Reca-assisted cloning of DNA
AssigneeThe United States of America as represented by the Secretary of Health and Human Services
InventorsLance Joseph Ferrin, R. Daniel Camerini-Otero
Life of this patent
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Abstract
DNA is cloned and labeled in a sequence-specific manner. The DNA is digested with one or more restriction enzymes which produce 3' recessed ends. A desired fragment is protected from elongation by DNA polymerase by addition of E. coli RecA protein and oligonucleotides about 30 to 60 bases in length complementary to the 3' recessed ends of the digested fragment. RecA and DNA polymerase are then inactivated, leaving only the desired fragment with 3' recessed ends which is then ligated into a vector containing complementary 3' recessed ends.
Claims26
A method of cloning a DNA fragment containing a predetermined DNA sequence, comprising the steps of:
- (a) digesting DNA coning a predetermined DNA sequence with at least one restriction enzyme which generates 3' recessed ends to produce DNA fragments having 3' recessed ends;
- (b) reacting said DNA fragments with RecA protein and two oligonucleotides, said oligonucleotides being complementary to the 3' recessed end of either DNA strand of the fragment containing the predetermined DNA sequence;
- (c) reacting the DNA fragments resulting from step (b) with a DNA polymerase, whereby all DNA fragments except the fragment coming the predetermined DNA sequence become blunt-ended;
- (d) dissociating said oligonucleotides from the ends of the fragment containing the predetermined DNA sequence;
- and
- (e) ligating said DNA fragments to a vector having 3' recessed ends complementary to those produced by the restfiction enzyme, whereby only the fragment containing the predetermined DNA sequence is incorporated into said vector.
A method of determining a genetic mutation in a mammal, comprising the steps of:
- (a) isolating genomic DNA containing said mutation from a mammal;
- (b) digesting said genomic DNA with one or more restriction enzymes which generate 3' recessed ends to produce genomic DNA fragments having 3' recessed ends;
- (c) reacting said genomic DNA fragments with RecA protein and two oligonucleotides, said oligonucleotides being complementary to the ends of the fragment containing the mutation;
- (c) reacting the genomic DNA fragments resulting from step (b) with a DNA polymerase, whereby all genomic DNA fragments except the fragment containing the mutation become blunt-ended;
- (d) dissociating said oligonucleotides from the ends of the fragment containing the mutation;
- (e) ligating said DNA fragments to a vector having 3' recessed ends complementary to those produced by the restriction enzyme(s), whereby only the fragment containing the mutation is incorporated into said vector;
- (f) amplifying the fragment containing the mutation;
- and
- (g) determining whether the mutation is present in the amplified fragments.
Dependencies are inferred from the claim text.