Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation

The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Va...

Full description

Saved in:
Bibliographic Details
Published in:Genome research 1999-04, Vol.9 (4), p.383-392
Main Authors: Heyman, J A, Cornthwaite, J, Foncerrada, L, Gilmore, J R, Gontang, E, Hartman, K J, Hernandez, C L, Hood, R, Hull, H M, Lee, W Y, Marcil, R, Marsh, E J, Mudd, K M, Patino, M J, Purcell, T J, Rowland, J J, Sindici, M L, Hoeffler, J P
Format: Article
Language:English
Subjects:
Animals
CHO Cells - physiology
Cloning, Molecular - methods
Cricetinae
DNA Topoisomerases, Type I - chemistry
DNA Topoisomerases, Type I - genetics
DNA Topoisomerases, Type I - metabolism
Gene Amplification
Gene Expression Regulation
Genetic Vectors
Genome
Humans
Mammals
Methods
Open Reading Frames
Plasmids - genetics
Polymerase Chain Reaction - methods
Protein Kinases - genetics
Saccharomyces cerevisiae - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.
ISSN:1088-9051
1549-5469
DOI:10.1101/gr.9.4.383