Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy

Abstract

The recent achievement of the human genome project has led to the identification of many disease genes in common hereditary conditions, in which patients and their relatives would benefit from genetic diagnosis. This has increased the need for simple, sensitive, and cost effective methods of mutation analysis. However, the “gold standard” of mutation analysis, direct sequencing, is still an expensive and labour intensive method for investigation of large genes, multi-allelic diseases, and large numbers of patients. Today the most frequently used methods for prescreening disease genes for mutations are single strand conformation polymorphism analysis (SSCP) and denaturing high performance liquid chromatography (DHPLC). Both methods accelerate specific diagnosis at the DNA level by limiting the need for direct sequencing to a few abnormal polymerase chain reaction (PCR) products identified in the prescreening procedure. The tecnique of SSCP analysis is based on the principle that changes in nucleic acid composition affect the conformation of single stranded DNA and thereby the mobility of the fragment when it is subjected to electrophoresis under non-denaturing conditions.1,2 Abnormal conformers identified in the prescreening procedure are subsequently investigated for the presence of sequence variants by direct sequencing. The tecnique was initially developed for manual gel electrophoresis in which the mobility of amplified PCR products was visualised by incorporation of radiolabelled nucleotides or by silver staining after electrophoresis.3 Recently, SSCP has been developed for automated capillary electrophoresis (CE) using buffered polymer solution as the sieving matrix for electrophoretic separation and fluorescence as the method of detection.4–7 Fluorescent capillary electrophoresis SSCP (F-SSCP) has the advantage of limited “post-PCR” handling of samples, which are loaded and analysed automatically with the possibility of a high throughput. Also, the requirements for reagent and sample volumes are small. Previous studies have reported a high sensitivity and specificity of …