Abstract
Polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) and PCR–restriction fragment length polymorphism (PCR-RFLP) are two independent methods used in the post-amplification genotyping of DNA variations. Both techniques are used in a wide range of screening applications to characterize single nucleotide polymorphisms (SNPs). The PCR-SSCP enables the identification of a potentially causative unknown SNP that could not be identified by PCR-RFLP. However, because complicated steps are not required to perform PCR-RFLP, it is used in many applications. On the other hand, PCR-RFLP is easier to process in terms of time and handover experience, the detection of a particular unknown SNP by PCR-SSCP has further chances. The simplicity of PCR-RFLP does not mean that it is better than PCR-SSCP. The reason is the limited ability of PCR-RFLP to detect nucleotide variations, which often go undetected because each restriction enzyme (RE) scans only a few recognition sequences, and other sequences are ignored. Furthermore, the efficacy of PCR-SSCP is sometimes hindered by many optimizations and also lack of experience. As PCR-SSCP allows other sequences within an amplicon to be separated and characterized, the choice between PCR-RFLP and PCR-SSCP is largely dependent on the reason for each genotyping experiment. This review provides a useful guide for comparing PCR-RFLP and PCR-SSCP in terms of their concepts, efficiency, ease of use, interpretation, and sensitivity as well as several other parameters. The comparison is extended to the practical applications of both techniques in terms of their utilization in molecular diagnostics and related applications.
Highlights
The gold-standard method for the identification of mutations in polymerase chain reaction (PCR) amplicons is direct sequencing
Several post-PCR genotyping techniques are available to identify the variations in nucleic acid sequences, such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), chemical mismatch cleavage (CMC) method, and amplification refractory mutation system (ARMS), which are applied to visualize the nucleic acid variations in a range of efficiencies and sensitivity
The concept of PCR-SSCP is based on the presence or absence of a particular mutation between normal and mutant amplicons as a result of the differences between their physical characteristics, which could be extended to include nucleotide sequences that exceed those detected by PCRRFLP
Summary
The gold-standard method for the identification of mutations in polymerase chain reaction (PCR) amplicons is direct sequencing. Parameter of Comparison · Based on the presence or absence of the recognition sequences · Based on physical and chemical differences between the normal and mutants single-strand DNA · Prior genomic knowledge requirements are mandatory · Detection of unknown mutation(s) is possible · It's costly in large-scale applications · Digestion with restriction enzymes is required · It’s always necessary to perform sequencing reactions · A considerable technical experience is required · Electrophoresis is usually performed on horizontal agarose-based gel format · Electrophoresis is usually performed on a vertical polyacrylamide-based gel format · Special optimizations, such as temperature, gel concentration, voltage, are required · Staining is usually simple and can be completed by only one step · Sensitivity is always high between normal and mutant nucleotides · Short time is sufficient for processing samples before conducting electrophoresis · Only a short time is needed for electrophoresis · Controlling temperature is required in electrophoresis · It is the favoured technique in working on a previously known SNP · Larger gel format is sometimes needed · Sizes of amplicons are essential in genotyping efficiency · Very low sample concentration is required for electrophoresis · Stained bands are easy to be read and interpret in almost all cases · Few specific nucleotides are targeted, while other sequences are ignored · It's suitable to simultaneously analyze many SNPs in the same amplicon · The exact conformation of genotyped bands could be predicted in electrophoresis
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