Related Experiment Videos
HLA typing for DR3 and DR4 using artificial restriction fragment length polymorphism PCR from archival DNA
V A Horton1, M Bunce, D R Davies
1Diabetes Research Laboratories, Radcliffe Infirmary, Oxford.
Aim:
To develop polymerase chain reaction based artificial restriction fragment length polymorphism (artificial RFLP PCR) assays for DR3 and DR4 alleles of the multiallelic DRB1 locus and to apply them to paraffin wax embedded archival material.
Methods:
Sixty five samples from DRB1 typed cell lines were analysed using the artificial RFLP PCR method to determine the specificity and sensitivity of the system.
Results:
The artificial RFLP PCR method for typing the DRB1 locus showed 100% accuracy in the 65 samples previously typed using allele specific PCR and serology. The samples included 18 combinations of alleles that included DR3, 18 that included DR4, four that were DR3/DR4 heterozygotes, and 10 samples that were neither DR3 nor DR4. Typing of 10 paraffin wax embedded samples using artificial RFLP PCR was in complete agreement with previous typing at the DRB1 locus.
Conclusion:
The application of artificial RFLP PCR for the analysis of multiallelic loci, such as those of the HLA system, in archival DNA samples has been achieved. Artificial RFLP PCR is a robust, easily implemented, non-isotopic system and may be useful for large retrospective studies.