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Evaluation of denaturing gradient gel electrophoresis (DGGE) as a method of mutation detection

Cadiou, Helene; (1995) Evaluation of denaturing gradient gel electrophoresis (DGGE) as a method of mutation detection. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The success in the isolation and characterisation of genes responsible for genetic disease, the considerable molecular heterogeneity at these loci has placed a growing emphasis on the importance of mutation detection techniques both in the coding and non-coding regions of human DNA. This thesis is principally concerned with the evaluation of one method of mutation analysis: Denaturing Gradient Gel Electrophoresis (DGGE), which has been used to identify sequence variation in three model systems: the well characterised alpha-1-antitrypsin (AAT) gene, with a range of known mutations; a segment of the Y chromosome where there is evidence for very little mutation and the Phenylalanine Hydroxylase (PAH) gene which mutated causes the autosomal recessive disorder Phenylketonuria (PKU), a disease characterised by severe mental retardation. The AAT gene was used as a test locus to evaluate the general applicability of DGGE for the detection of single base pair polymorphisms. The sequence on the Y chromosome was used to set up the computer programs associated with the technique. Subsequently the technique was used to screen for mutation in 5 exons and their splice site sequences of the PAH gene in 50 unrelated Phenylketonuria (PKU) patients. In the AAT gene, all six common point mutations were resolved. In contrast, no polymorphism was identified in the segment of Y chromosome investigated. In the PAH gene, the technique identified 10 different mutations, corresponding to 42% of affected chromosomes. Of these, three, K341R, L347F, and IVS10nt-3c-t were unique and not previously reported in the international data base. Two further sequence variants which caused no obvious alteration in the function of the PAH gene were also identified. A comparison of the efficiency of DGGE, in relation to single strand conformation polymorphism (SSCP) as a mutation detection system was also performed. Only four out of the twelve mutations identified using DGGE were resolved using SSCP analysis. Furthermore SSCP analysis did not identify any other mutations not observed in the initial DGGE screen. Overall, the study has evaluated the use of DGGE as a mutation detection system. It has resolved all the common point mutations in the AAT gene, confirmed the paucity of polymorphism in the Y chromosome and identified a number of mutations present in a South-East of England PKU population.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Evaluation of denaturing gradient gel electrophoresis (DGGE) as a method of mutation detection
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; DNA mutations
URI: https://discovery.ucl.ac.uk/id/eprint/10097911
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