A novel approach to DNA probe hybridization and heteroduplex analysis, termed directed heteroduplex analysis (DHDA) is presented here to illustrate its utility in simplification of human lymphocyte antigen (HLA)-typing. By strategic labeling of single-stranded probe sequences, DHDA allows the identification of specific heteroduplex structures that contribute to the differentiation of DQA1 and DQB1 alleles. Because of the high degree of polymorphism among major histocompatibility complex class II second exon sequences, this analysis of 50 different heteroduplex molecules provides evidence of the importance of unpaired bases and mismatched base pairs and their effect on heteroduplex electrophoretic-mobility differences. This strategy is further used to genotype accurately a family for DQA1 which was previously analyzed by sequence specific oligonucleotide (SSO) probe hybridization. To differentiate by SSO-typing among the DQA1 and DQB1 alleles analyzed in this study requires the use of 23 different probes. Equivalent results are obtained by DHDA using only three probes. Therefore, this study suggests that accurate HLA-typing can be simplified by DHDA. Additionally, DHDA may be useful for differentiation of DNA sequence polymorphisms in other genetic systems.
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| http://dx.doi.org/10.1093/nar/21.19.4541 | DOI Listing |
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