Creating Insertions or Deletions Using Overlap Extension Polymerase Chain Reaction (PCR) Mutagenesis
Abstract
Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA sequence of interest. It requires relatively little preparation compared with other mutagenesis methods and does not require the use of restriction enzymes. Because of its versatility, the method has become widely used. Unlike methods of random mutagenesis, directed mutagenesis requires that the researcher already have a specific mutation in mind to implement. Traditional overlap extension PCR mutagenesis protocols remain limited in several critical ways, especially when it comes to generating insertions and deletions. For example, traditional protocols require that all sequence alterations be embedded within the primer itself, which makes it difficult to make insertions >30 nt. This protocol describes an overlap extension PCR mutagenesis method that is more versatile than its predecessors. Using this method, one can essentially make insertions and deletions of any size at any position within a given DNA sequence. To generate an insertion mutation, first prepare an insertion fragment and two flanking fragments by PCR. In the secondary PCR, the insertion fragment is recombined with two flanking fragments derived from the original template. This method can also be used to generate deletions, which is discussed in the latter part of the protocol.
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Out of the 19 RBM-specific mAbs that showed reduced or loss of neutralization of the TM mutant, 16 showed complete or partial loss of binding to the E484K RBD mutant, consistent with E484K being an important viral escape mutation.
“Out of the 19 RBM-specific mAbs that showed reduced or loss of neutralization of TM mutant (Fig. 4d), 16 showed a complete or partial loss of binding to E484K RBD mutant (Fig. 4g-h), consistent with findings that E484K is an important viral escape mutation26, 39, 27.”
From Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies (2021) · cites this paper as [27]
The Δ140 mutant subsequently acquired E484K, which resulted in a further 4-fold drop in neutralization titre, indicating that a two residue change across NTD and RBD represents an effective pathway of escape that can dramatically inhibit the polyclonal response.
“This Δ140 mutant subsequently acquired E484K which resulted in a further 4-fold drop in neutralization titre indicating a two residue change across NTD and RBD represents an effective pathway of escape that can dramatically inhibit the polyclonal response.”
From Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies (2021) · cites this paper as [27]
Andreano et al. describe the emergence of Δ140 in virus co-incubated with potently neutralising convalescent plasma, causing a 4-fold reduction in neutralization titre.
“Evidence for the importance role of NTD deletions in combination with E484K in immune escape is provided by Andreano et al.27 who describe the emergence of Δ140 in virus co-incubated with potently neutralising convalescent plasma, causing a 4-fold reduction in neutralization titre.”
From Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies (2021) · cites this paper as [27]
Cites
Versatile PCR-mediated insertion or deletion mutagenesis
Lee, J., Lee, H.-J., Shin, M.-K., et al. (2004) · BioTechniques · doi:10.2144/04363bm04 · Cited by 96 · see citing works
Insertion and Deletion Mutagenesis by Overlap Extension PCR
Lee, J., Shin, M.-K., Ryu, D.-K., Kim, S., & Ryu, W. (2010) · Methods in molecular biology · doi:10.1007/978-1-60761-652-8_10 · Cited by 77 · see citing works
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SARS-CoV-2 B.1.1.7 sensitivity to mRNA vaccine-elicited, convalescent and monoclonal antibodies
Collier, D. A., De Marco, A., Ferreira, I. A. T. M., Meng, B., Datir, R., Walls, A. C., Kemp S, S. A., Bassi, J., Pinto, D., Fregni, C. S., et al. (2021) · doi:10.1101/2021.01.19.21249840 · Cited by 729 · see citing works
Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies
Collier, D. A., De Marco, A., Ferreira, I. A. T. M., Meng, B., Datir, R. P., Walls, A. C., Kemp, S. A., Bassi, J., Pinto, D., Silacci-Fregni, C., et al. (2021) · Nature · doi:10.1038/s41586-021-03412-7 · Cited by 728 · see citing works
Recurrent emergence and transmission of a SARS-CoV-2 spike deletion H69/V70
Kemp, S. A., Meng, B., Ferriera, I. A., Datir, R., Harvey, W. T., Papa, G., Lytras, S., Collier, D. A., Mohamed, A., Gallo, G., et al. (2020) · doi:10.1101/2020.12.14.422555 · Cited by 252 · see citing works
Chang, C. Y.-Y., Tseng, C.-C., Lai, M.-T., Chiang, A.-J., Lo, L.-C., Chen, C.-M., Yen, M.-J., Sun, L., Yang, L., Hwang, T., et al. (2021) · PLOS ONE · doi:10.1371/journal.pone.0248168 · Cited by 20 · see citing works
- Authors
- Forloni, M., Liu, A. Y., & Wajapeyee, N.
- Venue
- Cold Spring Harbor Protocols
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2018-08
- Volume
- 2018
- Issue
- 8
- Pages
- pdb.prot097758
- ISSN
- 1559-6095 (ISSN-L)
- 1940-3402
- OpenAlex
- W2887895672
- Cited by
- 20
- References
- 2
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 3 · 2023: 1 · 2022: 4 · 2021: 3 · 2020: 2 · 2019: 5
- Type
- posted content
- Language
- en
- Open access
- closed
- Locations
- Cold Spring Harbor Protocols (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- insertion mutation, deletion mutations, site-directed mutagenesis
- MeSH terms
- Polymerase Chain Reaction, Mutagenesis, Insertional, Sequence Deletion, DNA Primers
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