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Properties of an NAD+-dependent DNA ligase from the hyperthermophile Thermotoga maritima and its application in PCR amplification of long DNA fragments

Paper ID Volume ID Publish Year Pages File Format Full-Text
17717 42691 2010 5 PDF Available
Title
Properties of an NAD+-dependent DNA ligase from the hyperthermophile Thermotoga maritima and its application in PCR amplification of long DNA fragments
Abstract

The thermostable DNA ligase of Thermotoga maritima (Tma DNA ligase) was expressed in Escherichia coli, and purified by heat treatment followed by metal affinity chromatography. Purified Tma DNA ligase exhibited activity on DNA fragments with cohesive termini, and no activity was detected on blunt-end DNA. The ligase reaction required NAD+, and a divalent cation including Mg2+, Mn2+or Ca2+. The highest activity of Tma DNA ligase occurred at 60 °C and pH 8.0 when Hind III digested plasmid was used as substrate. The purified enzyme had a half-life of over 30 min at 95 °C, and retained over 80% of its activity after holding a pH ranging from 7.2 to 8.8 for 1 h at 80 °C. When the enzyme was employed in PCR cycles, Tma DNA ligase promoted the amplification of long DNA fragments from the genomic DNA of T. maritima.

Keywords
Amplification; Long DNA fragments; Thermostable DNA ligase; Thermotoga maritima
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Properties of an NAD+-dependent DNA ligase from the hyperthermophile Thermotoga maritima and its application in PCR amplification of long DNA fragments
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 46, Issue 2, 5 February 2010, Pages 113–117
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
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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