Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Single-stranded gaps as localized targets for in vitro mutagenesis.

D Shortle, D Botstein

    Basic Life Sciences
    |January 1, 1982
    PubMed
    Summary

    Researchers developed a novel "gap misrepair" method for targeted DNA mutagenesis. This technique precisely alters nucleotide sequences at specific sites in circular DNA molecules, advancing genetic engineering tools.

    Related Concept Videos

    You might also read

    Related Articles

    Articles linked to this work by shared authors, journal, and citation graph.

    Sort by
    Same author

    Editorial.

    Molecular biology of the cell·2006
    Same author

    Genomic perspective and cancer.

    Cold Spring Harbor symposia on quantitative biology·2004
    Same author

    The Gene Ontology (GO) database and informatics resource.

    Nucleic acids research·2003
    Same author

    Arrest, adaptation, and recovery following a chromosome double-strand break in Saccharomyces cerevisiae.

    Cold Spring Harbor symposia on quantitative biology·2003
    Same author

    Genome-wide scan of bipolar disorder in 65 pedigrees: supportive evidence for linkage at 8q24, 18q22, 4q32, 2p12, and 13q12.

    Molecular psychiatry·2003
    Same author

    The glycine allele of a glycine/arginine polymorphism in the beta2-adrenergic receptor gene is associated with essential hypertension in a population of Chinese origin.

    American journal of hypertension·2002

    Area of Science:

    • Molecular Biology
    • Genetic Engineering
    • Biotechnology

    Background:

    • Enzymatic generation of single-stranded gaps in circular DNA creates targets for in vitro mutagenesis.
    • Previous methods like sodium bisulfite deamination induced mutations at specific DNA sites (e.g., BglI, beta-lactamase gene).
    • Limitations existed in precisely controlling mutations at predetermined gap sites.

    Purpose of the Study:

    • To develop a new method for inducing mutations at specific gap sites in circular DNA.
    • To investigate the efficacy of this novel method for targeted DNA sequence alteration.
    • To explore the types of nucleotide substitutions achievable using the new technique.

    Main Methods:

    • Development of the "gap misrepair" technique for targeted mutagenesis.
    • Application of gap misrepair to induce mutations at HindIII and C1aI restriction sites on pBR322 DNA.
    • Utilized DNA polymerase I from Micrococcus luteus, T4 DNA ligase, and three deoxynucleoside triphosphates in the reaction.

    Main Results:

    • The gap misrepair method successfully induced mutations at the targeted HindIII and C1aI restriction sites.
    • This method resulted in specific nucleotide sequence alterations within or near the generated gaps.
    • Gap misrepair reactions yielded all three possible substitutions for adenine and cytosine residues.

    Conclusions:

    • The "gap misrepair" method is an effective strategy for targeted in vitro DNA mutagenesis.
    • This technique allows for precise control over nucleotide sequence changes at predetermined sites.
    • Gap misrepair expands the toolkit for genetic engineering and molecular biology research.

    Related Experiment Videos