Zahra Gholamrezaei,1,*Saeid Latifi-Navid,2Ezzat Nourizadeh,3Esmat Abdi,4
1. Department of Biology, Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran 2. Department of Biology, Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran 3. Department of Biology, Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran 4. Department of Biology, Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran
Introduction: The most frequent source of genetic diversity across individuals is single-nucleotide polymorphisms (SNPs). The most frequent source of genetic diversity across individuals is single-nucleotide polymorphisms (SNPs). SNPs can currently be found using a variety of techniques. DNA sequencing is a common method for detecting SNPs, but it requires a laboratory environment for sample processing as well as bulky, expensive, and slow DNA sequencing equipment. Here, we describe some easier and faster methods for identifying SNPs. It is also recommended to select a cost-effective technique. ARMS or As-PCR: The Allele-Specific PCR technique was created for allele analysis of clinically important alterations. The introduction of intentional mismatches inside the three bases at the 3' end of the primers may help with accurate discriminating between two alleles. Two complementary reactions make up a typical ARMS test that can identify a known SNP polymorphism: one contains an ARMS primer specific for the wild DNA sequence and cannot amplify the mutated DNA at a particular locus, and the other contains a specific mutated primer and does not amplify wild DNA. As a result, this strategy is one of several used to identify SNPs. HRM-PCR: A quick and easy method for genotyping, mutation scanning, and sequence matching is high-resolution melting (HRM) of DNA. This technique, which helps to overcome conventional approaches and prepares for high performance, is based on measuring the fluorescence change connected to the melting temperature of double-stranded DNA in the presence of a dye mixed with saturated DNA, known as intercalating dyes, in real-time PCR reactions. Due to the specificity and sensitivity of the DNA melting curve profiles created by HRM analysis, different nucleic acid species can be identified based on minute sequence variations. PCR-RFLP: This technique involves treating a PCR amplicon with a particular restriction enzyme that causes the DNA to be cut at a distinct restriction site known as the recognition site, resulting in numerous DNA fragments of various sizes. The digested amplicons are then placed on a gel and exposed to an electric field. Throughout the gel, bands of varying sizes move at varying intervals. The two main drawbacks of PCR-RFLP are the requirement for specialized RE and the challenge of accurately detecting changes when several SNPs are targeted at once. LAMP: loop-mediated isothermal amplification, is a quick and reliable technique for nucleic acid sequence-specific detection. The LAMP approach employs four sets of primers that are specifically designed to isolate six different areas of the target gene. The SNP-LAMP mismatch is caused by the SNP being at the 3' end of the LAMP primer, which results in the mismatch and hinders polymerase extension in the presence of a non-SNP sequence. This is how the LAMP method was established for SNP detection.
Methods: Articles required for the study were found using Google Scholar, PubMed, and MDPI searches.
Results: In this study, fast detection methods were investigated for identifying SNPs. ARMS-PCR, HRM-PCR, PCR-RFLP, and LAMP techniques were among those obtained in this study. According to research, ARMS-PCR is the most common and easiest approach for detecting SNPs. Specific primers are easily designed in this approach, after which PCR is done and the products are electrophoresed. In this technique, handling these processes and analyzing the results is easier and faster than in HRM-PCR, PCR-RFLP, and LAMP.
Conclusion: The analysis of the papers reveals that As-PCR is the most common, straightforward method for detecting SNPs. In many studies, this method has been developed to identify SNPs. Additionally, the comparison of various approaches demonstrates that, unlike other methods, it is more challenging to analyze HRM curves. Although AS-PCR involves more handling steps than HRM-PCR, it was the most time- and cost-efficient approach tested since it allows for straightforward and quick interpretation of the results and can be carried out using common laboratory equipment. PCR-RFLP techniques require more handling steps, have longer pause times, and cost more in materials. The LAMP method requires probes, which raises cost.
Keywords: SNP detection, As-PCR, ARMS-PCR, fast detection, simple method