High Resolution Melt (HRM) Based Quantitative Real-Time Polymerase Chain Reaction (qPCR) Primer Design Tool for Point Mutation Detection

Abstract

Single nucleotide polymorphisms (SNPs) are point mutations in the genetic material of the population which result different characteristics and disabilities in the population. Determining the abundance of these mutations may pave way for better clinical diagnostics and towards personalized medicine. Amplification Refractory mutation system – Polymerase Chain reaction (ARMS-PCR) and qPCR based Competitive amplification of differently melting amplicons (CADMA) are the most cost efficient techniques that can be used to detect these mutations. CADMA is a technique which incorporate additional mutations to increase Melting temperature (Tm) gap between different allele specific products. Tools were developed to design these different assays which facilitate the molecular biology researchers and healthcare professionals in Molecular diagnostics sector to design their HRM, ARMS-PCR and Allele specific PCR assays with just 2 clicks. These systems were developed using the PHP server site script which hides the inside functionality from the user to keep the methodology confidential. The NCBI e-utils were used to retrieve the DNA sequence and other information from the dbSNP server and soton.ac.uk server was used to address few primer design challenges. The combination of codes were used to design this effective tool which give wide range to information to the user and enhance the usability. WordPress framework and themes were used to identify the real users and their identities in order to get in touch with them to get feedbacks. Test cases were identified and manual grey-box testing and automated integrated system testing using Katalon studio were performed to ensure the smooth functioning of the tool system. Overall the tool works fine and makes the user easy and convenient in designing their own DNA tests for Single Nucleotide polymorphisms (SNPs) by just inserting the dbSNP ID of their interest into the tool.