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.
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.
Sheffield
V C
VC
Department of Pediatrics, University of California, San Francisco 94143.
Cox
D R
DR
Lerman
L S
LS
Myers
R M
RM
D000818:Animals; D001482:Base Composition; D003596:Cytosine; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005784:Gene Amplification; D005796:Genes; D005821:Genetic Techniques; D005914:Globins / Q000235:genetics; D006147:Guanine; D006801:Humans; D051379:Mice; D009154:Mutation; D011401:Promoter Regions, Genetic; D013824:Thermus / Q000201:enzymology
D006147:Guanine; D003596:Cytosine; D005914:Globins; D004259:DNA-Directed DNA Polymerase