The overall objective of this proposal is to develop a protocol to determine HLA class II genotypes using a combination of Denaturing Gradient Gel Electrophoresis (DGGE) of PCR products and automated sequencing of isolated PCR fragments. DGGE analysis of PCR fragments derived from the polymorphic exon (exon 2) of HLA class II genes will be used to distinguish most or all of the known alleles at each HLA class II locus. Our preliminary results demonstrate that DGGE is an excellent methodology for distinguishing HLA class II polymorphisms. DGGE analysis is rapid, inexpensive, and does not require the use of radioisotopes. This procedure is ideal for handling large sample sizes (a single technician could process 80 or more samples per day), and could be used to establish standard HLA-D region genotypes within populations. Furthermore, DGGE analysis has several advantages over oligonucleotide typing and AFLP for determining HLA class II genotypes. First, the genotypes of HLA heterozygotes can be determined unambiguously because each allele is directly visualized and identified. Second, new alleles can be readily detected on the basis of new mobilities in DGGE, regardless of where the base substitution occurs in the PCR product. Finally, PCR products can be excised from DGGE gels, assymetrically amplified, and directly sequenced using an automated DNA sequencer. Although DGGE genotyping may be sufficient for most applications, many clinicians contemplating transplantation protocols will prefer exact information about the compatibility of the donor and recipient, since single base substitutions in MHC genes can result in acute graft rejections in experimental systems. Our preliminary results demonstrate that PCR products isolated from DGGE gels are excellent samples for assymetric sequencing, and precise sequences can generally be obtained with 24 to 48 hours of additional laboratory time.
The specific aims of this proposal are: 1) To develop protocols and optimal DGGE conditions for the maximum resolution of allelic PCR products from DRB1, DQB1, DQA1, and DPB1; and 2) to develop methodologies for the rapid and accurate automated nucleotide sequencing of DGGE-resolved PCR products.
| She, J X (1996) Susceptibility to type I diabetes: HLA-DQ and DR revisited. Immunol Today 17:323-9 |
| Deng, G Y; Maclaren, N K; Huang, H S et al. (1996) No primary association between the 308 polymorphism in the tumor necrosis factor alpha promoter region and insulin-dependent diabetes mellitus. Hum Immunol 45:137-42 |
| Huang, H S; Peng, J T; She, J Y et al. (1995) HLA-encoded susceptibility to insulin-dependent diabetes mellitus is determined by DR and DQ genes as well as their linkage disequilibria in a Chinese population. Hum Immunol 44:210-9 |
| Morel, L; Rudofsky, U H; Longmate, J A et al. (1994) Polygenic control of susceptibility to murine systemic lupus erythematosus. Immunity 1:219-29 |
| She, J X; Bui, M M; Tian, X H et al. (1994) Additive susceptibility to insulin-dependent diabetes conferred by HLA-DQB1 and insulin genes. Autoimmunity 18:195-203 |
