Combinatorial recombination of gene fragments to construct a library of chimeras

Current Protocols in Protein Science
Michelle M MeyerF H Arnold

Abstract

Recombination of distantly related and nonrelated genes is difficult using traditional PCR-based techniques, and truncation-based methods result in a large proportion of nonviable sequences due to frame shifts, deletions, and insertions. This unit describes a method for creating libraries of chimeras through combinatorial assembly of gene fragments. It allows the experimenter to recombine genes of any identity and to select the sites where recombination takes place. Combinatorial recombination is achieved by generating gene fragments with specific overhangs, or sticky ends. The overhangs permit the fragments to be ligated in the correct order while allowing independent assortment of blocks with identical overhangs. Genes of any identity can be recombined so long as they share 3 to 5 base pairs of identity at the desired recombination sites. Simple adaptations of the method allow incorporation of specific gene fragments.

References

Jun 25, 1996·Proceedings of the National Academy of Sciences of the United States of America·K Back, J Chappell
Dec 1, 1999·Bioorganic & Medicinal Chemistry·M OstermeierS J Benkovic
Dec 10, 1999·Nature Biotechnology·M OstermeierS J Benkovic
May 1, 2001·Nature Biotechnology·V SieberF H Arnold
Sep 20, 2001·Proceedings of the National Academy of Sciences of the United States of America·S LutzS J Benkovic
Feb 28, 2002·Journal of Molecular Biology·John M JoernFrances H Arnold
Jun 26, 2003·Journal of Molecular Biology·Kaori Hiraga, Frances H Arnold
Jun 26, 2003·Methods in Molecular Biology·Peter MeinholdJonathan J Silberg
Jul 24, 2003·Protein Science : a Publication of the Protein Society·Michelle M MeyerFrances H Arnold
Feb 26, 2004·Proceedings of the National Academy of Sciences of the United States of America·Manish C SarafCostas D Maranas
Aug 28, 2004·Protein Engineering, Design & Selection : PEDS·Jeffrey B EndelmanFrances H Arnold
Apr 6, 2005·Proceedings of the National Academy of Sciences of the United States of America·D Allan DrummondFrances H Arnold
Jul 8, 2005·Proteins·Manish C SarafCostas D Maranas
Jul 16, 2005·Proteins·Griselda Hernández, David M LeMaster

❮ Previous
Next ❯

Citations

Dec 23, 2016·Nucleic Acids Research·Lauren B A WoodruffChristopher A Voigt
Jan 23, 2015·Protein Science : a Publication of the Protein Society·Deeptak VermaChris Bailey-Kellogg

❮ 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.