Detection of the common alpha‐1‐antitrypsin variants by denaturing gradient gel electrophoresis

Abstract

The well-characterized and highly polymorphic human alpha-1-antitrypsin (AAT) gene was used as a test locus to evaluate the general applicability of denaturing gradient gel electrophoresis (DGGE) for the detection of single base change polymorphisms. We report the resolution of all the major alleles, M1Ala, M1Val, M2, M3, S and Z and the identification of substantial genetic polymorphism in intron 3-4 by this technique. DGGE was found to be a quick and efficient method for the screening of multiple samples for the presence of genetic variation.

Mutation detection in the alpha-1 antitrypsin gene (PI) using denaturing gradient gel electrophoresis

“…For exon II however, three primer sets (II A , II B , and II C ) had to be designed because of the complex melting pattern of this exon. This was also observed by Johnson et al [1991]. While primers only comprised two-thirds of the exon, to obtain clear band separation the amplified product had to be cut at a Sty I restriction site.…”

“…In the patient population studied, DGGE provided a clear separation of the different normal phenotypes (P i M) and the entire mutation spectrum. Digestion of PCR fragments prior to DGGE to resolve variation at more than one site, as suggested by Johnson et al [1991], proved to be redundant. For exon II however, three primer sets (II A , II B , and II C ) had to be designed because of the complex melting pattern of this exon.…”

“…To detect the Z mutation only, Dubel et al [1991] developed a single primer set. To evaluate the applicability of DGGE, Johnson et al [1991], using the PI-gene as a test locus, covered a large part of the gene. However, some areas of exon II and V were not included, and primers were not at all suitable to detect splice mutations.…”

“…A panel of nine primer sets was designed allowing the specific amplification of all PI-exons and their exon-intron junctions, but not the PI-related gene [Bao et al, 1988] nor the PI-like pseudogene [Hofker et al, 1988]. Contrary to the method described by Johnson et al [1991], primer sets were chosen at a distance of 10 to 20 bp from the splice sites to be able to detect all potential changes and splice mutations. To accomplish equal amplification conditions for all exons, special notice was also taken of the annealing temperature.…”

“…genes involved in metabolic diseases, oncogenes, etc. Because of its simplicity and dependability, the method has been used for routine screening of defects of the PI-gene [Johnson et al, 1991;Dubel et al, 1991]. However, these setups did not detect mutations in the splice junction sites, which frequently occur in metabolic diseases such as phenylketonuria (PKU) [Michiels et al, 1996[Michiels et al, , 1998] but up to now only one has been detected in PI deficiency [Laubach et al, 1993].…”