Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review

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.

Genetic polymorphisms associated with polycystic ovary syndrome among Iranian women

“…Genotyping of SNPs can be done relatively easily and at reasonably low cost via polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). However, although the results of PCR–RFLP are easy to interpret, this approach may not be the most suitable for genotyping because it involves extensive manual handling and longer pause times 128 . Polymorphisms can also be analyzed via amplification refractory mutation system–polymerase chain reaction (ARMS‐PCR), which can be modified into a one‐step procedure based on real‐time PCR technology to reduce manual handling 129 …”

Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

“…Subsequently, the digested amplicons are loaded onto a gel, to which an electric field is applied. The differently sized bands will move at varying distances across the gel [51].…”

Sensitivity and applications of the PCR Single-Strand Conformation Polymorphism method

“…A detailed comparison of these methods is made in the work of Hashim et al [ 7 ]. Both of them have advantages and disadvantages.…”