Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis

Jan Norrander; Tomas Kempe; Joachim Messing

doi:10.1016/0378-1119(83)90040-9

Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis

Jan Norrander, Tomas Kempe, Joachim Messing

Genetics, Cell Biology and Development (TMED)

Research output: Contribution to journal › Article › peer-review

1651 Scopus citations

Abstract

The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M 13mp10 and M 13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.

Original language	English (US)
Pages (from-to)	101-106
Number of pages	6
Journal	Gene
Volume	26
Issue number	1
DOIs	https://doi.org/10.1016/0378-1119(83)90040-9
State	Published - Dec 1983

Keywords

DNA polymerase I Klenow fragment
M13 cloning
gene synthesis
phosphoramidites
synthetic DNA primers

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author = "Jan Norrander and Tomas Kempe and Joachim Messing",

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AU - Norrander, Jan

AU - Kempe, Tomas

AU - Messing, Joachim

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N2 - The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M 13mp10 and M 13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.

AB - The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M 13mp10 and M 13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.

KW - DNA polymerase I Klenow fragment

KW - M13 cloning

KW - gene synthesis

KW - phosphoramidites

KW - synthetic DNA primers

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Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis

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