Oligonucleotide-directed Construction of Mutations: a Gapped Duplex DNA Procedure Without Enzymatic Reactions in Vitro

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1988 Jul 25

PMID 3405755

Citations 5

Authors

H J Fritz

J Hohlmaier

W Kramer

A Ohmayer

J Wippler

Affiliations

Soon will be listed here.

Abstract

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.

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References

Nossal N . DNA synthesis on a double-stranded DNA template by the T4 bacteriophage DNA polymerase and the T4 gene 32 DNA unwinding protein. J Biol Chem. 1974; 249(17):5668-76. View

Smith M . In vitro mutagenesis. Annu Rev Genet. 1985; 19:423-62. DOI: 10.1146/annurev.ge.19.120185.002231. View

Fritz H, Belagaje R, Brown E, Fritz R, Jones R, Lees R . High-pressure liquid chromatography in polynucleotide synthesis. Biochemistry. 1978; 17(7):1257-67. DOI: 10.1021/bi00600a020. View

Burgers P, Eckstein F, Hunneman D . Stereochemistry of hydrolysis by snake venom phosphodiesterase. J Biol Chem. 1979; 254(16):7476-8. View

Bryant F, Benkovic S . Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom. Biochemistry. 1979; 18(13):2825-8. DOI: 10.1021/bi00580a022. View

Burgers P, Sathyanarayana B, Saenger W, Eckstein F . Crystal and molecular structure of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester (Sp diastereomer). Substrate stereospecificity of snake venom phosphodiesterase. Eur J Biochem. 1979; 100(2):585-91. DOI: 10.1111/j.1432-1033.1979.tb04205.x. View

Messing J, Crea R, Seeburg P . A system for shotgun DNA sequencing. Nucleic Acids Res. 1981; 9(2):309-21. PMC: 326694. DOI: 10.1093/nar/9.2.309. View

Baas P, Teertstra W, van Mansfeld A, Jansz H, van der Marel G, Veeneman G . Construction of viable and lethal mutations in the origin of bacteriophage 'phi' X174 using synthetic oligodeoxyribonucleotides. J Mol Biol. 1981; 152(4):615-39. DOI: 10.1016/0022-2836(81)90120-0. View

Bonura T, Schultz R, Friedberg E . An enzyme activity from Escherichia coli that attacks single-stranded deoxyribopolymers and single-stranded deoxyribonucleic acid containing apyrimidinic sites. Biochemistry. 1982; 21(10):2548-56. DOI: 10.1021/bi00539a039. View

10.

Kramer W, Schughart K, Fritz H . Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments. Nucleic Acids Res. 1982; 10(20):6475-85. PMC: 326937. DOI: 10.1093/nar/10.20.6475. View

11.

Potter B, Connolly B, Eckstein F . Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction. Biochemistry. 1983; 22(6):1369-77. DOI: 10.1021/bi00275a008. View

12.

Messing J . New M13 vectors for cloning. Methods Enzymol. 1983; 101:20-78. DOI: 10.1016/0076-6879(83)01005-8. View

13.

Kunkel T, Loeb L, Goodman M . On the fidelity of DNA replication. The accuracy of T4 DNA polymerases in copying phi X174 DNA in vitro. J Biol Chem. 1984; 259(3):1539-45. View

14.

Shi J, Fersht A . Fidelity of DNA replication under conditions used for oligodeoxynucleotide-directed mutagenesis. J Mol Biol. 1984; 177(2):269-78. DOI: 10.1016/0022-2836(84)90456-x. View

15.

Connolly B, Potter B, Eckstein F, Pingoud A, Grotjahn L . Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site. Biochemistry. 1984; 23(15):3443-53. DOI: 10.1021/bi00310a010. View

16.

Kramer B, Kramer W, Fritz H . Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984; 38(3):879-87. DOI: 10.1016/0092-8674(84)90283-6. View

17.

Kramer W, Drutsa V, Jansen H, Kramer B, PFLUGFELDER M, Fritz H . The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 1984; 12(24):9441-56. PMC: 320472. DOI: 10.1093/nar/12.24.9441. View

18.

Eckstein F . Nucleoside phosphorothioates. Annu Rev Biochem. 1985; 54:367-402. DOI: 10.1146/annurev.bi.54.070185.002055. View

19.

Stec W, Zon G, Uznanski B . Reversed-phase high-performance liquid chromatographic separation of diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides and other backbone-modified congeners of DNA. J Chromatogr. 1985; 326:263-80. DOI: 10.1016/s0021-9673(01)87452-5. View

20.

Burke D, Olson M . Oligodeoxynucleotide-directed mutagenesis of Escherichia coli and yeast by simple cotransformation of the primer and template. DNA. 1986; 5(4):325-32. DOI: 10.1089/dna.1986.5.325. View