SARS-CoV-2 Receptor Binding Domain Deoxy Fragment Sequencing

Abstract

Since first reporting of SARS-CoV-2 case in China, there were urgent need to report and monitor emerging variants and keep track circulating mutation, in this methodology, we used simple, cost-effective method to monitor the circulating mutations which could be used to predict the SARS-CoV-2 variants based on accumulating mutations in the receptor binding domain.

Steps

RNA Extraction

The RNA of SARS-CoV-2 extracted using Automated Extraction System ExiPrep^TM 96 Lite from Bioneer (South Korea) using the Exiprep 96 Viral DNA/RNA kit using the standard recommended procedure by the manufacturer.

cDNA synthesis

The extracted RNA used as a template to construct complementary DNA, cDNA synthesis considered using the GoScript^TM Reverse Transcription Mix, Random Primers, Promega (USA) using manufacturer recommended procedure. this will ensure synthesis of the first DNA strand only.

Concentration of synthesized DNA measured by Quantus Fluorometer (Promega, USA).

Amplicon Amplifications

RBD specific primers, amplification regions, and other details listed below:

This set of forward and reverse primers is designed as part of NGS primer pool, the current procedure chooses one set to generate a different length that it designed for that is sufficient to detect all basic mutations in the receptor binding domain that uses frequently for variants discriminations.

Reaction setups include:

1- GoTag Green Master Mix (Promega, USA)

2- Ladder gel Marker (100-1500), (Promega, USA)

the following program used for amplification:

Amplicon Sequencing

Amplicon Sequencing referred to Macrogen, South Korea to be sequenced using the ABI3730XL sequencing Machine.