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

Abstract:

: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.

journal_name

Genome Res

journal_title

Genome research

authors

Heyman JA,Cornthwaite J,Foncerrada L,Gilmore JR,Gontang E,Hartman KJ,Hernandez CL,Hood R,Hull HM,Lee WY,Marcil R,Marsh EJ,Mudd KM,Patino MJ,Purcell TJ,Rowland JJ,Sindici ML,Hoeffler JP

subject

Has Abstract

pub_date

1999-04-01 00:00:00

pages

383-92

issue

4

eissn

1088-9051

issn

1549-5469

journal_volume

9

pub_type

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