Generation of unidirectional deletion mutants

Details Generation of unidirectional deletion mutants Generation of unidirectional deletion mutants

1991-09-17

1994-10-18

Schwartz, Richard A.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 911, 4351723, 4353201, 536 243, C12Q 168

Patent

active

053567730

ABSTRACT:
A novel method that allows introduction of unidirectional deletions into cloned DNA is described. This method is based on the use of a mixture of oligodeoxynucleotide primers that have fixed 5' (or 3') ends defining the end point of the deletion and variable 3' (or 5') ends composed of mixtures of all four nucleotides at six positions. The 5' ends of the oligodeoxynucleotides are hybridized to a fixed location of the M13K11RX templates and the 3' ends are hybridized randomly to the DNA to be analyzed. Such oligodeoxynucleotide primers when extended with DNA polymerase can direct deletions of intervening parts of the single-stranded DNA that by design contains multiple Eco K sites; the deletion products are selected on a host strain with the Eco K restriction system (e.g., using JM101 cells). This method is an efficient way of generating a nested set of deletion mutants useful for dideoxy-sequencing. It can also be used for creating a set of deletion mutants with a particular codon at the 5' or 3' end point.

REFERENCES:
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patent: 4582788 (1986-04-01), Erlich
Hasan et al., "A Novel Multistep Method for . . . " Gene, 50 (1986) pp. 55-62.
Barcak et al. "A Method for Unidirectional Deletion Mutagenesis . . . " Gene, 49 (1986), pp. 119-128.
Waye et al. Nucl. Acids Res. vol. 13 pp. 8561-8571, 1985 "EcoK selection vectors for shotgun cloning into M13 and deletion mutagenesis".
Carter et al. Nucl. Acids Res. vol. 13, pp. 4431-4443 1985 "Improved oligonucleotide site-directed mutagenesis using M13 vectors".
Poncz et al. Proc Natl. Acad Sci vol. 79 pp. 4298-4302 1982 "Nonrandom DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method".

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