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KMID : 0578319950050010052
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Molecules and Cells
1995 Volume.5 No. 1 p.52 ~ p.57
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Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis for Screening New Alleles in HLA-DQα
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Nam, Hyun Suk
Kim, Kyun Eun/Lee, Kong-Joo/Kim, Eun-Hee
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Abstract
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The sequences of HLA-DQα alleles are relatively polymorphic and HLA-DQ typing methods developed to date sometimes fail to discern minute base differences among alleles. Thus thiss study tried to apply the denaturing gradient gel electrophoresis (DGGE) metHod for screening new alleles in HLA-DQMα typing. DGGE is a method in which double stranded, equal length DNA molecules with one base difference can be resolved because of their different melting behavior. 242 bp of the second exon of HLA-DQα gene was amplified by polymerase chain reaction with a GC-clamped primer and subsequently subjected to DGCE. The optimum polymerase chain reaction condition for HLA-DQα with a 39 bp GC-clamped primer was investigated. A computer algorithm to simulate melting behavior was used in order to optimize DGGE condition. Also extensive experimental adjustment was done regarding the denaturant gradient range and gel running time. Computationl simulation and experimental results were compared. Tentative new alleles were discovered among alleles which were typed as the same alleles using the sequence specific oligonucleotide probe method. This study demonstrated the superior sensitivity of DGGE as an HLA-DQα allele typing method.
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