HLA-Cw allele analysis by PCR-restriction fragment length polymorphism: study of known and additional alleles.
We describe a technique for HLA-Cw genotyping by digestion of PCR-amplified genes with restriction endonucleases. Locus-specific primers selectively amplified HLA-Cw sequences from exon 2 in a single PCR that avoided coamplification of other classical and nonclassical class I genes. Amplified DNAs were digested with selected enzymes. Sixty-three homozygous cell lines from International Histocompatibility Workshop X and 113 unrelated individual cells were genotypes for HLA-Cw and compared with serology. The present protocol can distinguish 23 alleles corresponding to the known HLA-Cw sequences. Genotyping of serologically undetectable alleles (HLA-Cw Blank) and of heterozygous cells was made possible by using this method. Six additional HLA-Cw alleles were identified by unusual restriction patterns and confirmed by sequencing; this observation suggests the presence of another family of allele-sharing clusters in the HLA-B locus. This PCR-restriction endonuclease method provides a simple and convenient approach for HLA-Cw DNA typing, allowing the definition of serologically undetectable alleles, and will contribute to the evaluation of the biological role of the HLA-C locus.
Tatari
Z
Z
Laboratoire d'Immunologie et d'Histocompatibilité, Hôpital Saint Louis, Paris, France.
Fortier
C
C
Bobrynina
V
V
Loiseau
P
P
Charron
D
D
Raffoux
C
C
D000483:Alleles; D001402:B-Lymphocytes / Q000166:cytology; D001483:Base Sequence; D002460:Cell Line; D005091:Exons / Q000235:genetics; D005787:Gene Frequency; D005838:Genotype; D015236:HLA-C Antigens / Q000145:classification / Q000235:genetics*; D006579:Heterozygote; D006650:Histocompatibility Testing / Q000379:methods*; D006720:Homozygote; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D044465:White People / Q000235:genetics
D015236:HLA-C Antigens