Polymerase chain reaction based genotyping for characterization of SLA‐DQB and SLA‐DRB alleles in domestic pigs

Abstract

Summary Molecular genotyping of swine major histocompatibility complex SLA‐DQB and SLA‐DRB genes using polymerase chain reaction (PCR)‐based amplification is described. Locus‐specific oligonucleotide primers were designed for the analysis of expressed SLA genes by reverse transcription‐polymerase chain reaction (RT‐PCR). RT‐PCR products were sequenced, and the information gained was used to design primers for PCR genotyping of the exon 2 (β1) region from genomic DNA templates. A single segregating amplification product was detected for both DQB and DRB in all animals. PCR products were digested with restriction enzymes. Seven SLA‐DQB PCR‐restriction fragment length polymorphism (RFLP) pattern types were observed for both HaeIII and Rsal that defined 14 SLA‐DQB alleles. A total of seven SLA‐DRB PCR‐RFLP pattern types were defined using Mspl (3 RFLP pattern types) and Rsal (6 RFLP pattern types). In order to demonstrate their universal utility, the primers were tested on genomic DNA samples from 10 different swine breeds. No breed‐specific alleles were observed. These results show that locus‐specific oligonucleotide primers and RFLP analysis provide a simple and rapid method for genotyping expressed SLA‐DQB and SLA‐DRB from genomic DNA.

A new method of genotyping MDX4CV mice by PCR‐RFLP analysis

“…PCR‐RFLP has been used for genotyping different organisms over several decades, and here, we have optimized and applied this protocol to genotype the mdx 4cv mouse. Briefly, a primer set was identified where the forward primer binds within intron 52 of the dystrophin gene (position 84575312 of X chromosome), and the reverse primer binds within exon 53 (position 84575771 of X chromosome).…”

Irradiation before and donor splenocyte infusion immediately after transplantation induce tolerance to lung, but not heart allografts in miniature swine

“…Male donors and female recipients were mismatched for the major histocompatibility complex (MHC) class I DC 80 and H04 haplotypes and for reactivity with the SLA I haplotype d‐specific Ab 74‐11‐10 . SLA II mismatch was confirmed by reverse transcription PCR (RT‐PCR) and sequencing of SLA‐DQB .…”

Splenocyte Infusion and Whole-Body Irradiation for Induction of Peripheral Tolerance in Porcine Lung Transplantation: Modifications of the Preconditioning Regime for Improved Clinical Feasibility

“…cDNA was amplified with HotStar Taq polymerase (QIAGEN) using oligonucleotide primers ScDQB5a: 5′-GGC TGT GTT GAC TAC CAT TA-3′ and ScDQB3a: 5′-AGA CCA GCA GGT TGT GG-3′ for the second exon (β1 domain) of the MHC II molecule. 12 Clean up of DNA was facilitated by NucleoSpin Extract II (Macherey & Nagel, Dueren, Germany). Sequencing of both strands was done by SeqLab (Goettingen, Germany).…”