Abstract
Background and Objective: High resolution melting (HRM) analysis is a technique to measure decrease of fluoresce, caused by discharge of the dye, throughout DNA thermal melting gradient process. STK11 gene encodes one of cellular serine-threonine kinase proteins that regulates cellular polarity. It also acts as tumor suppressor protein. Mutations of germline in the gene is coincident with Peutz-Jeghers syndrome and potential of developing a variety of neoplasia. Methodology: In an experimental study, genome DNA of 56 patients with digestive system cancer was extracted. Afterward, nucleotide changes over the gene were examined using Real-time PCR and High resolution melting (HRM). Findings: Nucleotide screening using HRM technique revealed two types of SNP in introns 6 & 7 in 10 patients. Four patients showed homozygous C/T nucleotide changes [cluster id/dsSNP/rs9282860] in intron 6 and six patients showed heterozygous C/G nucleotide changes [cluster id/dsSNP/rs2075607] in intron 7. Comparison of HRM results with sequencing results indicated 100% conformity. Conclusion: Although no mutation was observed in exon section of the gene, primary screening of STK11 gene to diagnose unknown nucleotide changes of germline and somatic in patients with neoplasia using HRM was feasible, easy, and cost effective.
Highlights
Following PCR proliferation, high resolution melting (HRM) is a new and homogenous method, which is performed in a closed tube
The process is plotted in a melt curve with high resolution that is highly variable for sequences of DNA so that it is possible to distinguish amplicons that are different even only in one unbound pair
High resolution melting (HRM) analysis can be used for detecting small genetic changes such as insertion, deletion, and substitution of single nucleotide polymorphism (SNP) [1]
Summary
Following PCR proliferation, high resolution melting (HRM) is a new and homogenous method, which is performed in a closed tube. This method is feasible in PCR products for analyzing genetic changes (SNPs, mutation, and methylation). HRM differentiates nucleic acid samples based on sequence, length, and GC volume It is featured with proliferation of the gene in 80-250bp pieces in a reaction in which florescent dyes are bound to two-stranded DNA. STK11 gene demonstrates serine/threonine kinase activity in 19p13.3 chromosome It expanses in 23kbase region with 9 encoder exons and one non-encoding exon. Activation of AMPK-like kinases by STK11 plays a key role in preserving cellular polarity, which results in suppression of tumor cells. Heating plan included primary denaturation (5min, 95oC) followed by 40 thermal cycles (25s-95oC and 20s-50oC) along with adsorbing the dye at green channel (30s-72oC) and HRM (75oC – 93oC) with temperature shift of 0.1oC/s
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