Braun A, Meyer P, Cleve H, Roscher A A
Dr. von Haunersches Kinderspital, Ludwig-Maximilians-Universität München, Abteilung für Klinishce Chemie und Biochemie, Germany.
Eur J Clin Chem Clin Biochem. 1996 Sep;34(9):761-4. doi: 10.1515/cclm.1996.34.9.761.
We describe a simple DNA-based method to assign the two common alpha 1-proteinase inhibitor (alpha 1-antitrypsin) deficiency alleles in the Pi-system (PiZ and PiS). Two sets of mutated primers are used in the polymerase chain reaction (PCR), followed by a restriction enzyme digest of the products. The mutated forward primers create a Taq I site only if the wildtype alleles (mostly M or subtypes) are present and not in the presence of the PiZ or PiS alleles. The reverse primers are mutated for an invariant Taq I site which serves as an internal control site in order to assure the completion of the restriction enzyme digest. The digested PCR products can be clearly resolved by 2.5% MetaPhore-agarose gel electrophoresis. This simple PCR probing of the most common alpha 1-antiproteinase deficiency alleles can be routinely applied either to samples showing quantitatively decreased alpha 1-antiproteinase values in serum or to blood spots of Guthrie cards used for mass screening purposes. In addition, this method may provide the opportunity for a simple, rapid, and reliable prenatal diagnosis of alpha 1-antiproteinase deficiency in special cases.
我们描述了一种基于DNA的简单方法，用于在Pi系统（PiZ和PiS）中鉴定两种常见的α1-蛋白酶抑制剂（α1-抗胰蛋白酶）缺陷等位基因。在聚合酶链反应（PCR）中使用两组突变引物，随后对产物进行限制性酶切。只有在存在野生型等位基因（主要是M或亚型）而不存在PiZ或PiS等位基因时，突变的正向引物才会产生一个Taq I位点。反向引物针对一个不变的Taq I位点进行了突变，该位点用作内部对照位点，以确保限制性酶切的完成。消化后的PCR产物可以通过2.5%的MetaPhore琼脂糖凝胶电泳清晰地分辨出来。这种对最常见的α1-抗蛋白酶缺陷等位基因进行简单的PCR检测方法，可以常规应用于血清中α1-抗蛋白酶值定量降低的样本，或用于大规模筛查的Guthrie卡片上的血斑。此外，这种方法可能为特殊情况下α1-抗蛋白酶缺陷的简单、快速和可靠的产前诊断提供机会。
