Towards development of techniques to observe mutational spectra in human tissue samples.
We have devised a protocol with the resolving power to uncover mutational spectra within DNA sequences from human genomic DNA. The protocol is a combination of: (a) in vitro DNA amplification of the desired DNA sequence; (b) denaturing gradient gel electrophoresis of the amplified DNA to separate the wild type and mutant sequences; (c) isolation of the mutant sequence(s); (d) determination of the nature of the base-pair change(s). Data based on reconstruction experiments using exon 3 of the human hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene suggest that the protocol can detect mutant sequences present at mutant fractions equal to or greater than 10(-4).
Keohavong
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Thilly
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WG
D001483:Base Sequence; D004252:DNA Mutational Analysis; D006801:Humans; D009154:Mutation