High‐resolution DNA melting curve analysis to establish HLA genotypic identity

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Abstract
High‐resolution melting curve analysis is a closed‐tube fluorescent technique that can be used for genotyping and heteroduplex detection after polymerase chain reaction. We applied this technique at the HLA‐A locus and suggest that this method can be used as a rapid, inexpensive screen between siblings prior to living‐related transplantation. At any locus, there are seven general cases of shared alleles among two individuals, ranging from identical homozygous genotypes (all alleles shared) to two heterozygous genotypes that share no alleles. We studied each case using previously typed cell lines to show that identity or non‐identity can be determined in all cases by high‐resolution melting curve analysis. HLA genotype identity is suggested when two individuals have the same melting curves. Identity is confirmed by comparing the melting curve of a 1 : 1 mixture with the individual melting curves. Non‐identity at the amplified locus changes the heteroduplexes formed in the mixture compared with the original samples and alters the shape of the melting curve. The technique was tested on DNA from a 17‐member CEPH family. High‐resolution melting curve analysis revealed six different genotypes in the family. The genotype clustering was confirmed by sequence‐based typing. Although this technique does not sequence or determine specific HLA alleles, it does rapidly establish identity at highly polymorphic HLA loci. The technique may also prove useful for confirmation of HLA genotypic identity between unrelated individuals prior to allogeneic hematopoietic stem‐cell transplantation.