Detection of DNA sequence variation via deliberate heteroduplex formation from genomic DNAs amplified en masse in "population tubes".
PCR Methods Appl
; 3(4): 225-31, 1994 Feb.
Article
en En
| MEDLINE
| ID: mdl-8173512
ABSTRACT
We have developed the population tube (poptube) system for sensitive detection and large-scale sampling of DNA sequence variation in several human populations of wide geographic distribution. In this methodology, genomic DNAs from five individuals in a population are PCR amplified en masse to maximize deliberately the chances of forming heteroduplexes among allelic variants. Interpopulation mixing is performed in a separate set of tubes containing one individual from each of five populations as well as a reference chimpanzee sample deliberately chosen to be different from all humans. Mismatches at sites of allelic variation retard the electrophoresis and reduce the stability of heteroduplex molecules. The products are electrophoresed on denaturing gradient gels where detection of heteroduplexes is accomplished readily. Using poptubes, we have discovered a rare variant in an otherwise highly conserved 440-bp segment in the long intron of the glucose-6-phosphate dehydrogenase (G6PD) gene. The polymorphism at this X-chromosome locus could be only detected in males by mixing samples, as homoduplexes for both alleles co-focus on denaturing gradient electrophoresis.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Variación Genética
/
ADN
/
Reacción en Cadena de la Polimerasa
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Ácidos Nucleicos Heterodúplex
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
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Female
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Humans
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Male
Idioma:
En
Revista:
PCR Methods Appl
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
1994
Tipo del documento:
Article