Rapid DNA analysis of alpha 1-antitrypsin deficiency: application of an improved method for amplifying mutated gene...

The gene coding for alpha 1-antitrypsin (AAT) is mutated in some individuals to code for a protein, the PiZ variant, associated with alpha 1-antitrypsin serum deficiency. Homozygotes for this defect are at high risk for various liver diseases and for early onset emphysema. Here, we employ an enzymatic amplification procedure for diagnosis of a Z-specific gene mutation in cellular DNA, and we describe a new procedure for selective enhancement of the mutated sequence. We have synthesized 2 oligonucleotides which prime amplification of a 156 bp region of exon V including a single base mutation specific for the PiZ variant. Amplification of 2 to 6 X 10(5)-fold is obtained allowing rapid detection of the Z mutation, using selective oligonucleotide probes, in unseparated genomic DNA of cells from family members homozygous or heterozygous for the defect. New methodology allows enzymatic amplification at elevated temperatures. To take advantage of this, we have developed a method using discriminating oligonucleotides as primers that allows enhanced sensitivity of hybridization. These methods allow diagnosis of the Z form of AAT deficiency within a single day using submicrogram quantities of DNA.