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A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme

Paper ID Volume ID Publish Year Pages File Format Full-Text
24833 43540 2008 7 PDF Available
Title
A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme
Abstract

Type IIS restriction enzymes have been successfully used as “universal” restriction enzymes in DNA manipulations. We took a step further to develop a rapid technique for recombining DNA fragments, fully automatic single-tube recombination (FASTR), which enables multiple-fragment DNA recombination in a single step. Crude PCR products are directly mixed with both type IIS restriction endonuclease and DNA ligase to initiate a spontaneous and one-way recombination reaction. Highly efficient DNA recombination can be achieved by an inhibition of DNA polymerase with aphidicolin and a selective digestion of template DNAs by DpnI, a restriction enzyme to digest hemi-methylated DNA in the reaction solution; thereby the entire procedure takes less than 15 min. Owing to its simplicity, efficiency and rapidity, one-step FASTR can be applied to a wide range of DNA manipulations including those involving high-throughput applications where significant reduction in time and cost is expected.

Keywords
DNA manipulations; DNA recombination; DNA cloning; Type IIS restriction endonuclease; High-throughput
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A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 137, Issues 1–4, 10 October 2008, Pages 1–7
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us