Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes

Val C Sheffield; D R Cox; L S Lerman; R M Myers

doi:10.1073/pnas.86.1.232

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Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes

Journal article

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Peer reviewed

Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes

Val C Sheffield, D R Cox, L S Lerman and R M Myers

Proceedings of the National Academy of Sciences - PNAS, Vol.86(1), pp.232-236

01/1989

DOI: 10.1073/pnas.86.1.232

PMCID: PMC286438

PMID: 2643100

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Abstract

Denaturing gradient gel electrophoresis (DGGE) can be used to distinguish two DNA molecules that differ by as little as a single-base substitution. This method detects approximately 50% of all possible single-base changes in DNA fragments ranging from 50 to approximately 1000 base pairs. To increase the number of single-base changes that can be distinguished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human beta-globin genes. We show that this GC-clamp allows the detection of mutations, including the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human beta-globin gene, that were previously indistinguishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from less than 5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.

Genes

Mutation

Promoter Regions, Genetic

Thermus - enzymology

Humans

Genetic Techniques

Guanine

Globins - genetics

Animals

Gene Amplification

Base Composition

Cytosine

Mice

DNA-Directed DNA Polymerase - metabolism

Details

Title: Subtitle: Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes
Creators: Val C Sheffield - Department of Pediatrics, University of California, San Francisco 94143
D R Cox
L S Lerman
R M Myers
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.86(1), pp.232-236
DOI: 10.1073/pnas.86.1.232
PMID: 2643100
PMCID: PMC286438
NLM abbreviation: Proc Natl Acad Sci U S A
ISSN: 0027-8424
eISSN: 1091-6490
Publisher: National Academy of Sciences; United States
Grant note: GM07085 / NIGMS NIH HHS
Language: English
Date published: 01/1989
Academic Unit: Stead Family Department of Pediatrics; Iowa Neuroscience Institute; Medical Genetics and Genomics; Ophthalmology and Visual Sciences
Record Identifier: 9984065493202771

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