Genotyping of human platelet antigens 1 to 6 and 15 by high-resolution amplicon melting and conventional hybridization probes.
High-resolution melting techniques are a simple and cost-effective alternative to other closed-tube genotyping methods. Here, we genotyped human platelet antigens (HPAs) 1 to 6 and 15 by high-resolution melting methods that did not require labeled probes. Conventional melting analysis with hybridization probes (HybProbes) was also performed at each locus. HybProbe assays were performed individually, whereas amplicon melting (HPAs 1 to 5 and 16) and unlabeled probe (HPA 6) assays were duplexed when possible. At all loci for each method, both homozygous and heterozygous genotypes were easily identified. We analyzed 100 blinded clinical samples (33 amniotic fluid, 12 cultured amniocytes, and 55 blood samples) for all 7 single-nucleotide polymorphisms (SNPs) by each method. Genotype assignments could be made in 99.0% of the SNPs by high-resolution melting and in 98.7% of the SNPs with HybProbes with an overall genotype concordance of 98.8%. Errors included two sample misidentifications and six incorrect assignments that were all resolved by repeating the analysis. Advantages of high-resolution melting include rapid assay development and execution, no need for modified oligonucleotides, and similar accuracy in genotyping compared with other closed-tube melting methods.
Liew
Michael
M
ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108-1221, USA. liewm@aruplab.com
Nelson
Lesa
L
Margraf
Rebecca
R
Mitchell
Sheri
S
Erali
Maria
M
Mao
Rong
R
Lyon
Elaine
E
Wittwer
Carl
C
D016824:Antigens, Human Platelet / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D016824:Antigens, Human Platelet; D005456:Fluorescent Dyes