Single-strand conformation polymorphism (SSCP) assays for major histocompatibility complex B genotyping in chickens.
We have developed a DNA-based method for defining MHC B system genotypes in chickens. Genotyping by this method requires neither prior determination of allele-specific differences in nucleotide sequence nor the preparation of haplotype-specific alloantisera. Allelic differences at chicken B-F (class I) and B-L (class II) loci are detected in PCR single-strand conformation polymorphism (SSCP) assays. PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the two class I and two class I loci of the B-complex and used to generate DNA fragments that are heat- and formamide-denatured and then analyzed on nondenaturing polyacrylamide gels. PCR primer pairs were tested for the capacity to produce SSCP patterns allowing the seven B haplotypes in the MHC B congenic lines, and seven B haplotypes known to be segregating in two commercial broiler breeder lines to be distinguished. Primer pairs were further evaluated for their capacity to reveal the segregation of B haplotypes in a fully pedigreed family and in a closed population. Concordance was found between SSCP patterns and previously assigned MHC types. B-F and B-L SSCP patterns segregated in linkage as expected for these closely linked loci. We conclude that this method is valuable for defining MHC B haplotypes and for detecting potential recombinant haplotypes especially when used in combination with B-G (class IV) typing by restriction fragment pattern.
Goto
R M
RM
Division of Molecular Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
Afanassieff
M
M
Ha
J
J
Iglesias
G M
GM
Ewald
S J
SJ
Briles
W E
WE
Miller
M M
MM
D000818:Animals; D002645:Chickens / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005838:Genotype; D006239:Haplotypes; D008285:Major Histocompatibility Complex / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D016415:Sequence Alignment
C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific