Faculty Collaboration Database

Use of GC clamps in DHPLC mutation scanning. Clin Med Res 2003 Apr;1(2):111-8

Date

06/03/2005

Pubmed ID

15931297

Pubmed Central ID

PMC1069033

DOI

10.3121/cmr.1.2.111

Scopus ID

2-s2.0-27744461448 (requires institutional sign-in at Scopus site)   9 Citations

Abstract

OBJECTIVE: Development of a systematic mutation detection assay strategy for denaturing high performance liquid chromatography (DHPLC).

DESIGN: Adaptation of Guanine and Cytosine (GC)-clamping from denaturing gradient gel electrophoresis (DGGE) to DHPLC.

METHODS: Three target sequences harboring known allelic variants were studied to develop a general DHPLC assay design strategy. These were exon 10 of the human RET (REarranged during Transfection) gene, exon 52 of the mouse Col1a2 gene, and exon 9 of the human FAS (APO-1, CD-95) gene. Available software was used to analyze melting curves and determine assay conditions. GC clamps of 20 bp or 36 bp were added to polymerase chain reaction (PCR) primers to introduce a high melting temperature (T(m)) domain to each of the target molecules. DHPLC was performed under partially denaturing conditions.

RESULTS: DHPLC assays of PCR-amplified sequences can be developed using a personal computer. The following three steps allowed for mutation detection in all three targets. The target sequence should have a uniform T(m)GC clamps of length sufficient to introduce a second melting domain with a T(m) > or = 8 degrees above that of the target sequence should be appended to one of the primers. The DHPLC assay should be performed at the highest temperature at which the target sequence is predicted to be > or = 90% double stranded

CONCLUSION: Addition of GC-clamps to primers facilitates mutation detection by DHPLC. The theoretical basis for this observation is identical to that underlying the utility of GC-clamps in DGGE.

Author List

Wurzburger RJ, Gupta R, Parnassa AP, Jain S, Wexler JA, Chu JL, Elkon KB, Blank RD

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

Animals
Base Composition
Chromatography, High Pressure Liquid
Cytosine
DNA
DNA Mutational Analysis
Electrophoresis
Guanine
Humans
Mice
Mutation, Missense
Nucleic Acid Denaturation