Jan 22, 2024

Public workspaceSARS-CoV-2 Receptor Binding Domain Deoxy Fragment Sequencing 

DOI
dx.doi.org/10.17504/protocols.io.261gedbp7v47/v1
  • 1University of Baghdad
  • Noor Saber Jawad: Currently Mustansiriyah University
Noor Saber Jawad
Open access
DOI: dx.doi.org/10.17504/protocols.io.261gedbp7v47/v1
Protocol Citation: Noor Saber Jawad, Nuha Joseph Kandala 2024. SARS-CoV-2 Receptor Binding Domain Deoxy Fragment Sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.261gedbp7v47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 22, 2024
Last Modified: January 22, 2024
Protocol Integer ID: 93906
Keywords: SARS-CoV-2, Sanger Sequencing, RBD
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.
RNA Extraction
RNA Extraction
The RNA of SARS-CoV-2 extracted using Automated Extraction System ExiPrep‱ 96 Lite from Bioneer (South Korea) using the Exiprep 96 Viral DNA/RNA kit using the standard recommended procedure by the manufacturer.
cDNA synthesis
cDNA synthesis
The extracted RNA used as a template to construct complementary DNA, cDNA synthesis considered using the GoScript‱ 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
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
Amplicon Sequencing referred to Macrogen, South Korea to be sequenced using the ABI3730XL sequencing Machine.