Comprehensive, serologically equivalent DNA typing for HLA‐B by PCR using sequence‐specific primers (PCR‐SSP)

Abstract

Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.

A Functional Genetic Variation of the Serotonin (5-HT) Transporter Affects 5-HT1AReceptor Binding in Humans

“…Genomic DNA was extracted from buffy coat lymphocytes using a standard sodium chloride-chloroform technique and stored in sterile distilled water at Ϫ20°C. The genotyping assay was performed as described by Bunce et al (1995), using sequence-specific primers. PCRs were performed for both the common and variant alleles, and each reaction contained control primers to detect a conserved sequence in the adenomatosis polyposis coli gene, thereby eliminating the possibility of false-negative results.…”

Association between the DRD2 gene Taq1A (C32806T) polymorphism and alcohol consumption in social drinkers

“…Genotyping was carried out by PCR using sequence specific primers using methods previously described. 29 Reactions were carried out for both common and variant alleles, each containing control primers which detected a conserved sequence in the adenomatosis polyposis coli (APC) gene, and eliminated the possibility of false negative results.…”

Identification of CYP2B6 Sequence Variants by Use of Multiplex PCR with Allele-Specific Genotyping

“…The PCR reaction was set up in a volume of 8 L containing 1ϫ ammonium buffer, 1.9 mM magnesium chloride, 400 M deoxynucleotide triphosphates, and 0.125 U of Taq DNA polymerase (23 ). We added 1 L of a 1:100 dilution of PCR product from the multiplex round to 60 L of this buffer and then added 5 L of the DNA-buffer mixture to each well, which contained 10 L of mineral oil and 3 L of allele-specific primer mixture (see Table 2 in the online Data Supplement).…”