73 GATA 9(3): 73-79, 1992
REVIEW
PCR-SSCP: A Method for Detection of Mutations KENSHI HAYASHI PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) analysis is one of the simplest and perhaps one of the most sensitive methodsfor detection of mutations based on PCR technology. The principles of PCR--SSCP, guidelines for experiments, and applications of this technique in various fields are reviewed.
Introduction The development of PCR (polymerase chain reaction) enabled specific segments of genomic DNA to be obtained without the complicated and skill-demanding procedures of cloning in vivo [1]. A genomic region specified by a pair of primers can be easily amplified from different sources and sequence changes, that is, mutations, can be found. Of several PCR-based techniques for detection of mutations, PCR-SSCP (single-strand conformation polymorphism), analysis is perhaps the easiest and one of the most sensitive [2, 3]. The original method required radioactivity, but nonradioactive methods have recently been developed. Since its first report, this technique has been widely used for the detection of mutations in genes responsible for various hereditary diseases, somatic mutations of oncogenes or tumor-suppressor genes in cancer tissues. This technique has also been useful for detecting polymorphisms that are linked to various genes or that can serve as locus markers in linkage mapping of the human genome.
Principle Detection of mutations by mobility shift of singlestranded DNA in nondenaturing polyacrylamide gel electrophoresis was first demonstrated by Kanazawa From the Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan. Address correspondence to Dr. K. Hayashi, Oncogene Division, National Cancer Center Research Institute, 1-1 Tsukiji 5chome, Chuo-ku, Tokyo 104, Japan. Received 5 February 1992; revised and accepted 29 June 1992.
and his colleagues in studies on Escherichia coli F1ATPase [4]. The method became versatile with the advent of PCR [2, 5]. In PCR-SSCP analysis, the target sequence in genomic DNA or cDNA is simultaneously amplified and labeled by using radioactive-labeled primers or nucleotides. The amplified product is then denatured to a single-stranded form and subjected to nondenaturing polyacrylamide gel electrophoresis. Bands of the single-stranded DNA at different positions in the autoradiogram indicate the presence of mutations. In nondenaturing conditions, single-stranded DNA has a folded structure that is determined by nucleotide sequence. The sensitivity of PCR-SSCP (that is, its ability to detect mutation) depends on how mutation affects the folding and how the folding affects electrophoretic mobility of the target sequence. No theory can either predict the exact folded structure of single-stranded DNA or accurately estimate the effects of structure on mobility in gel electrophoresis [6]. Therefore, the sensitivity of PCR-SSCP cannot be predicted physicochemically. An empirical estimation of sensitivity is also difficult because it depends on the neighboring sequences that interact with the mutated sites, and too few data are available to enable prediction of the sensitivity of this technique in the context of all possible sequences. Still, judging from the limited experience of our group and others, PCR-SSCP seems to detect >90% of all single-base substitutions in 200 nucleotide fragments and >80% in 400 nucleotide fragments [3, 7, 8]. The sensitivity of PCR-SSCP tends to decrease with increasing fragment length [3]. Therefore, long sequences must be divided into shorter segments before analysis by SSCP. This can be done either by amplifying the target sequence in overlapping subfragments separately or by amplifying the intact fragments in one unit and then digesting them with suitable restriction enzymes. Kovar et al. proposed separation of the digested fragments in a two-dimensional gel [9]. It should be stressed, however, that most exons in mammalian genomes are
REVIEW
PCR-SSCP: A Method for Detection of Mutations KENSHI HAYASHI PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) analysis is one of the simplest and perhaps one of the most sensitive methodsfor detection of mutations based on PCR technology. The principles of PCR--SSCP, guidelines for experiments, and applications of this technique in various fields are reviewed.
Introduction The development of PCR (polymerase chain reaction) enabled specific segments of genomic DNA to be obtained without the complicated and skill-demanding procedures of cloning in vivo [1]. A genomic region specified by a pair of primers can be easily amplified from different sources and sequence changes, that is, mutations, can be found. Of several PCR-based techniques for detection of mutations, PCR-SSCP (single-strand conformation polymorphism), analysis is perhaps the easiest and one of the most sensitive [2, 3]. The original method required radioactivity, but nonradioactive methods have recently been developed. Since its first report, this technique has been widely used for the detection of mutations in genes responsible for various hereditary diseases, somatic mutations of oncogenes or tumor-suppressor genes in cancer tissues. This technique has also been useful for detecting polymorphisms that are linked to various genes or that can serve as locus markers in linkage mapping of the human genome.
Principle Detection of mutations by mobility shift of singlestranded DNA in nondenaturing polyacrylamide gel electrophoresis was first demonstrated by Kanazawa From the Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan. Address correspondence to Dr. K. Hayashi, Oncogene Division, National Cancer Center Research Institute, 1-1 Tsukiji 5chome, Chuo-ku, Tokyo 104, Japan. Received 5 February 1992; revised and accepted 29 June 1992.
and his colleagues in studies on Escherichia coli F1ATPase [4]. The method became versatile with the advent of PCR [2, 5]. In PCR-SSCP analysis, the target sequence in genomic DNA or cDNA is simultaneously amplified and labeled by using radioactive-labeled primers or nucleotides. The amplified product is then denatured to a single-stranded form and subjected to nondenaturing polyacrylamide gel electrophoresis. Bands of the single-stranded DNA at different positions in the autoradiogram indicate the presence of mutations. In nondenaturing conditions, single-stranded DNA has a folded structure that is determined by nucleotide sequence. The sensitivity of PCR-SSCP (that is, its ability to detect mutation) depends on how mutation affects the folding and how the folding affects electrophoretic mobility of the target sequence. No theory can either predict the exact folded structure of single-stranded DNA or accurately estimate the effects of structure on mobility in gel electrophoresis [6]. Therefore, the sensitivity of PCR-SSCP cannot be predicted physicochemically. An empirical estimation of sensitivity is also difficult because it depends on the neighboring sequences that interact with the mutated sites, and too few data are available to enable prediction of the sensitivity of this technique in the context of all possible sequences. Still, judging from the limited experience of our group and others, PCR-SSCP seems to detect >90% of all single-base substitutions in 200 nucleotide fragments and >80% in 400 nucleotide fragments [3, 7, 8]. The sensitivity of PCR-SSCP tends to decrease with increasing fragment length [3]. Therefore, long sequences must be divided into shorter segments before analysis by SSCP. This can be done either by amplifying the target sequence in overlapping subfragments separately or by amplifying the intact fragments in one unit and then digesting them with suitable restriction enzymes. Kovar et al. proposed separation of the digested fragments in a two-dimensional gel [9]. It should be stressed, however, that most exons in mammalian genomes are
