Simple and comprehensive SLA-DQB1 genotyping using genomic PCR and direct sequencing

Abstract

To enable the efficient analysis of a highly polymorphic swine major histocompatibility complex (MHC) class II gene, swine leukocyte antigen (SLA)-DQB1, we developed a simple and comprehensive high-resolution genotyping protocol. To obtain sufficient sequence information to design a set of common genotyping primers for SLA-DQB1, we cloned SLA-DQB1 introns 1 and 2 from 11 alleles with official four-digit allelic designations and sequenced the regions directly surrounding the SLA-DQB1 exon 2. Significant intronic nucleotide variations, including several deletions, were identified. Based on 733-bp assembled genomic sequences including introns 1 and 2 and exon 2 from 11 different alleles, a primer set was identified that allowed the ubiquitous amplification and analysis of the complete SLA-DQB1 exon 2 sequence. We then developed a method to directly sequence the amplified polymerase chain reaction (PCR) products without further experimental steps. We especially focused on avoiding superimposed peaks, which arose from the presence of allelic deletions, in the sequencing electropherogram of SLA-DQB1 heterozygous animals. The genotyping accuracy was evaluated by comparing the results of genomic sequence-based typing (GSBT) with those of other available methods, including cDNA sequence-based typing (SBT), low-resolution PCR typing with sequence-specific primers, allelic segregation analysis, and heterozygote simulation typing. In all cases, the results were consistent between SLA-DQB1 GSBT and previously reported methods or expected results. We applied it to genotype 350 animals from seven pig breeds. The observed level of heterozygosity from our genotyping was ∼51%, reflecting that a large portion of the animals were inbred miniature pigs. Among the seven pig breeds tested, the allelic diversity of SLA-DQB1 was highest in Berkshire pigs. In conclusion, we have developed a simple and effective SLA-DQB1 GSBT method by combining simple genomic DNA PCR and direct sequencing. Our new method may aid in the study of SLA diversity and disease resistance and susceptibility.

Sequence variations of the locus‐specific 5′ untranslated regions of SLA class I genes and the development of a comprehensive genomic DNA‐based high‐resolution typing method for SLA‐2

“…We previously confirmed that direct sequencing of PCR products is a suitable method based on our SLA class II typing results (18)(19)(20).…”

“…We are interested in the genetic polymorphisms of the SLA genes, and we previously reported the development of a high-resolution genotyping method for the SLA class II genes, DQB1 (18), DRB1 (19), and DQA (20) along with the results of our analysis. However, an equivalent typing method for SLA class I genes has not been reported.…”

Establishment of major histocompatibility complex homozygous gnotobiotic miniature swine colony for xenotransplantation

“…Swine leukocyte antigen (SLA) typing was conducted at the Laboratory of Mammalian Genetics in KU by genomic PCR and direct sequencing methods essentially as described (Park et al 2010). …”

Swine Leukocyte Antigen-<italic>DQA</italic> Gene Variation and Its Association with Piglet Diarrhea in Large White, Landrace and Duroc

“…SLA-DRB1 and SLA-DQB1 loci display a very high degree of polymorphism, although the SLA-DQA gene has been reported as a moderately polymorphic gene, and SLA-DRA was reported to exhibit only limited polymorphism (Lunney et al, 2009; Ho et al, 2009; Ho et al, 2010). Research utilizing SLA class II genes primarily focus on exon 2 of the DRB and DQB because these loci code for the peptide binding region (PBR) which is in direct contact with the processed peptide (Babik et al, 2005; Luetkemeier et al, 2009; Park et al, 2010). However, there is currently no study on the relationship between SLA-DQA gene and piglet diarrhea.…”