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

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.

Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping

“…Melting curve analysis has grown in sophistication from genotyping single-base variants with fluorescence resonance energy transfer probes (1 ) to inferring and differentiating sequence homologies through high-resolution amplicon melting with DNA binding dyes (2)(3)(4). These advanced techniques are already being adapted to existing real-time PCR instruments.…”

High-resolution DNA melting analysis: advancements and limitations

“…That is, about 90% of the time different heterozygotes will follow different melting paths that are distinguished using current technology. One method to verify the identity of 2 heterozygotes is to mix them together either before or after PCR and compare the melting curve of the mixture to the individual samples (Zhou et al, 2004). Tindall et al (2009) also demonstrate for the first time that some, but not all, cis/trans haplotypes are distinguishable by high resolution melting.…”

High resolution melting as an alternative method to genotype diacylglycerol O-acyltransferase 1 (DGAT1) K232A polymorphism in cattle

“…HRM allows melt profiles of 96 or 384 different PCR products to be achieved in minutes, compared to at least 12 h for most gelbased methods (Tindall et al, 2009). HRM analysis has primarily been used for the discovery and genotyping of single nucleotide polymorphisms (SNPs) (Graham et al, 2005), but it has also been used for precise amplicon verification (Erali et al, 2006), sequence matching applications such as HLA identity (Zhou et al, 2004) and generating STS markers in linkage mapping studies (Croxford et al, 2008).…”