Analysis of large deletions in theHPRT gene of primary human fibroblasts using the polymerase chain reaction
- 1 January 1993
- journal article
- research article
- Published by Springer Nature in Somatic Cell and Molecular Genetics
- Vol. 19 (1) , 9-19
- https://doi.org/10.1007/bf01233950
Abstract
Spontaneous and X-ray-induced mutants of theHPRT gene were isolated from two primary human fibroblast lines. The limited life-span of the mutants restricted the use of methods requiring large quantities of DNA, and the polymerase chain reaction (PCR) was used in particular to check for the presence of multiple genomic sites in mutant analysis. Robust PCR primers were designed to amplify sites of up to 1 kb, mostly with 1-kb spacings between sites, over the entire 56-kbHPRT gene region. Using PCR, large deletions were found in 43% of independent X-ray-induced mutants, and their breakpoints were localized where these fell within the gene. Anonymous DNA sites in the Xq26 chromosomal region containingHPRT (covering ≥1.5 Mb) were also amplified by PCR to assess codeletion withHPRT; sites up to 1 Mb distal to the gene (DXS86, DXS10) were codeleted in some mutants, but no mutant was found with loss of a proximal site (DXS79).Keywords
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