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A method for filling in the cohesive ends of double‐stranded DNA using Pfu DNA polymerase
Author(s) -
Yang Shaohui,
Li Xin,
Ding Dongfeng,
Hou Jianhua,
Jin Zhaoxia,
Yu Xinchun,
Bo Tao,
Li Weidong,
Li Minggang
Publication year - 2005
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20040195
Subject(s) - klenow fragment , dna polymerase i , dna , transformation (genetics) , dna polymerase , polymerase , transformation efficiency , microbiology and biotechnology , sticky and blunt ends , ligation , biology , polymerase chain reaction , chemistry , genetics , dna ligase , exonuclease , reverse transcriptase , gene , agrobacterium
The present paper reports a highly efficient method of making blunt ends from cohesive ends of double‐stranded DNA. Klenow fragment and Pfu DNA polymerases were used to fill in the cohesive ends. Since the transformation efficiency can directly reflect the filling‐in efficiency, similar ligation and transformation conditions were used, and the filling‐in efficiency was compared with the corresponding transformation efficiency. The results indicate that the filling‐in efficiency of Pfu DNA polymerase was 1.96 times that of Klenow fragment and its efficiency was markedly higher than that of Klenow fragment ( P <0.01). The optimization experiments on reaction conditions indicate, when the pH is 8.5 and the temperature is 74 °C, that the filling‐in efficiency was highest upon using a buffer containing 3 mM MgSO 4 and 300 μM dNTP.