Single‐strand Conformation Polymorphism (Sscp) Analysis of Hla‐drb1*1101‐06

Abstract

Single-strand conformation polymorphism (SSCP) has been developed as a method for detecting the presence of mutations in a segment of DNA. We applied it to the subtyping of the DRII group of alleles. The SSCP patterns of DRB I-DR52 group-specific products were defined in cell lines representing the DRBI*1101-06 alleles, using non-denaturing acrylamide gel electrophoresis and silver staining. Only one set of gel electrophoresis conditions was able to discriminate the DRII alleles tested. The protocol was validated in an analysis of 105 DRll-positive individuals previously typed by oligonucleotides probing. The study demonstrates the suitability of the SSCP technique to define the DRB 1*1101-06 alleles, the technique being particularly valuable in confirming and extending the oligotyping of DRBI-DR52 heterozygous samples.analysis and heteroduplex analysis (Sorrentino et al., 1992;D' Amato & Sorrentino, 1994;Savage et al., 1995). SSCP is based on the different mobilities of DNA single strands in non-denaturing electrophoresis conditions, depending on their sequences and, consequently, on the secondary structures they assume during the migration (Orita et al., 1989). This powerful technique allows the discrimination of a single mutation in a fragment of DNA under the right conditions, even if it is not able to define its

Resolution of cis‐trans ambiguities between HLA‐DRB1 alleles using single‐strand conformation polymorphisms and sequencing

“…SSCP has been applied previously as a typing strategy for HLA genes, by comparing the electrophoretic patterns between alleles (18, 19), or by establishing identity between two samples based on similar migration rates (20, 21). SSCP has not achieved widespread use in this regard, however, likely due to difficulty in achieving reproducibility between gels, and multiple conformers produced by the same sequence.…”

Molecular prenatal diagnosis of ataxia telangiectasia heterozygosity by direct mutational assays

“…Detailed information on these markers can be found in the Genome Data Base. Moreover, polymorphic loci HLA-DRB1 and HLA-DQA1 were analysed by PCR-SSP and SSCP methods respectively (for details, see Mora et al (1996)).…”