Method for in vivo recombination and mutagenesis

• US 5,286,632 A
• Filed: 01/09/1991
• Issued: 02/15/1994
• Est. Priority Date: 01/09/1991
• Status: Expired due to Term

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America)
• Original Assignee: United States Department Of Health And Human Services (Government of the United States of America)
• Inventors: Jones, Douglas H.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US07/638,512
• Time in Patent Office: 1,133 Days
• Field of Search: 435/91, 435/172.3
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites

Abstract

The subject invention relates to a method referred to as recombination PCR (RPCR). In the method, the polymerase chain reaction is utilized to add double-stranded homologous ends to DNA. These homologous ends undergo recombination in vivo following transfection of host cells. The placement of these homologous ends, by the amplifying primers permits the rapid cloning of the desired mutant or recombinant, with a minimal number of steps and primers.

Claims

1. A method for synthesizing a double-stranded circular DNA molecule, using the polymerase chain reaction (PCR) process, comprising the steps of:

(i) amplifying a double-stranded DNA segment by means of PCR, wherein two primers effect said amplification and add nucleotide sequences to said segment, several of which are homologous to the ends of a linear, second, double-stranded, extra-chromosomal DNA molecule;

(ii) transfecting the resulting product of step (i) into a host cell comprising said linear, second, double-stranded, extra-chromosomal DNA molecule; and

(iii) allowing the added nucleotide sequences of the product of step (i), which are homologous to the ends of said linear, second, double-stranded, extra-chromosomal DNA molecule, to recombine therewith, thereby producing a double-stranded circular DNA molecule.

2. The method of claim 1 wherein 3 basepairs of each of the nucleotide sequences which are added to said double-stranded DNA segment are homologous to at least one end of said linear, second, double-stranded, extrachromosomal DNA molecule.

3. The method of claim 1 wherein the primers of step (i) are 10 nucleotides or more in length.

4. The method of claim 1 wherein said linear, second, double-stranded, extrachromosomal DNA molecule is co-transfected into said host cell.

5. A method of producing one or more site-specific mutations in a double-stranded circular DNA molecule comprising the steps of:

in a first container means containing a first aliquot of said double-stranded DNA molecule;

(i) contacting said double-stranded circular DNA molecule with a first and second primer wherein said first primer contains at least 10 nucleotides that are complementary to strand 1 of said double-stranded DNA, and said second primer contains at least 10 nucleotides that are homologous to said strand;

(ii) producing non-circular copies of a portion of said double-stranded circular DNA molecule by means of the polymerase chain reaction;

in a second container means containing a second aliquot of said double-stranded circular DNA molecule;

(iii) contacting said double-stranded circular DNA molecule with a third and fourth primer

wherein said third primer contains at least 10 nucleotides that are complementary to strand 2 of said double-stranded circular DNA molecule and said third primer contains at least three nucleotides that are complementary to said first primer,

fourth primer contains at least 10 nucleotides that are homologous to a portion of said strand 2 of said double-stranded circular DNA, and said fourth primer contains at least 3 nucleotides that are complementary to said second primer;

(iv) producing non-circular copies of a portion of said double-stranded circular DNA molecule by means of the polymerase chain reaction using said third and fourth primers;

(v) co-transfecting a host cell with the products of steps (ii) and (iv); and

(vi) isolating said double-stranded circular DNA molecule containing at least one mutation, resulting from the co-transfection of step (v).

6. A method of producing a site-specific mutation in a double-stranded circular DNA molecule comprising the steps of:

(i) contacting said double-stranded circular DNA molecule with a first and second primer wherein said first primer contains at least 10 nucleotides that are complementary to one strand of said double-stranded DNA, said second primer contains at least 10 nucleotides that are homologous to said one strand, and said first primer contains at least 3 nucleotides that are complementary to said second primer;

(ii) producing non-circular copies of said double-stranded circular DNA molecule by means of the polymerase chain reaction;

(iii) transfecting a host cell with the product of step (ii);

(iv) isolating said double-stranded circular DNA molecule, resulting from the transfection of step (iii).

7. A method of DNA recombination, with or without concurrent mutagenesis, resulting in the production of circular DNA, comprising the steps of:

in a first container means containing an aliquot of double-stranded donor template DNA;

(i) contacting said donor template DNA with a first and second primer wherein said first primer contains at least 10 nucleotides that are complementary to a portion of one strand of said double-stranded donor template DNA, and wherein said second primer contains at least 10 nucleotides that are homologous to a portion of said one strand of said donor template DNA;

(ii) producing non-circular copies of a portion of said donor template DNA by means of the polymerase chain reaction;

in a second container means containing an aliquot of double-stranded recipient DNA;

(iii) contacting said double-stranded recipient DNA with a third and fourth primer wherein said third primer contains at least 10 nucleotides that are complementary to a portion of one strand of said recipient DNA, and wherein said fourth primer contains at least 10 nucleotides that are homologous to a portion of said one strand of said recipient DNA,

(iv) producing non-circular copies of a portion of said recipient DNA by means of the polymerase chain reaction using said third and fourth primers;

wherein said first primer contains at least three nucleotides that are complementary to a region of a first strand of the product of said third and fourth primers and said second primer contains at least three nucleotides that are complementary to a region of a second strand of the product of said third and fourth primers or,

alternatively, wherein said third primer contains at least three nucleotides that are complementary to a region of a first strand of the product of step (ii) and said fourth primer contains at least three nucleotides that are complementary to a region of a second strand of the product of step (ii),

(v) co-transfecting a host cell with the product of steps (ii) and (iv); and

(vi) isolating said circular DNA, resulting from the co-transfection of step (v).

8. The method of claim 7 wherein a restriction enzyme fragment is substituted for the product of step (ii) or step (iv).