Analysis of complex genetic systems by ARMS-SSCP: application to HLA genotyping.
We have developed a new method for analysis of complex genetic systems by a combination of the Amplification Refractory Mutation System (ARMS) and Single-Strand Conformation Polymorphism (SSCP) analysis: ARMS-SSCP. Thus, a complex allelic series is subdivided into a number of groups by ARMS followed by the identification of specific alleles using SSCP analysis. We have shown that the HLA alleles at the DRB3 and DQB1 loci were distinguishable from each other using ARMS-SSCP. In 36 individuals typed for the DRB3 and 48 individuals typed for the DQB1 loci, ARMS-SSCP results were in complete agreement with those obtained using the established method of sequence-specific oligonucleotide (SSO) hybridisation. With silver staining, ARMS-SSCP is a rapid, non-radioactive and reliable method which also offers the possibility for detecting new HLA alleles. We have demonstrated that ARMS-SSCP can be performed using fluorescent PCR primers, a feature which gives the method potential for automation.
Lo
Y M
YM
Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, Oxford, UK.
Patel
P
P
Mehal
W Z
WZ
Fleming
K A
KA
Bell
J I
JI
Wainscoat
J S
JS
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000235:genetics*; D005456:Fluorescent Dyes; D005802:Genes, MHC Class II / Q000235:genetics*; D005838:Genotype; D006683:HLA-DQ Antigens / Q000235:genetics*; D059866:HLA-DQ beta-Chains; D006684:HLA-DR Antigens / Q000235:genetics*; D059826:HLA-DRB3 Chains; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D016622:Silver Staining
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D006683:HLA-DQ Antigens; D059866:HLA-DQ beta-Chains; C069051:HLA-DQB1 antigen; D006684:HLA-DR Antigens; D059826:HLA-DRB3 Chains; D009838:Oligodeoxyribonucleotides