Hla‐dqa1 and ‐dqb1 Genotyping by Pcr‐rflp, Heteroduplex and Homoduplex Analysis

Abstract

PCR-RFLP typing methods for DQA1 and DQB1 in conjunction with the analysis of heteroduplex and homoduplex patterns have allowed a simple method for typing all of the major DQA1 and DQB1 alleles. This method has advantages over PCR amplification with sequence-specific primers (PCR-SSP), PCR hybridization with sequence-specific oligonucleotide probes (PCR-SSO) and other PCR-RFLP strategies for typing DQ alleles. The analysis of heteroduplex and homoduplex patterns can be used in conjunction with other PCR typing systems such as PCR-SSP as a confirmatory step with little additional work. In addition, a PCR-RFLP strategy was designed for resolving the DQB1*0602 and DQB1*0603 alleles, which involved the use of a primer containing a base mutation, creating a new restriction site which distinguished the two alleles. These techniques have enabled resolution of the major homozygous and heterozygous combinations of these DQA1 and DQB1 alleles. The PCR-RFLP technique does not require the large number of oligonucleotides that are necessary for both the PCR-SSP and PCR-SSO techniques and is thus both time and cost effective for infrequent or small numbers of samples.

Association Between HLA-DQ Genotypes and Haplotypes vs Helicobacter pylori Infection in an Indonesian Population

“…The primer pair used to amplify DQA1 consisted of DQA1-5' (5'-CAC GGA TCC GGT AGC AGC GGT AGA GTT G-3') and DQA1-3' (5'-ATG GTG TAA ACT TGA ACC AGT-3') (Maeda et al, 1989;Steck et al, 1995). DQA1 PCR products were digested with FokI, HaeIII, DdeI, ScrFI and RsaI Teutsch et al, 1996) . The exon of DQB1 with DQw1 specificity was amplified using PCR primers GH28NL and QB202 (DQB1 202 primer 1: 5'-CTC GGA TCC GCA TGT GCA ACT TCA CCA ACG, primer 2 5'-CAC CTG CAG ATC CCG CGG TAC GCC ACC TC).…”

“…When digested with restriction enzyme, 5% agarose gel was used. Cleavage or no cleavage of amplified fragments was detected by staining with ethidium bromide Teutsch et al, 1996).…”

Genetic control of autoantibody expression in autoimmune myasthenia gravis: role of the self-antigen and of HLA-linked loci

“…42 Subsequently, PCR products were typed by analysis of heteroduplexes. 43 Statistical analysis of data The influence of genetic markers and of thymus histopathology on autoantibody peak titers was tested by a multi-way analysis of variance (ANOVA), after normalization of the distribution of autoantibody titers by logarithmic transformation. Genetic markers were coded as binary variables, depending on the presence or absence of the associated allele.…”

New Diagnostic Methods in Oncology and Hematology

“…The techniques used to analyze the amplified fragments include the restriction fragment length polymorphism (PCR-RFLP) [72][73][74][75][76][77][78], the single strand conformation polymorphism (PCR-SSCP) [79][80][81][82][83][84][85][86][87][88][89], the heteroduplex formation (PCR-HDF) [90][91][92][93][94][95][96][97], the use of sequence-specific oligonucleotide probing (PCR-SSOP) , the use of sequence-specific priming (PCR-SSP) , and the determination of the nucleotide sequence of the PCR amplified DNA by sequencing based typing (PCR-SBT) . The techniques used to analyze the amplified fragments include the restriction fragment length polymorphism (PCR-RFLP) [72][73][74][75][76][77][78], the single strand conformation polymorphism (PCR-SSCP) [79][80][81][82][83][84][85][86][87][88][89]…”