Polymerase chain reaction—single strand conformation polymorphism or how to detect reliably and efficiently each sequence variation in many samples and many genes

Abstract

A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. to clarify the genetic background of individuals with multifactorial diseases. We evaluated polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis to identify MPSV in several genes, which are thought to be involved in the pathogenesis of multifactorial autoimmune diseases like multiple sclerosis. The method is based on the property, that the electrophoretic mobility of single-stranded nucleic acids depends not only on its size but also on its sequence. The target sequence was amplified, digested into fragments ranging from 50-200 bp, heat-denatured and analyzed on native gels. The analysis of 55 PCR systems, including a total of 145 fragments demonstrates, that the detection rate of MPSV depends primarily on the fragment lengths. Appropriate dilutions of samples enhances the proportion of ssDNA compared to dsDNA. Changing the gel conditions, glycerol concentrations and/or the addition of urea may increase fragment resolution in some cases. In general, the detection of MPSV is neither influenced by their location within the fragment nor by the type of substitution, i.e. transitions or transversions. The standard PCR-SSCP system described here provides high reliability and detection rates and allows the efficient analysis of many samples and many genes.

The interferon gene cluster: a candidate region for MS predisposition?

“…No functionally relevant polymorphisms were identified. The high efficiency of SSCP analysis to detect MPSV has been reviewed recently, 26 in fact a polymorphism within the coding region of the IFNB gene, albeit non-functional, could be identified by this method. Excluding polymorphism(s) within the IFNB gene in the Caucasian population indicates that NAB production towards IFN␤-1a could not be attributed to sequence variation in the recombinant protein.…”

“…26 The gel matrix consists of 5% polyacrylamide and 2-5% glycerol. Single stranded DNA was detected by radioactively labeling PCR fragments during the amplification process and autoradiography after gel electrophoresis and drying.…”

“…Confirmation of polymorphisms detected by SSCP analysis was achieved by directly sequencing the PCR product of selected DNA's as described previously. 26 Cycle sequencing was carried out utilizing the Thermo Sequenase™ Dye Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech, Freiburg, Germany). Samples were applied to an ABI 373 automated sequencer and analyzed with the ABI 373 software, version 2.1 (Applied Biosystems GmbH, Weiterstadt, Germany).…”

Electrophoresis of DNA in human genetic diagnostics – state‐of‐the‐art, alternatives and future prospects

“…Sequence variations such as small deletions or insertions and even single base substitutions can cause a conformational change and thereby result in a detectable band shift in PAGE as compared to other samples not harboring the genetic variation. The sensitivity of detection of sequence variations with SSCP can be close to 100% under optimized conditions [15] and the use of different gel and electrophoresis conditions can further increase the sensitivity for detection [16]. In some diagnostic laboratories, the exonic sequence of a target gene is screened by SSCP in all patient samples and only the amplicons showing band shifts are subsequently sequenced in order to identify the specific underlying mutation.…”

Identification of the four most common β‐globin gene mutations in Greek β‐thalassemic patients and carriers by PCR‐SSCP: advantages and limitations of the method

“…In this study we investigated whether PCR-SSCP, a method that satisfies most of these criteria and has been used extensively for mutation detection (10,(14)(15)(16), could also be employed for the presumptive identification of known mutations, e.g., the four most common b-globin gene mutations present in Greek patients and carriers.…”