Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates

Analytical Biochemistry
S N SlilatyS Lebel

Abstract

An approach for generating structures capable of directing full-length complementary-strand synthesis for double-stranded plasmid DNA is described. The structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing" oligonucleotide. Consisting of a sequence complementary to the free ends of one of the two plasmid strands, the closing oligonucleotide functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently closed heteroduplex molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.

References

May 5, 1976·Journal of Molecular Biology·L A Sherman, M L Gefter
Sep 1, 1978·Proceedings of the National Academy of Sciences of the United States of America·A RazinA D Riggs
Jan 1, 1979·Cold Spring Harbor Symposia on Quantitative Biology·J G Sutcliffe
Jun 25, 1985·Nucleic Acids Research·P CarterG Winter
Sep 20, 1985·Science·D Botstein, D Shortle
Jan 1, 1985·Annual Review of Genetics·M Smith
Oct 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·W Mandecki
Jul 1, 1986·The Biochemical Journal·P Carter
Jan 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·T A Kunkel
Jan 1, 1985·Annual Review of Biochemistry·G K Ackers, F R Smith
Dec 28, 1983·Biochemical and Biophysical Research Communications·S J GargerL K Grill
Jul 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·D DiMaioT Maniatis
Jun 5, 1983·Journal of Molecular Biology·D Hanahan
Sep 19, 1980·Science·R B WallaceJ Abelson

❮ Previous
Next ❯

Citations

Aug 15, 2002·The Journal of Biological Chemistry·Marianne L JensenPhilip K Ahring

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.