Method of targeting DNA
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsRafael D. Camerini-Otero, Margaret McIntosh, Carol S. Camerini-Otero, Lance J. Ferrin
Life of this patent
Estimating the term…
Abstract
The present invention relates to a method of forming a three-stranded DNA molecule wherein each strand of the three-stranded DNA molecule is hybridized (that is, non-covalently bound) to at least one other strand of the three-stranded DNA molecule. The method comprises: contacting a recombination protein with a double-stranded DNA molecule and with a single-stranded DNA molecule sufficiently complementary to one strand of the double-stranded DNA molecule to hybridize therewith, which contacting is effected under conditions such that the single-stranded DNA molecule hybridizes to the double-stranded molecule so that the three stranded DNA molecule is formed.
Claims18
A method of selecting for a specific double-stranded DNA molecule present in a sample comprising:
- i) contacting a recombination protein with said double-stranded DNA molecule and with a single-stranded DNA molecule, which single-stranded DNA molecule is sufficiently complementary to a specific sequence present in said double-stranded DNA molecule to hybridize therewith, wherein said contacting is effected in the presence of ADP and ATP-gamma-S and under conditions such that said single-stranded DNA molecule hybridizes to said double-stranded molecule so that a three-stranded DNA molecule is formed,
- wherein said single-stranded DNA molecule has a first member of a binding pair bound thereto;
- ii) separating said three-stranded DNA molecule from unhybridized single-stranded DNA molecules;
- iii) contacting said three-stranded DNA molecule with a second member of said binding pair;
- and
- iv) isolating said three-stranded DNA molecule bound to said second member of said binding pair.
A method of effecting cleavage of a double-stranded DNA molecule containing at least two restriction endonuclease recognition sites at a first of said sites by a restriction enzyme specific for said sites comprising:
- i) contacting a recombination protein with the double-stranded DNA molecule and with a single-stranded DNA molecule, which single-stranded DNA molecule is sufficiently complementary to a portion of one strand of said double-stranded DNA molecule that includes the first of said restriction endonuclease recognition sites to hybridize therewith,
- wherein said contacting is effected under conditions such that said single-stranded DNA molecule hybridizes to said double-stranded molecule at said first of said restriction endonuclease recognition sites so that a three-stranded DNA molecule is formed, and
- ii) methylating the at least one other of said sites so as to render it protected from said restriction enzyme;
- iii) dissociating said single-stranded DNA molecule from said double stranded molecule;
- and
- iv) cleaving said double-stranded molecule resulting from step (iii) at said first of said restriction sites.
A method of protecting a sequence of a double-stranded DNA molecule from modification by a methylase comprising:
- i) contacting a recombination protein with the double-stranded DNA molecule and with a single-stranded DNA molecule, which single-stranded DNA molecule is sufficiently complementary said sequence of said double-stranded DNA molecule to hybridize therewith,
- wherein said contacting is effected under conditions such that said single-stranded DNA molecule hybridizes to said sequence of said double-stranded molecule so that a three-stranded DNA molecule is formed, and
- ii) contacting said three-stranded DNA molecule with said methylase under conditions such that said three-stranded DNA molecule remains intact, whereby said sequence is protected from modification.
A method of inhibiting transcription or replication of a specific sequence present on one strand of a double-stranded DNA molecule comprising contacting a recombination protein with said double-stranded DNA molecule and with a single-stranded DNA molecule, which single-stranded DNA molecule is sufficiently complementary to said sequence to hybridize therewith,
- wherein said contacting is effected under conditions such that said single-stranded DNA molecule hybridizes to said gene sequence so that a three-stranded DNA molecule is formed, whereby transcription and replication of said sequence is inhibited.
A method of protecting a double-stranded DNA molecule containing at least two restriction endonuclease recognition sites from cleavage by a restriction endonuclease at a first of said restriction endonuclease recognition sites comprising:
- i) contacting a recombination protein with the double-stranded DNA molecule and with a single-stranded DNA molecule, which single-stranded DNA molecule is sufficiently complementary to a portion of one strand of said double-stranded DNA molecule that includes said first restriction endonuclease recognition site to hybridize therewith,
- wherein said contacting is effected in the presence of ATP-gamma-S and ADP and under conditions such that said single-stranded DNA molecule hybridizes to said double-stranded molecule at said first restriction endonuclease recognition site so that a three-stranded DNA molecule is formed;
- and
- ii) contacting said three-stranded DNA molecule with said restriction endonuclease under conditions such that said three-stranded DNA molecule remains intact, whereby said first restriction endonuclease recognition site is protected from cleavage.
Dependencies are inferred from the claim text.