Single strand conformation polymorphism sscp analysis of polymerase chain reaction pcr products is a genetic screening technique for rapid detection of nucleotide substitutions in pcr. A novel genetic approach for classifying the species of aspergillus section flavi is described here. The principle of sscp analysis is based on the fact that single stranded dna has a defined conformation. Pcr single strand conformation polymorphism analysis for detection of bovine.
Single strand conformation polymorphism sscp technique is a simple and efficient means to detect any small alteration in pcramplified product. Singlestrand conformation polymorphism analysis of pcr. Single strand conformation polymorphism sscp sscp relies on the fact that within a short dna segment usually no more than 300 base pairs bp, a single base change in the sequence can cause major changes in single stranded conformation that is a reflection of the secondary structure of single stranded. Use of a singlestrand conformation polymorphism analysis. Highthroughput, highsensitivity genetic mutation detection by. Single strand conformation polymorphism method for initial detection dna sequences homogeneity topik hidayat 1, adi pancoro 2 1department of biology education, faculty of mathematic and science education, indonesia university of education, jalan dr. Highthroughput, highsensitivity genetic mutation detection by tandem single strand conformation polymorphismheteroduplex analysis. Rapid identification of bacteria by pcr single strand conformation polymorphism. Singlestrand conformation polymorphism sscp description sscps are dna fragments of about 200800 bp amplified by pcr using specific primers of 2025 bp. Screening for mutations by rna singlestrand conformation polymorphism.
This application note describes the use of cap on the applied. Mutation scanning methods, including pcrcoupled singlestrand conformation polymorphism sscp, have significant advantages over many other nucleic acid techniques for the accurate analysis of. Sscp single strand conformation polymorphism is a technique used for the detection of single nucleotide polymorphism snps. Single strand conformation polymorphism analysis targeting the v3 region of 16s rdna revealed between four to six distinct archaeal peaks per digester. Single strand conformation polymorphism an overview. Pcrsinglestrand conformational polymorphism method for. Single strand conformation polymorphism sscp analysis on. Detection of cyclospora in captive chimpanzees and. Single strand conformation polymorphism based snp and.
Nucleic acid chains have a strong tendency for base pairing. Single strand conformation polymorphism sscp youtube. Hence, the size of pcr fragments investigated are usually in the range of 175250 bp. Bray glanzmann thrombasthenia gt is the most common in herited disorder of platelets. Pcrbased singlestrand conformation polymorphism sscp analysis to clone nine aquaporin genes in cucumber jiahua xie,1 todd c. This can be detected by highly sensitive electrophoresis methods, such as capillary electrophoresis ce. This property allows sequences to be distinguished by means of gel electrophoresis, which. Under nondenaturing conditions, single stranded dnas will fold into secondary structures conformations according to their nucleotide sequences and their phys. Jci a single strand conformation polymorphism study of. Singlestrand conformational polymorphism analysis springer. Singlestrand conformation polymorphism sscp is a conformational polymorphism on a single strand.
Mapping singlestrand conformation polymorphism analysis. Singlestrand conformation polymorphism an overview. Singlestrand conformation polymorphism analysis coupled. Because of the lack of a complementary strand, rna and single stranded dna pair with themselves and thereby assume strange conformations, which are highly complex and cannot be determined beforehand. Highthroughput single nucleotide polymorphism typing by fluorescent single strand conformation polymorphism analysis. We have found that single strand conformation polymorphism analysis of sequences that are potentially less subject to. Committee on molecular biology techniques, single strand conformation polymorphism analysis with high throughput modifications, and its use in mutation detection in familial hypercholesterolemia, clinical. Four methods for detecting mutations based on sscp are described here. Comparison of conformation sensitive gel electrophoresis and singlestrand conformation polymorphism analysis for detection of mutations in the. Detection of single nucleotide mutations in wheat using single strand conformation polymorphism gels. Pcr and single strand conformational polymorphism for recognition of medically important opportunistic fungi. Singlestrand conformation polymorphism sscp for the. Nested pcrlinked capillary electrophoresis and single.
Get a printable copy pdf file of the complete article 1. Steve e humphries, vilmundur gudnason, ros whittall, ian n m day, for the international federation of clinical chemistry scientific divison. The principle reasons for the popularity of these two methods are their technical simplicity and their relatively high sensitivity for the detection of mutations. Land rb and robinson dw eds genetics of reproduction in. Gel electrophoresis of single strand dna is used to detect nucleotide sequence variation among the amplified fragments. Singlestrand conformation polymorphism analysis with high. One widely used technique is single strand conformation polymorphism sscp analysis 1. Single strand conformation polymorphism method for initial.
A brief introduction to single strand conformational. To identify efficiently mutations in the gene for cd40 ligand, eight pairs of pcr primers that could be used to screen genomic dna by single strand conformation polymorphism sscp were designed. Dna polymorphism produced by differential folding intramolecular interaction of single stranded dna harboring mutations. This property allows sequences to be distinguished by means of gel electrophoresis, which separates fragments according to. Pdf link pdf citation y jin, hc dietz, a nurden, pf bray. Singlestrand conformation polymorphism sscp analysis. Pdf singlestrand conformation polymorphism sscp for. Pcrbased singlestrand conformation polymorphism sscp. Conkling3 departments of horticultural science and genetics, north carolina state university, raleigh, nc. It is important to optimise the pcr reaction to minimise. A method for the molecular analysis of genetic diseases kakavas v.
The method is based on the fact that the electrophoretic mobility of. Single stranded conformation polymorphism sscp analysis is a widely used screening method that allows you to identify different genomic variants in a large number of samples and in a broad range of organisms, from microorganisms to humans. Pmc free article walsh tj, francesconi a, kasai m, chanock sj. Applications of polymerase chain reactionsingle stranded. It is based on the assumption that subtle nucleic acid change affects the migration of single stranded dna fragment and, therefore, results in visible mobility shifts across a nondenaturing. Cyclospora is a protistan parasite that causes enteritis in several species of animals including humans. The reference standard methods for drug susceptibility testing of mycobacterium tuberculosis, such as culture on lowensteinjensen or middlebrook 7h1011 medium, are very slow to give results. The aim of this study was to investigate the presence of cyclospora in captive nonhuman primates. Nikiforov, in diagnostic immunohistochemistry third edition, 2011. Among the techniques capable of detecting a single base change, the singlestrand conformation polymorphism sscp analysis is most commonly used due to its simplicity and effectiveness orita et al. Singlestrand conformation polymorphism analysis with high throughput modifications, and its use in mutation detection in familial hypercholesterolemia.
A method for mutant detection and snp identification based on pcr without the need of sequencing. Singlestrand conformational polymorphism sscp analysis is a simple and sensitive technique for mutation detection and. Singlestrand conformation polymorphism sscp analysis is a postpcr technique that can be used to screen for mutations that are not limited to a single hot spot but are randomly distributed throughout the exons. Singlestrand conformation polymorphism sscp analysis pcr sscp analysis is generally considered to be most suitable for the detection of mutations in short stretches of dna. Singlestrand conformation polymorphism sscp for the analysis of. The single strand conformation polymorphism sscp technique is based on changes in the conformation of single stranded dna ssdna that are induced by a single base mutation.
This approach consists of pcr amplification of the 5. Sscp is a technique designed to detect mutations in oncogenes and corresponding author. Pdf analysis of singlestrand conformation polymorphism. Highthroughput single nucleotide polymorphism typing by fluorescent single strand conformation polymorphism analysis with capillary electrophoresis. Eijkman winkler institute for medical andclinical microbiology, university hospital utrecht, roomg04. The use of pop conformational analysis polymer cap on the applied biosystems 3 and 3xl genetic analyzers is an efficient, convenient, and costeffective solution for performing sscp. Mutation scanning methods, including pcrcoupled singlestrand conformation polymorphism sscp, have significant advantages over many other. A total of 119 faecal samples from pan troglodytes, macaca sylvanus, cercopithecus cephus, erythrocebus patas, chlorocebus aethiops and macaca. Singlestrand conformational polymorphism sscp analysis is a simple and sensitive technique for mutation detection and genotyping.
Single strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein iiia. The subsequent sequence analysis of polymorphic bands isolated from gels is also detailed. Analysis of single strand conformation polymorphism by capillary electrophoresis with laserinduced fluorescence detection using shortchain polyacrylamide as sieving medium. Single strand conformation polymorphism sscp thermo fisher.
Comparison of conformationsensitive gel electrophoresis. Singlestrand conformation polymorphism sscp, or single strand chain polymorphism, is defined as a conformational difference of single stranded nucleotide sequences of identical length as induced by differences in the sequences under certain experimental conditions. Singlestrand conformation polymorphism analysis is a. Single strand confirmation polymorphism sscp detection. A new approach to utilize pcrsinglestrandconformation.
Single strand conformation polymorphism sscp is an electrophoretic technique which has been developed, like dgge 10, for the detection of mutations, mainly in human genetics, 34. Sscp technique is a method capable of identifying most sequence variations in a single strand of dna, typically between 150. Single strand conformation polymorphism sscp analysis. Sscp analysis permits the detection of mutations in very small amounts of genomic dna or cdna. Singlestrand conformation polymorphism analysis fig. A sscp assays are performed using heatdenatured pcramplified dna on. The diversity of dominant archaea in the 44 digesters was estimated as 23 different 16s rdna sequences. Single strand conformational polymorphism sscp analysis is a simple and sensitive technique for mutation detection and genotyping.
286 581 87 1511 1190 820 596 419 859 982 1525 1532 93 140 727 723 1350 792 1147 287 650 1486 1146 795 448 1586 839 734 274 621 389 703 86 936 83 1226 472 204 215 390 1004