Faculty Collaboration Database

Scanning the beta-globin gene for mutations in large populations by denaturing capillary and gel electrophoresis. Electrophoresis 2005 Jun;26(13):2531-8

Date

06/11/2005

Pubmed ID

15948210

DOI

10.1002/elps.200410431

Scopus ID

2-s2.0-22244480969 (requires institutional sign-in at Scopus site)   12 Citations

Abstract

Separation of mutant from nonmutant DNA sequences of 100 bp may be accomplished by using defined denaturing conditions of chemical denaturant and/or elevated temperature during electrophoresis on either polyacrylamide slab gels (denaturing gradient gel electrophoresis, DGGE) or capillary gels (constant denaturant capillary electrophoresis, CDCE). In analysis of mutant directly from a polymerase chain reaction (PCR) product mixture, both have detection sensitivities of approximately 1%. CDCE that facilitates an intermediate mutant enrichment step permits detection of mutants at fractions as low as 2 x 10(-6). Here we report the successful application of both approaches to scan for mutations of the human beta-globin gene (HBB) in two human population samples of approximately 5000 persons in the HBB. Using DGGE, the coding region and flanking intronic splice sites of HBB were scanned in a population of 4949 Han Chinese individuals in pool sizes of 48 individual DNA samples. Four point mutations ranging in mutant frequency from 0.5 to 0.0002 were identified. Using CDCE with a mutant enrichment step, these same sequences were scanned in a population of 5028, predominantly African-American juveniles (<9 years) as a single pooled DNA sample. Three point mutations were identified ranging in mutant frequency from 0.13 to 0.0005. This study shows that both the DGGE/small pool and the CDCE/large pool approaches offer the means to define the fine structure map of genetic variation in large population samples, and with appropriately engineered facilities to provide high throughput, should be useful in pangenomic scans to discover genes carrying casual mutations for common diseases.

Author List

Li-Sucholeiki XC, Hu G, Perls T, Tomita-Mitchell A, Thilly WG

Author

Aoy Tomita Mitchell PhD Professor in the Surgery department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Adult
Child
China
DNA Mutational Analysis
Electrophoresis, Capillary
Electrophoresis, Polyacrylamide Gel
Gene Frequency
Genetics, Population
Globins
Humans
Nucleic Acid Denaturation
Open Reading Frames
Polymerase Chain Reaction
RNA Splice Sites