Circularisable Ligation Probes: Synthesis and Use For Estimating the Frequency of Recombination at the 21-Hydroxylase Locus

Abstract

This thesis describes the development of circularisable ligation probes (CLiPs) and the use of these probes to examine the frequency of meiotic and mitotic recombination in the 2l-hydroxylase genes. CLiPs are single stranded probes that consist of target-specific ends separated by a non-complementary 'linker' sequence. When hybridised to target, the CLiP forms a nicked circle which may be sealed by a DNA ligase only if the 5' and 3' ends show perfect Watson-Crick base-pairing complementarity, thus enabling the discrimination of single nucleotide variation. In this work, a PCR-based (enzymatic) method for the synthesis of CLiPs is described, which overcomes many of the limitations associated with standard synthetic phosphoramidite systems. The enzymatically prepared CLiPs were readily able to distinguish between allelic variants. The effects of design and synthesis parameters on CLiP ligation were systematically investigated. 21-hydroxylase deficiency is an autosomal recessive disease that results in impaired cortisol biosynthesis and is due to mutations in the gene CYP21. Approximately 95% of 2l-hydroxylase deficiency alleles are thought to be the result of unequal crossing-over or apparent gene conversion between CYP21 and a nearly identical pseudogene (CYP21P). Previous studies have indicated that the frequency of recombination between the two genes is relatively high, however, measurement of de novo recombination in matched blood and sperm cells indicates that this may not be the case. A CLiP assay and an allele-specific PCR assay were developed and used to estimate the frequency of recombination between CYP21 and CYP21P from genomic DNA isolated from blood and sperm (mitotic and meiotic cells). Data is presented to show that previous studies are likely to be erroneous due to PCR artifacts, and that the frequency of unequal crossing-over is rarer than 1 in 1 x l05, and the frequency of gene conversion is rarer than 1 in 1 x l04.