Open AccessDiagnosis of α1antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products
Author(s) -
Cambridge Newton,
Noor Kalsheker,
Alexander Graham,
Stephen Joseph Powell,
A. Gammack,
John Riley,
Alexander F. Markham
Publication year - 1988
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/16.17.8233
Subject(s) - biology , polymerase chain reaction , genetics , locus (genetics) , multiple displacement amplification , genomic dna , microbiology and biotechnology , dna sequencing , primer dimer , dna , minisatellite , inverse polymerase chain reaction , gene , dna profiling , multiplex polymerase chain reaction , dna extraction , microsatellite , allele
We have compared sequencing of cloned "polymerase chain reaction" (PCR) products and the direct sequencing of PCR products in the examination of individuals from six families affected with alpha 1-antitrypsin (AAT) deficiency. In families where paternity was in question we confirmed consanguinity by DNA fingerprinting using a panel of locus-specific minisatellite probes. We demonstrate that direct sequencing of PCR amplification products is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes and homozygotes in a single, rapid and facile assay. Furthermore, we demonstrate the reproducibility of the PCR and a rapid DNA isolation procedure. We have also shown that two loci can be simultaneously amplified and that the PCR product from each locus can be independently examined by direct DNA sequencing.