APPLICATION OF RSCA FOR HLA-C TYPING,
K Mark, A McWhinnie, JA Argüello, A-M Little, and J.A Madrigal,
The Anthony Nolan Research Institute, London, UK.
The influence of HLA-C matching on the outcome of bone marrow transplantation with unrelated donors is not well defined. However, an increasing number of studies suggest that HLA-C matching may be as important as other HLA loci, with evidence to indicate that HLA-C matching increases graft survival1 and mismatching contributes towards GVHD2. This has lead to the development and improvement of various molecular strategies including single strand conformation polymorphism (SSCP), sequence specific oligonucleotide probes (SSOP), sequence specific PCR (SSP), direct sequencing and more recently reference strand conformation analysis (RSCA). The latter method, RSCA, measures, for individual HLA loci, differences in the mobility of DNA duplexes formed between a fluorescently labelled sense strand of a fluorescent labelled reference (FLR) DNA which has been hybridised to the anti-sense strands of the alleles to be tested within a sample. The duplexes are separated by non-denaturin g gel electrophoresis in an Alfexpress DNA sequencer and mobility values are obtained for the various fluorescent duplex signals. We have applied RSCA to HLA type and/or crossmatch HLA-C alleles3,4. To further improve on the resolution of RSCA for HLA-C typing, 66 different FLRs were tested on a panel of genomic DNAs representative of 26 different HLA-C alleles. Three suitable FLRs have been chosen that differentiate between all the C-alleles tested. After selection of optimum FLR’s a HLA-C duplex ladder was generated. This ladder was prepared by pooling independent hybridisations of DNA controls with one of the FLRs. Enhanced resolution was obtained using the three FLR’s along with the duplex ladder as an external control on each gel, thereby significantly improving the quality and resolution of the HLA-C allelic level typing performed by RSCA.
1Nagler et al. 1996. BMT, 18: 891. 2 Sasazuki et al. 1998. N. Engl. J. Med 339: 1177.
3 Arguello et al. 1998 Tissue Antigens 52: 57. 4 Arguello et al. 1998 BMT, 22: 527.