A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase.
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 degrees C, that the filling-in efficiency was highest upon using a buffer containing 3 mM MgSO4 and 300 microM dNTP.
Yang
Shaohui
S
The Key Laboratory of Bioactive Materials, Ministry of Education/Institute for Molecular Biology, Nankai University, Tianjin 300071, People's Republic of China.
Li
Xin
X
Ding
Dongfeng
D
Hou
Jianhua
J
Jin
Zhaoxia
Z
Yu
Xinchun
X
Bo
Tao
T
Li
Weidong
W
Li
Minggang
M
D002021:Buffers; D004247:DNA; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008278:Magnesium Sulfate / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D002021:Buffers; D009711:Nucleotides; D008278:Magnesium Sulfate; D004247:DNA; C072146:Pfu DNA polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase