Selective cloning of homoduplex nucleic acids
AssigneeStratagene
InventorsAlan L. Greener, Lisa Joy Hexdall, Carsten-Peter Carstens, Joseph A. Sorge
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Abstract
The subject invention provides for a method of selectively cloning homoduplex nucleic acid molecules, in particular, by using a strain of host cells that contains a conditionally expressed and/or conditionally active mismatch-recognizing enzyme, e.g., a temperature sensitive variant of the gene encoding the endonuclease VII from phage T4. Using this host strain, the invention features a novel cloning method that selects for PCR products that are devoid of PCR-generated mutations.
Claims38
A host cell for selectively cloning homo duplex nucleic acid molecules, wherein the host cell is:
- (a) competent;
- (b) contains a gene encoding a resolvase that is conditionally expressed and/or conditionally active;
- and
- (c) can be maintained under conditions under which the resolvase is not expressed or is repressed and propagated in inactive form.
A method of selectively cloning homoduplex nucleic acid molecules, the method comprising:
- (a) providing a host cell containing a gene encoding a resolvase that is conditionally expressed and/or conditionally active;
- (b) transforming said host cell with a cloned nucleic acid molecule, thereby producing a transformed host cell;
- and
- (c) maintaining the transformed host cell under conditions suitable for expression of the resolvase, wherein, if the host cell contains a heteroduplex cloned nucleic acid molecule, the heteroduplex cloned nucleic acid molecule is cleaved by the resolvase, and if the host cell contains a homoduplex cloned nucleic acid molecule, the homoduplex cloned nucleic acid molecule is not cleaved by the resolvase;
- thereby selectively cloning homoduplex nucleic acid molecules.
A method of selectively cloning amplified nucleic acid molecules possessing a reduced number of heteroduplexes, the method comprising:
- (a) providing a host cell containing a gene encoding a resolvase that is conditionally expressed and/or conditionally active;
- (b) transforming said host cell with a cloned amplified nucleic acid molecule, thereby producing a transformed host cell;
- and
- (c) maintaining the transformed host cell under conditions suitable for expression of the resolvase, wherein, if the host cell contains a heteroduplex cloned nucleic acid molecule, the heteroduplex cloned nucleic acid molecule is cleaved by the resolvase, and if the host cell contains a homoduplex cloned nucleic acid molecule, the homoduplex cloned nucleic acid molecule is not cleaved by the resolvase;
- thereby selectively cloning amplified nucleic acid molecules possessing a reduced number of heteroduplexes.
A kit comprising a competent host cell containing a gene encoding a resolvase that is conditionally expressed and/or conditionally active, and packaging materials therefore.
A kit comprising a competent host cell containing a gene encoding a conditionally expressed resolvase, wherein the host cell can be maintained under conditions under which the resolvase is expressed, and packaging materials therefore.
A kit comprising a host cell containing an expression vector encoding the temperature sensitive mutant of bacteriophage T4 Endonuclease VII of SEQ ID NO:2, PCR reagents, helper phage and packaging materials therefore.
A kit comprising an isolated DNA encoding the temperature sensitive mutant of bacteriophage T4 Endonuclease VII of SEQ ID NO:2 and packaging materials therefore.
A kit comprising an expression vector encoding the temperature sensitive mutant of bacteriophage T4 Endonuclease VII of SEQ ID NO:2 and packaging materials therefore.
A kit comprising a host cell containing an expression vector encoding the temperature sensitive mutant of bacteriophage T4 Endonuclease VII of SEQ ID NO:2 and packaging materials therefore.
An isolated nucleic acid encoding a temperature sensitive mutant of bacteriophage T4 Endonuclease VII, wherein said isolated nucleic acid has the nucleic acid sequence of SEQ ID NO:1.
The isolated nucleic acid of claim 37, wherein said isolated nucleic acid is cloned into an expression vector.
An isolated protein of a temperature sensitive mutant of bacteriophage T4 Endonuclease VII, wherein said isolated protein has the amino acid sequence of SEQ ID NO:2.
Dependencies are inferred from the claim text.