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Use of GC Clamps in DHPLC Mutation Scanning

Research Division, Hospital for Special Surgery, New York, New York

Rajarsi Gupta, BA^*

Laboratory for Fluorescence Dynamics, Physics Department, University of Illinois, Urbana-Champaign, Urbana, Illinois

Andrew P. Parnassa, BA^*

Department of Microbiology and Immunology, Weill Medical College, Cornell University, New York, New York

Sargam Jain, BA^*

State University of New York at Stony Brook, School of Medicine, Stony Brook, New York

Jason A. Wexler, MD^*

Division of Endocrinology, Clinical Nutrition and Vascular Medicine, University of California, Davis Medical Center, Sacramento, California

Jia Li Chu, MD^*

Division of Rheumatology, University of Washington, Seattle, Washington

Keith B. Elkon, MD^*

Division of Rheumatology, University of Washington, Seattle, Washington

Robert D. Blank, MD, PhD^*

Section of Endocrinology, Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin and Geriatrics Research, Education and Clinical Center, William S. Middleton Veterans Hospital, Madison, Wisconsin

REPRINT REQUESTS: Robert D. Blank, MD, PhD, Section of Endocrinology, Department of Medicine, University of Wisconsin-Madison, H4/556 CSC (5148), 600 Highland Avenue, Madison, Wisconsin 53792, Telephone: 608-263-7780, Fax: 608-263-9983, Email: rdb{at}medicine.wisc.edu

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 Colla2 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.

1. The target sequence should have a uniform T_m
   
2. GC clamps of length sufficient to introduce a second melting domain with a T_m 8° above that of the target sequence should be appended to one of the primers.
   
3. The DHPLC assay should be performed at the highest temperature at which the target sequence is predicted to be 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.

Key Words: Heterozygote detection • Nucleic acid denaturation • Genetic screening • Polymerase chain reaction