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A single nucleotide polymorphism or SNP is a single nucleotide variation at a specific genomic position in a large population. It is the most prevalent type of sequence variation found in the human genome. Point mutations that occur in more than 1% of the population qualify as SNPs. These are present once every 1000 nucleotides on an average in the human genome. Replacement of a purine with another purine (A/G) or a pyrimidine with another pyrimidine (C/T) is known as a transition. In contrast,...

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A Routine Sanger Sequencing Target Specific Mutation Assay for SARS-CoV-2 Variants of Concern and Interest.

¹Affiliation Milford Molecular Diagnostics Laboratory, 2044 Bridgeport Avenue, Milford, CT 06460, USA.

|December 28, 2021

Summary

This summary is machine-generated.

A new protocol accurately sequences SARS-CoV-2 variants by targeting the ACE2 receptor binding domain (RBD) and N-terminal domain (NTD) of the spike protein. This method aids in precise mutation detection for vaccines, therapeutics, and diagnostics.

Keywords:

ACE2 RBD Delta variant N-terminal domain (NTD)SARS-CoV-2 Sanger sequencing amino acid mutations nested RT-PCR spike protein variants of concern variants of interest

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Area of Science:

Virology
Molecular Biology
Genetics

Background:

SARS-CoV-2 genetic mutations, including amino acid changes and deletions, impact viral countermeasures like vaccines and therapeutics.
Accurate identification of SARS-CoV-2 variants is crucial for understanding viral behavior and developing effective interventions.
Current whole genome sequencing methods for variant detection are limited in scope and precision.

Purpose of the Study:

To introduce a generic protocol for routine sequencing of specific SARS-CoV-2 genetic regions.
To enable accurate detection of amino acid mutations in the ACE2 receptor binding domain (RBD) and N-terminal domain (NTD) of the spike protein.
To facilitate precise identification of all SARS-CoV-2 variants of concern and interest.

Main Methods:

Development of a generic protocol for nested RT-PCR amplification of a 437-bp amplicon from the ACE2 RBD.
Development of a generic protocol for nested RT-PCR amplification of a 490-bp amplicon from the S gene's NTD.
Application of Sanger sequencing on amplified cDNA targets from clinical specimens.

Main Results:

The protocol successfully amplified both target nucleic acid regions (ACE2 RBD and S gene NTD) into cDNA amplicons.
Sanger sequencing was performed on the cDNA amplicons from all 16 SARS-CoV-2 positive clinical specimens.
The method demonstrated capability for precise determination of mutations essential for variant classification.

Conclusions:

The presented protocol offers a reliable method for routine sequencing of key SARS-CoV-2 genetic regions.
This approach supports accurate variant identification, aiding in the monitoring of SARS-CoV-2 evolution.
The protocol is valuable for assessing the impact of mutations on viral countermeasures and public health strategies.