• Simple and Fast One-step PCR Method for Detection of a HOTAIR SNP
  • Zahra Gholamrezaei,1,* Saeid Latifi-Navid,2 Ezzat Nourizadeh,3 Esmat 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
    4. Department of Biology, Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran


  • Introduction: HOTAIR, or HOX transcript antisense RNA, is an oncogenic long non-coding RNA (lncRNA), and multiple studies have demonstrated that HOTAIR is increased in a wide range of human malignancies. Single nucleotide polymorphisms (SNPs) are well known for directly regulating lncRNA expression and altering their activities. As a result, detecting single nucleotide polymorphisms (SNPs) is critical for understanding human diseases, identifying pathogenic variations, and implementing genetic modification programs. Here, we examine an easy and fast method to detect an SNP (rs17720428) related to the HOTAIR gene. Rs17720428 is one of the SNPs that increases the risk of gastric cancer.
  • Methods: Initially, 50 human DNA pools previously genotyped by Infinium HTS platform SNP array (Illumina Infinium GSA BeadChip—a robust, high quality assay) were examined using this approach. The primers were designed following Chen et al's strategy. The strategy makes use of the differential efficiency of genomic PCR using a primer that has a single mismatch with the chromosome that contains the SNP to be identified (usually the variant allele) against two mismatches with the corresponding alternative allele (often the wild type allele). The primer TM was set at 59°C. The cycling protocol was the same for all PCRs. Initial denaturation of genomic DNA took 5 minutes at 95 °C. This was followed by 35 repetitions of the steps: denature at 95 °C for 40 seconds, annealing at 59 °C for 40 seconds, and extension at 72 °C for 40 seconds. The last extension took 7 minutes at 72 °C. Before loading the PCR samples onto an agarose gel, no extra preparation was necessary.
  • Results: In this study, two PCR reactions were performed on each DNA sample, where one PCR reaction uses a primer with mismatches to detect the variant but not the wild type allele and the second PCR reaction employs a primer with mismatches to detect the wild type allele but not the variant. The forward primer has two crucial characteristics. First, the primer's 3' nucleotide is at the SNP and pairs with the SNP residue to be identified. However, it is mismatched with the other allele residues. As a result, the nucleotides at the 3′ end of the variant and wild type primers differ. Second, two bases upstream of the SNP location, an additional alteration that is a mismatch with both the variant and wild type alleles was inserted to further differentiate the two alleles in PCR. In both cases, the reverse primer is the same. The variant primer has one mismatch relative to the variant allele, whereas the wild type primer contains two mismatches. As a result, the variant primer amplifies the variant allele preferentially. Likewise, the wild type primer identifies only the wild type allele. We successfully identified the wild type/mutant allele for lncRNA HOTAIR rs17720428 among 50 human DNA samples. The results were completely consistent with those from the SNP array.
  • Conclusion: In summary, this primer design method was evaluated on 50 human DNA samples. Result accuracy was 100%, as demonstrated by the SNP array. It thus indicates that this primer design strategy can be applied to known SNPs. It also saves time/effort and costs on reagents and makes SNP detection and genotyping by PCR substantially easier.
  • Keywords: SNP detection, PCR, one-step PCR, fast detection, Primer design