的M、Z和S等位基因的鉴别。围绕Z突变位点扩增90个核苷酸以及围绕S突变位点扩增120个核苷酸，可显著提高等位基因特异性寡核苷酸（ASO）杂交直接检测这些等位基因的灵敏度和可靠性。使用与19个碱基的寡核苷酸杂交的Southern印迹或斑点印迹进行分析，并进行差异洗涤以实现等位基因特异性。Z和S突变位点的扩增可在一次PCR中合并，以便在通过凝胶电泳和Southern转移分析时检测到两种突变。使用少于0.1微克的基因组DNA或少于100个羊膜细胞或白细胞就能可靠地进行这项技术。这项技术已用于对一名有ZZ Pi型AAT缺乏风险胎儿的绒毛膜绒毛样本（CVS）进行产前诊断。
