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

The gold-standard method for the identification of mutations in polymerase chain reaction (PCR) amplicons is direct sequencing. Unfortunately, sequencing the reactions of all PCR amplicons is expensive, laborious, and time-consuming, particularly in large-scale applications.1 Nucleic acid-based techniques are mainly used to access and explore phenotype variations between analyzed individuals. In this process, genomic DNA is extracted, a particular genetic locus is targeted, and PCR is performed. Hence, 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. Similarly, the requirements for creating a gradient gel have reduced the availability of both DGGE and TGGE. Furthermore, the necessity of using high-cost fluorescence-labeled probes in CMC has reduced its popularity,2 and both time and cost may be increased when PCR-ARMS is applied.3 For these reasons, the widespread adoption of these post-PCR genotyping techniques has been restricted to use in a limited number of applications. In contrast with the abovementioned techniques, PCR-RFLP and PCR-SSCP have been widely used to genotype amplified products, which has increasingly been reported in the literature.4-6 Despite the development of high-throughput next-generation sequencing and whole exon sequencing,7 the accumulated data on both techniques have been continuously reported.8,9 It is worth mentioning that in addition to being valuable for the determination of intraspecies variation,10 both techniques have been employed in species identification and differentiation. Furthermore, both techniques have been used to differentiate many organisms to species level by the amplification of a conserved region of the mitochondrial D-loop,11,12 ribosomal regions,13,14 or other genetic loci.15,16 However, although PCR-SSCP can be applied to any gene in any organism, PCR-RFLP has less spectrum superiority. Nevertheless, PCR-RFLP has attracted researchers’ attention worldwide because of its low costs and does not require advanced instruments.17 Despite the wide Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review


Introduction
The gold-standard method for the identification of mutations in polymerase chain reaction (PCR) amplicons is direct sequencing. Unfortunately, sequencing the reactions of all PCR amplicons is expensive, laborious, and time-consuming, particularly in large-scale applications. 1 Nucleic acid-based techniques are mainly used to access and explore phenotype variations between analyzed individuals. In this process, genomic DNA is extracted, a particular genetic locus is targeted, and PCR is performed. Hence, 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. Similarly, the requirements for creating a gradient gel have reduced the availability of both DGGE and TGGE. Furthermore, the necessity of using high-cost fluorescence-labeled probes in CMC has reduced its popularity, 2 and both time and cost may be increased when PCR-ARMS is applied. 3 For these reasons, the widespread adoption of these post-PCR genotyping techniques has been restricted to use in a limited number of applications. In contrast with the above-mentioned techniques, PCR-RFLP and PCR-SSCP have been widely used to genotype amplified products, which has increasingly been reported in the literature. [4][5][6] Despite the development of high-throughput next-generation sequencing and whole exon sequencing, 7 the accumulated data on both techniques have been continuously reported. 8,9 It is worth mentioning that in addition to being valuable for the determination of intraspecies variation, 10 both techniques have been employed in species identification and differentiation. Furthermore, both techniques have been used to differentiate many organisms to species level by the amplification of a conserved region of the mitochondrial D-loop, 11,12 ribosomal regions, 13,14 or other genetic loci. 15,16 However, although PCR-SSCP can be applied to any gene in any organism, PCR-RFLP has less spectrum superiority. Nevertheless, PCR-RFLP has attracted researchers' attention worldwide because of its low costs and does not require advanced instruments. 17 Despite the wide versatility of both techniques, no comprehensive review has been reported to have compared PCR-SSCP and PCR-RFLP in terms of sensitivity, efficiency, technical requirements, time, costs, and other related aspects. For this reason, little is known about when and how to select one of these techniques to address genotyping issues. Many researchers need guidelines for selecting between PCR-RFLP and PCR-SSCP in terms of their intended applications. Accordingly, in the present study, a direct comparison has been made between both techniques to enlighten researchers who plan to genotype PCR amplicons by helping them decide which one should be utilized in a specific application. Therefore, the objective of this review is to provide a technical comparison between PCR-RFLP and PCR-SSCP and to determine which one is the most suitable for post-PCR screening.

The Concepts of the Techniques
The PCR-RFLP, which is also known as cleaved amplified polymorphic sequence, was invented by Botstein et al. 18 In this technique, a PCR amplicon is treated by a certain RE that cuts the DNA in a unique restriction site, which is known as the recognition site, to generate several DNA fragments in various sizes. Subsequently, the digested amplicons are loaded onto a gel, and an electric field is applied. The differently sized bands will move at varying distances across the gel. 19 The PCR-SSCP technique was originally applied by Orita et al 20 to identify possible point mutations within PCR amplicons. The main concept of PCR-SSCP is based on the initial separation (melting) of the double-stranded forms by heat into singlestranded forms. In the separated state, the molecules tend to fold into a three-dimensional conformation according to their nucleic acid sequences. Thus, in the polyacrylamide gel electrophoresis, the separated single strands of DNA molecules occupy the same size but accommodate different three-dimensional structures with different mobilities. Such structural conformations are affected by a mutation at a particular nucleotide position in the primary sequence, which can alter the physical conformation of the denatured singlestrand bands. This alteration often leads to the slightly tilted positioning of the mutant single-strand bands with respect to their normal counterparts in the neutral polyacrylamide gels. 21

Simplicity of Use
The most powerful aspect of PCR-RFLP is its simplicity. The PCR-RFLP can be performed without the need for considerable experience in molecular biology. However, despite the ease of use and extreme simplicity of PCR-RFLP, it is confined only with the recognition site of RE (Figure 1), and other sequences are ignored unless double digestion is used with another RE. Thus, the main limitations of PCR-RFLP are the requirement for specific RE and the difficulty of identifying the exact variation in the event in which several SNPs are being targeted at the same time. However, the mixing of two enzymes in one reaction mixture can partially solve this problem. 22 Nevertheless, regarding digestion, there are further complications because of the different types of cofactors and the concentrations needed for each RE to undertake its scheduled task of standardized digestion. 17 In addition, the higher costs of PCR-RFLP resulting from the higher costs of double or triple digestion have added another inevitable limitation that could not be excluded from post-PCR screening experiments.
With regard to PCR-SSCP, several previous reports indicated the simplicity of this technique. [23][24][25] Compared with PCR-RFLP, there is considerable difficulty in PCR-SSCP. Although the electrophoresis of PCR amplicons is carried out in neutral conditions, they should be prepared before being loaded onto polyacrylamide gels, including denaturation with an appropriate SSCP loading dye for 5-10 minutes and chilling in ice for at least 10 minutes. Another difficulty in PCR-SSCP is that it cannot be used to predict the exact conformation of a DNA fragment under different parameters. 26 Therefore, optimizing the conditions of PCR-SSCP electrophoresis for each specified type of PCR product is necessary to control the porosity of the gels, amplicons sizes, loaded amplicons, the voltage applied, and other variable parameters. 27 Therefore, several optimization steps should be conducted to circumvent the undesired low resolutions that might occur in PCR-SSCP. The optimization of PCR-SSCP is achieved in a series of experiments that should be performed to increase the resolution of PCR-SSCP, such as polyacrylamide gel concentration (8%-14%), temperature (4-20°C), and voltage (5-10 V/cm). Other optimizations are required in common PCR-SSCP experiments, including the choice of gel dimension format and the possibility of glycerol being mixed with the neutral gel. 28 Moreover, it is sometimes necessary to perform pre-electrophoresis before loading PCR amplicons onto gels. Although several parameters of PCR-SSCP could be arranged, some optimizations may increase the difficulty of these experiments. However, in some PCR experiments, it is not necessary to conduct all the optimizations in all amplicons, as many of them may yield reasonable SSCP bands by relying on only one procedure. Therefore, such procedures are mandatory when no differences are observed among the analyzed PCR products.

Staining Requirements
With regard to staining amplicons in PCR-RFLP, any commercially available dye is sufficient to stain the digested amplicons. This step can be further simplified by adding the staining dye to the agarose gel before it is polymerized. 29 However, this simplification could slightly halt the movement of the digested DNA molecules. However, in all cases, the dyes used in agarose gel, including the commonly used ethidium bromide, are less sensitive than silver nitrate by about 100fold. Similarly, the agarose gel that is commonly used in PCR-RFLP does not have a high sieving ability compared to the polyacrylamide gel, which is usually used in PCR-SSCP experiments.
In contrast to simple staining methods described in PCR-RFLP, PCR-SSCP is associated with complicated silver-staining procedures. 30 Because of the sensitivity of silver staining reagents, stringent precautions should be taken into account to obtain the best results. These precautions are not only taken in the preparation of the staining kit but also extended to the procedures used to develop the bands. 31 There are two types of bands in polyacrylamide gels: double-stranded, nondenatured bands (dsDNA), and single-stranded, denatured bands (ssDNA). The ssDNA is the highest concern of all optimizations, as it is the region at which a researcher expects to see a possible variation between the normal and mutant ssDNA bands. When such slight differences are observed between the wild type and the altered DNA in the ssDNA region, the task of PCR-SSCP is completed.

Time Requirement
The time required to process the samples is divided into two stages: in vitro digestion of amplicons with RE and electrophoresis. In digestion with REs, the variable times of incubation are required, which vary according to the type of used RE. However, for some enzymes, such as HinfI, the incubation time is 30 min, whereas the standard incubation time is 60 minutes, which is required by almost all enzymes to digest their target recognition sequences. Nevertheless, the incubation time may be extended overnight (or about 17 hours). Because of the extended period of incubation with endonucleases, PCR-RFLP could be considered, in these cases, as a time-consuming method. 32 It is noteworthy that although the brand of RE is a key factor, it is sometimes not considered. However, the quality of synthesized enzymes in terms of the type and the proficiency of the recombinant DNA technology used to generate such enzymes may vary from manufacturer to manufacturer. Therefore, the same RE produced by two manufacturers may differ in cost, expiry date, transportation conditions, efficiency of digestion, time needed for incubation, and other parameters. In addition, in some experiments, incubation times are extended to ensure efficient digestion. Nevertheless, there is no guarantee that acceptable results will be achieved in all cases because some REs tend to lose efficiency after being incubated with amplicons for extended periods. 33 Moreover, any change in the RE and the buffer concentration may lead to undesirable outcomes. 34 Therefore, optimization is needed for both the incubation time and the brand of RE used in the incubation. 35 Regardless of the time required for incubation, the time needed to run the digested amplicons is only 20-30 minutes at 7 V/cm, which usually yields the intended results. In previous protocols, different periods were used to perform electrophoresis in PCR-SSCP, which differed in terms of the duration required to obtain adequate separation among the genotyped samples. Some protocols relied on only 4 hours of electrophoresis with applying high constant voltage, 36 while in other procedures, extended periods up to 20 hours were used to run amplicons. In these periods, a lower voltage was applied, and constant temperature control was achieved by recirculating chillers. 37 However, several optimizations are highly recommended before the application of each procedure.

Recommended Sizes of Amplicons
The size of the amplicons in PCR-RFLP does not limit the successful performance of its use. The reason is that PCR-RFLP does not rely on the physical status of the amplicon. Instead, the presence or absence of a recognition sequence is the only rate-limiting step; otherwise, the intended RE does not pay attention to the length of its corresponding amplicon. The PCR-RFLP is usually conducted on horizontal agarose gels. However, to conduct a successful PCR-RFLP, a high concentration of amplicons is needed 38 because of the limited capability of agarose gels to separate molecules compared with the highly sensitive polyacrylamide gels. It is worth mentioning that PCR-RFLP can be conducted on a vertical polyacrylamide gel, and the amplicons can be detected by highly sensitive silver-staining kits. 39 However, this protocol is not usually applied in PCR-RFLP because almost all procedures take place in horizontal agarose formats. The PCR-SSCP can detect polymorphism up to 500 bp fragments, which, however, are optimized between a capacity of 200-600 bp. To accommodate as many nucleic acids as possible without being affected by the low efficiency of PCR-SSCP detection, the optimal size of amplicons should range between 330 and 380 bp. However, PCR-SSCP requires only a small number of amplicons to undergo optimal electrophoretic separation on polyacrylamide gels. This feature is derived from the ability of the post-electrophoresis silver-staining technique to detect extremely low concentrations of DNA. Thus, PCR-SSCP is usually performed by applying amplicons that are 2 µL, which saves residual amounts for other applications. Moreover, loading small sizes of amplicons enhances the staining sensitivity of silver nitrate.

The Choice Between PCR-RFLP and PCR-SSCP
The PCR-SSCP is sometimes difficult to understand because of the multitude of interacting factors and outcomes of each genotype amplicon. However, the interpretation of PCR-RFLP results is easy compared to PCR-SSCP because of previously designed amplicons, recognition sequences, and the expected sizes of the digested fragments. However, this does not mean that the superiority of PCR-RFLP over PCR-SSCP as PCR-SSCP has significant advantages over PCR-RFLP in providing the accurate detection of nucleic acid variations. 21 Therefore, the choice of the most appropriate method could depend on the targeted purpose of genotyping. If only one SNP is being genotyped in a certain population, the choice will usually be PCR-RFLP. In contrast, PCR-SSCP is favored when the largescale screening of all amplicons is required in searching for previously unknown data (Table 1).

Applications
Both the PCR-RFLP and PCR-SSCP techniques have been recently in several species, ranging from humans to microorganisms. The apparent superiority of PCR-RFLP was observed regarding its broad utilization in several aspects of medical human genetics, such as the diagnosis of carcinogenesis, parasitic infection, gastritis, urinary tract infection, arthrosclerosis, infertility, and blood grouping. [40][41][42][43][44][45][46][47][48] This higher reliability on PCR-RFLP may be attributed to its simplicity compared with PCR-SSCP, because of which it has been used in previous SNP-specified applications. In contrast to medical applications, PCR-RFLP has not exhibited superiority compared with PCR-SSCP. This alteration has been well documented in several domestic animals in which many genotype-phenotype studies were performed. Several productive and reproductive traits that depend on SNPs were detected by PCR-SSCP, such as wool characteristics, milk synthesis, carcass weight, meat tenderness, and biochemical parameters [49][50][51][52][53][54] (Table 2). However, PCR-RFLP applications were used in the detection of pork contamination in frozen meat products. 55 The same results observed in domestic animals using PCR-RFLP were obtained in assessing the possible adulteration of sausage products made with chicken. 56 Moreover, PCR-RFLP has also been considered in the study of some growth and performance traits. 57 However, PCR-SSCP was considered as a cornerstone in recent post-PCR genotyping studies on poultry, such as egg-production traits, body weight characteristics, and intramuscular fat content. [58][59][60] Because of its powerful ability to identify unknown SNP(s),  However, both techniques have been employed to study several mechanisms in plants with a variety of genetic polymorphism purposes that ranged from splicing alterations to fingerprinting and diagnostic markers. [62][63][64] With respect to fish and amphibians, species identification and the detection of environmental samples and closely related organisms were recently conducted using PCR-RFLP. [56][57][58][59][60][61][62][63][64][65][66][67][68] The PCR-SSCP was used in the detection of parasite species. 69 The ease of the use of PCR-RFLP was demonstrated in its successful implementation in species identification and discrimination in algae, bacteria, and fungi. [70][71][72]

Future Perspectives
The future uses of PCR-RFLP and PCR-SSCP techniques are now being challenged because of the massive development of high-throughput DNA sequencing protocols. 25 Nevertheless, such highly efficient protocols are not available in mediocratic laboratories because of budget limitations. Both techniques are highly valuable in genotyping, including wide applications in agricultural, 73 medical, 74 and microbiological 75 genotyping research.

Conclusions
The concept of PCR-RFLP is based on the presence or absence of a particular recognition site in the target sequence, which usually does not exceed eight nucleotides in length. 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 PCR-RFLP. Moreover, each technique has advantages that the other technique does not provide. The PCR-SSCP method has been characterized by its ability to detect unknown mutations, but more laboratory skills are required to optimize it before it can exhibit this feature. In contrast to PCR-SSCP, PCR-RFLP is easy to use and has highly specific characteristics, but it does not have the ability to detect unknown mutations in the amplified locus. Therefore, it can be stated that when the main purpose of a particular genotyping experiment is the detection of an unknown SNP, the best technique is PCR-SSCP. However, when a specified locus is targeted, and there is no need to determine the neighboring sequences, PCR-RFLP is the best technique. Despite the simplicity of PCR-RFLP, it does not have the ability to identify unknown mutations. In contrast, the complexity of PCR-SSCP is usually accompanied by the ability of this technique to identify unknown mutations. Therefore, there is an urgent need for a robust, efficient, and affordable technique that combines the simplicity of PCR-RFLP and the high sensitivity of PCR-SSCP.