# US 5,707,811 A · Reca-assisted cloning of DNA

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 5,707,811 A
- Document ID: US5707811A
- Status: Granted
- Kind: A
- Application number: 08/682,305
- Filed: 1996-07-17
- Granted: 1998-01-13
- Earliest claimed: 1996-07-17
- Assignees: The United States of America as represented by the Secretary of Health and Human Services
- Inventors: Lance Joseph Ferrin; R. Daniel Camerini-Otero
- Web page: https://patentgrep.com/patent/US5707811A

## CPC

- C12Q 1/6853: using modified primers or templates
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The method of claim 1, wherein said oligonucleotides are between 30 and 60 bases in length.

### Claim 3 (depends on 1)

The method of claim 1, wherein said restriction enzyme is EcoRI.

### Claim 4 (depends on 1)

The method of claim 1, wherein two restriction enzymes are reacted in step (a).

### Claim 5 (depends on 4)

The method of claim 4, wherein said restriction enzymes are EcoRI and BamHI.

### Claim 6 (depends on 1)

The method of claim 1, wherein said DNA polymerase is the Klenow fragment of E. coli DNA polymerase I.

### Claim 7 (depends on 1)

The method of claim 1, wherein said vector is a plasmid.

### Claim 8 (depends on 7)

The method of claim 7, wherein said plasmid is pBC SK.sup.+ or pBS SK.sup.+.

### Claim 9 (depends on 1)

The method of claim 1, wherein said vector is a yeast artificial chromosome, bacterial artificial chromosome or P1 phage derived artificial chromosome.

### Claim 10 (depends on 1)

The method of claim 1, further comprising the step of size fractionating said DNA fragments of step (a) to enrich for the fragment containing the predetermined DNA sequence.

### Claim 11 (depends on 10)

The method of claim 10, further comprising, prior to the ligating step, ligating the enriched DNA fragments to a biotinylated duplex containing complementary 3' recessed ends, wherein said biotinylated duplex is bound to streptavidin-coated beads.

### Claim 12 (depends on 1)

The method of claim 1, further comprising amplifying the DNA fragment containing the predetermined DNA sequence.

### Claim 13 (depends on 12)

The method of claim 12, wherein said amplifying step comprises transfection into bacteria.

### Claim 14 (depends on 12)

The method of claim 12, wherein said amplifying step comprises PCR.

### Claim 15

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.

### Claim 16 (depends on 15)

The method of claim 15, wherein step (f) comprises growth of said vector in a suitable microorganism.

### Claim 17 (depends on 15)

The method of claim 15, wherein step (f) comprises PCR.

### Claim 18 (depends on 15)

The method of claim 15, wherein said oligonucleotides are between 30 and 60 bases in length.

### Claim 19 (depends on 15)

The method of claim 15, wherein step (g) comprises sequencing said fragment.

### Claim 20 (depends on 15)

The method of claim 15, wherein step (d) comprises treating with sodium dodecyl sulfate or phenol/chloroform.

### Claim 21 (depends on 15)

The method of claim 15, wherein said mammal is a human.

### Claim 22 (depends on 15)

The method of claim 15, wherein said DNA polymerase is the exonuclease-deficient Klenow fragment of E. coli DNA polymerase I.

### Claim 23 (depends on 15)

The method of claim 15, wherein said vector is a plasmid.

### Claim 24 (depends on 15)

The method of claim 15, wherein said vector is a yeast artificial chromosome, bacterial artificial chromosome or P1 phage derived artificial chromosome.

### Claim 25 (depends on 15)

The method of claim 15, further comprising the step of size fractionating said DNA fragments of step (a) to enrich for the fragment containing the mutation.

### Claim 26 (depends on 25)

The method of claim 25, further comprising prior to the ligating step, ligating the enriched DNA fragments to a biotinylated duplex containing complementary cohesive ends, wherein said biotinylated duplex is bound to streptavidin-coated beads.