Rapid denaturing high-performance liquid chromatography (DHPLC) for mutation scanning of the transforming growth factor beta3 gene using a novel proof-reading polymerase.
We have utilized a novel variation on the conventional denaturing high-performance liquid chromatography (DHPLC) technology, which we term rapid DHPLC, combining changes in instrumentation, cartridge technology and analysis conditions to enable significant increases in throughput to be achieved. In addition, the use of a novel proof-reading polymerase for sample amplification with a low misincorporation rate enables simplification of the DHPLC patterns and hence enhanced mutation detection recognition. This scheme for increasing DHPLC throughput has been tested by scanning the transforming growth factor (TGF) beta3 gene for the presence of mutations for which there is limited published or on-line data available regarding the presence of gene polymorphisms. TGFbeta isoforms have multiple roles in cell division, growth, proliferation, transformation and differentiation. TGFbeta3 is a TGFbeta cytokine isoform, and has an important role in embryogenesis, cell differentiation and wound healing. The TGFbeta3 gene consists of seven exons and six introns spanning 43 000 bp of the human genome on chromosome 14q23-24. The rapid DHPLC approach enabled scanning of all seven exons and part of the promoter region (1000 bp upstream from exon 1 in the 5'-flanking regions) of the TGFbeta3 gene in 95 Caucasian individuals in only 8 days, in comparison to the 17 days it would have previously taken. Mutations were clearly identified in the promoter region of the TGFbeta3 gene but were absent from the exonic regions. Understanding the genetic variations affecting the TGFbeta3 gene is important as this molecule has multiple regulatory functions on a variety of cell types.
Bayat
A
A
Centre for Integrated Genomic Medical Research, University of Manchester, Manchester, UK. ardeshir.bayat@man.ac.uk
Walter
J M
JM
Lamb
H
H
Ferguson
M W J
MW
Ollier
W E R
WE
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase; D005260:Female; D006801:Humans; D008297:Male; D009154:Mutation; D011401:Promoter Regions, Genetic; D016212:Transforming Growth Factor beta / Q000235:genetics*; D053782:Transforming Growth Factor beta3
D016212:Transforming Growth Factor beta; D053782:Transforming Growth Factor beta3; D004259:DNA-Directed DNA Polymerase