Arguello-Astorga, Jesus Rafael; (1999) Development of methods for the identification of genetic polymorphism. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text out.pdf Download (9MB) | Preview

Abstract

A unique feature of the human Major Histocompatibility Complex genes is their extensive polymorphism which is localised mainly within those regions encoding the groove of the HLA molecules. Comparison of HLA allelic sequences reveals a patchwork pattern in which an individual allele comprises a unique combination of sequence motifs, each of which is shared with other alleles, and only a few alleles have a unique sequence that is not present elsewhere in the HLA region. This feature of the HLA polymorphism has complicated the application of DNA-based methods that rely on sequence identification to type the HLA genes. To overcome this problem, two novel high resolution HLA typing methods were developed. The first utilises a set of only 40 probes, which identify the recombinational motifs present in exon 2 and 3 of the HLA class I genes, allowing a unique hybridisation pattern for each allele. The unambiguous identification of the alleles is achieved by the use of a new allelic separation technique called Complementary Strand Analysis. The second method, Reference Strand mediated Conformation Analysis (RSCA), differs from conventional sequence based typing methodologies in that the HLA type is assigned on the basis of accurate measurement of conformation dependent DNA mobility in polyacrylamide gel electrophoresis. RSCA utilises a fluorescent labelled locus specific reference DNA to selectively modify the molecular conformation of the tested DNA. The use of laser based instrumentation and computer software, in addition to internal DNA markers for correction of gel variability, allows the discrimination of HLA alleles which differ by one nucleotide in a DNA fragment nearly as large as a kilobase in length. RSCA has been successfully applied in blind studies of HLA typing demonstrating that is reproducible, able to identify new alleles, and to resolve ambiguous heterozygous combinations.

Download activity - last month

Export as JSONXMLCSV

Download activity - last 12 months

Export as JSONCSVXML

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item