A comparison of fluorescent SSCP and denaturing HPLC for high throughput mutation scanning

Abstract

We examined 67 different mutations in 16 different amplicons in a comparison of mutation detection by fluorescent single strand conformation polymorphism (F‐SSCP) and by denaturing HPLC (DHPLC). F‐SSCP was used to analyze fluorescent amplicons with internal size standards and automated fragment analysis (GeneScan, PE Applied Biosystems, Foster City, CA). In DHPLC, unlabelled amplicons were analyzed by reverse phase HPLC with fragment detection by absorbance at 260nm. Both methods had high sensitivity (95–100%) and specificity (100%). Overall, F‐SSCP with external temperature control was the more sensitive method, but DHPLC was particularly useful for the rapid analysis of novel fragments. Hum Mutat 15:556–564, 2000. © 2000 Wiley‐Liss, Inc.

Validation of Denaturing High Performance Liquid Chromatography as a Rapid Detection Method for the Identification of Human INK4A Gene Mutations

“…24,32 It has been estimated that the sensitivity of the detection is greater than 95%; 30 similar or superior to that obtained by SSCP. 24,30,33,34 The sensitivity of the method is dependent on the temperature at which the analysis is completed, and to partially circumvent the operator's experience, software has been developed for predicting the optimal temperature for DHPLC analysis. For the analyses of INK4A mutations we have relied on the software-based predictions and we also selected overlapping temperatures to increase the chance of heteroduplex detection.…”

Simultaneous (AC)n microsatellite polymorphism analysis and single‐stranded conformation polymorphism screening is an efficient strategy for detecting ankyrin‐1 mutations in dominant hereditary spherocytosis

“…This may be due to the less than 100% yield of SSCP reported previously (Michaud et al, 1992;Hayashi & Yandell, 1993;Sheffield et al, 1993;Liu & Sommer, 1994;Jordanova et al, 1997;Ellis et al, 2000;Liu et al, 2000), or due to the fact that the mutations are located outside the regions screened by SSCP. Accordingly, approximately 50% dHS patients are likely to carry ANK1 mutations.…”

Denaturing High-Performance Liquid Chromatography Detection of Ribosomal Mutations Conferring Macrolide Resistance in Gram-Positive Cocci

“…It is not clear why the point mutation in the S. pneumoniae rplD gene was missed. Other detection failures were also reported, but the mutations not detected at the T P were all found at the optimal temperature, except for one case, in which C3G and G3C transversions could not be discriminated successfully (5,10,22).…”

“…A technique recently developed in human genetic studies, denaturing high-performance liquid chromatography (DHPLC), allows the rapid automated detection of single base substitutions as well as small insertions or deletions, employing a combination of temperature-dependent denaturation of DNA and ion pair chromatography (5,6,12,22). DHPLC has been used in its early stages for bacterial identification, typing, and molecular epidemiology and has been recently developed for the analysis of resistance to quinolones in laboratory strains of S. aureus and Yersinia pestis and clinical isolates of Salmonella (3,4,7,8,9,28).…”