An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites—a case study with Omicron Spike DNA

  1. Rita Rani
  2. Kishore V L Parsa
  3. Kiranam Chatti
  4. Aarti Sevilimedu

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Site-directed mutagenesis is an invaluable technique that enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations, is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost-effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis. We demonstrate this method using the Omicron Spike DNA construct as an example, and create a construct bearing 37 mutations (as compared to wild-type Spike DNA), as well as 4 other constructs bearing subsets of the full spectrum of mutations. We believe that this method can be an excellent alternative to gene synthesis, especially when three or more variants are required.

Article activity feed

  1. Version published to 10.1093/biomethods/bpac037
  2. ScreenIT

    SciScore for 10.1101/2022.02.10.480017: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    Additional fragments to complete the assembly of each clone were also amplified using the pCMV-Spike (wild-type) template (labeled fragments 17-23).
    pCMV-Spike
    suggested: None
    Construct assembly Part 1- Gibson assembly of Clones 1,2,3 and 4: pCDNA3.1 was digested with BamHI and HindIII, treated with Antarctic Phosphatase and purified using a DNA cleanup column, and checked for background by transformation before using in the assembly reaction.
    pCDNA3.1
    suggested: RRID:Addgene_79663)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Despite these caveats, we believe that this strategy will be of great use in resource-constrained settings.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2022.02.10.480017v1 on bioRxiv