PMID- 15931297
OWN - NLM
STAT- MEDLINE
DCOM- 20051107
LR  - 20240314
IS  - 1539-4182 (Print)
IS  - 1554-6179 (Electronic)
IS  - 1539-4182 (Linking)
VI  - 1
IP  - 2
DP  - 2003 Apr
TI  - Use of GC clamps in DHPLC mutation scanning.
PG  - 111-8
AB  - 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.
FAU - Wurzburger, Robert J
AU  - Wurzburger RJ
AD  - Research Division, Hospital for Special Surgery, New York, New York, USA.
FAU - Gupta, Rajarsi
AU  - Gupta R
FAU - Parnassa, Andrew P
AU  - Parnassa AP
FAU - Jain, Sargam
AU  - Jain S
FAU - Wexler, Jason A
AU  - Wexler JA
FAU - Chu, Jia Li
AU  - Chu JL
FAU - Elkon, Keith B
AU  - Elkon KB
FAU - Blank, Robert D
AU  - Blank RD
LA  - eng
GR  - AR45482/AR/NIAMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Clin Med Res
JT  - Clinical medicine & research
JID - 101175887
RN  - 5Z93L87A1R (Guanine)
RN  - 8J337D1HZY (Cytosine)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Animals
MH  - *Base Composition
MH  - Chromatography, High Pressure Liquid/*methods
MH  - Cytosine
MH  - DNA/isolation & purification
MH  - DNA Mutational Analysis/*methods
MH  - Electrophoresis
MH  - Guanine
MH  - Humans
MH  - Mice
MH  - Mutation, Missense
MH  - Nucleic Acid Denaturation
PMC - PMC1069033
EDAT- 2005/06/03 09:00
MHDA- 2005/11/08 09:00
PMCR- 2003/04/01
CRDT- 2005/06/03 09:00
PHST- 2003/01/10 00:00 [received]
PHST- 2003/02/24 00:00 [accepted]
PHST- 2005/06/03 09:00 [pubmed]
PHST- 2005/11/08 09:00 [medline]
PHST- 2005/06/03 09:00 [entrez]
PHST- 2003/04/01 00:00 [pmc-release]
AID - 1539-4182-1-2-111 [pii]
AID - 10.3121/cmr.1.2.111 [doi]
PST - ppublish
SO  - Clin Med Res. 2003 Apr;1(2):111-8. doi: 10.3121/cmr.1.2.111.