Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro.
The gapped duplex DNA approach to oligonucleotide-directed construction of mutations (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) has been developed further. A procedure is described that makes in vitro DNA polymerase/DNA ligase reactions dispensable. Direct transfection of host bacteria with gdDNA molecules of recombinant phage M13 plus mutagenic oligonucleotide results in marker yields in excess of 50% (gap size 1640 nucleotides). An important feature incorporated into the mutagenic oligonucleotide is the presence of one or two internucleotidic phosphorothioate linkages immediately adjacent to the 5'-terminus. Automated preparation and biochemical properties of such compounds are described as well as their performance in oligonucleotide-directed mutagenesis. A systematic study of the following parameters influencing marker yield is reported: Gap size, length of oligonucleotide, chemical nature of oligonucleotide termini and heatshock temperature during transformation.
Fritz
H J
HJ
Max-Planck-Institut für Biochemie, Abteilung Zellbiologie, Martinsried bei München, FRG.
Hohlmaier
J
J
Kramer
W
W
Ohmayer
A
A
Wippler
J
J
D001483:Base Sequence; D004252:DNA Mutational Analysis; D005822:Genetic Vectors; D006358:Hot Temperature; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D009946:Organothiophosphorus Compounds; D013329:Structure-Activity Relationship; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D009946:Organothiophosphorus Compounds