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

Efficient saturation mutagenesis of a pentapeptide coding sequence using mixed oligonucleotides.

S A Goff1, S R Short-Russell, J F Dice

  • 1Department of Physiology, Tufts University School of Medicine, Boston, MA 02111.

DNA (Mary Ann Liebert, Inc.)
|August 1, 1987
PubMed
Summary
This summary is machine-generated.

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

A molecular chaperone complex at the lysosomal membrane is required for protein translocation.

Journal of cell science·2001
Same author

Sequence and analysis of the tomato JOINTLESS locus.

Plant physiology·2001
Same author

Protein translocation across membranes.

Biochimica et biophysica acta·2001
Same author

Regulation of gene expression by small molecules in rice.

Novartis Foundation symposium·2001
Same author

Regulation of lamp2a levels in the lysosomal membrane.

Traffic (Copenhagen, Denmark)·2001
Same author

Unique properties of lamp2a compared to other lamp2 isoforms.

Journal of cell science·2000

This study introduces a cost-effective method for site-directed mutagenesis, enabling the creation of diverse protein mutations. This technique efficiently generates multiple amino acid substitutions in a single step, simplifying genetic engineering.

Area of Science:

  • Molecular Biology
  • Genetic Engineering
  • Protein Biochemistry

Background:

  • Targeting cytosolic proteins to lysosomes is crucial for cellular processes, especially during serum withdrawal.
  • Existing methods for site-directed mutagenesis can be complex and costly, limiting the generation of diverse mutation sets.

Purpose of the Study:

  • To develop an economical and efficient method for site-directed mutagenesis.
  • To generate a diverse set of mutations within a pentapeptide sequence for lysosomal targeting.
  • To facilitate the study of protein localization and function under specific cellular conditions.

Main Methods:

  • Utilized site-directed mutagenesis with degenerate oligonucleotides at a specific codon.
  • Employed low-temperature annealing of mixed oligonucleotides to single-stranded phage DNA.

Related Experiment Videos

  • Implemented a genetic selection strategy to isolate DNA strands containing desired mutations.

    • Main Results:

    • Successfully constructed a set of substitution mutations in the target pentapeptide sequence.
    • Demonstrated that mixed oligonucleotides provide an economical way to generate a large number of mutations.
    • Showcased the ability to change a single codon to most other amino acids in one step.

    Conclusions:

    • The described technique offers an efficient and cost-effective approach for generating diverse substitution mutations.
    • This method eliminates the need for flanking restriction enzyme sites, increasing its applicability to various DNA fragments.
    • The approach is valuable for researchers needing to rapidly create and study mutant proteins, particularly for investigating protein localization.