title: A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase.
abstract: 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.
authors.affiliation: The Key Laboratory of Bioactive Materials, Ministry of Education/Institute for Molecular Biology, Nankai University, Tianjin 300071, People's Republic of China.
authors.forename: Shaohui
authors.initials: S
authors.lastname: Yang
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Dongfeng
authors.initials: D
authors.lastname: Ding
authors.forename: Jianhua
authors.initials: J
authors.lastname: Hou
authors.forename: Zhaoxia
authors.initials: Z
authors.lastname: Jin
authors.forename: Xinchun
authors.initials: X
authors.lastname: Yu
authors.forename: Tao
authors.initials: T
authors.lastname: Bo
authors.forename: Weidong
authors.initials: W
authors.lastname: Li
authors.forename: Minggang
authors.initials: M
authors.lastname: Li
mesh_terms: 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
chemical_list: D002021:Buffers; D009711:Nucleotides; D008278:Magnesium Sulfate; D004247:DNA; C072146:Pfu DNA polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase