Cloning method and kit

• US 5,525,493 A
• Filed: 05/19/1992
• Issued: 06/11/1996
• Est. Priority Date: 11/21/1989
• Status: Expired due to Term

• Current Assignee: Life Technologies AS (Perspecta, Inc.)
• Original Assignee: Dynel Comapny
• Inventors: Uhlen, Mathias, Hornes, Erik
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/849,389
• Time in Patent Office: 1,484 Days
• Field of Search: 435/6, 435/91, 435/91.2, 435/172.3, 935/77, 935/78
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method of amplifying target DNA is disclosed wherein said DNA is first amplified by PCR, the amplified DNA then being contacted with a single stranded linearised plasmid vector having terminal regions which are complementary to terminal regions of the PCR amplified DNA, whereby a cyclic product is formed comprising single stranded sequences from said target DNA and said vector and two double stranded regions from the overlapping terminal regions of the vector and the PCR amplified DNA; the cyclic product then being introduced into a host organism. Two-stage PCR may be performed and site-specific mutagenesis may be effected between PCR amplification and formation of the cyclic product. The single-stranded linearised plasmid vector and/or the target DNA may be immobilised. Kits are disclosed for performing various aspects of the method which can be used in a method of diagnosis wherein the target DNA is characteristic of a physiological condition.

Claims

1. A method of cloning a target DNA, wherein

(a) said target DNA is amplified by PCR to obtain single-stranded amplified target DNA,

(b) the single-stranded, amplified target DNA is contacted with a single-stranded, linear vector DNA having terminal regions which are complementary to respective terminal regions of said amplified target DNA, wherein said complementary terminal regions overlap and hybridize to form a cyclic DNA product comprising single-stranded target DNA, single-stranded vector DNA, and two double-stranded regions formed by hybridization of said overlapping complementary terminal regions of the single stranded vector and the single stranded amplified target DNA, wherein said double-stranded regions are separated from each other by a region of single-stranded target DNA, and wherein said cyclic DNA product is essentially single-stranded apart from said double-stranded complementary regions,

(c) said cyclic DNA product is introduced into a host organism, and

(d) said cyclic DNA product is cloned by replication of said host organism.

2. A method of cloning target DNA as claimed in claim 1, wherein the PCR is two-stage and uses nested primers, each primer of the second stage being complementary to a sequence of the target DNA between the sites complementary to the first stage primers.

3. A method as claimed in claim 2, wherein said second stage primers comprise single-stranded 5'"'"' nucleotide extensions which form terminal regions in one strand of the amplified target DNA that are complementary to the terminal regions of said single stranded vector.

4. A method as claimed in claim 1, wherein each of said double-stranded regions of the cyclic DNA product comprises at least one restriction site.

5. A method as claimed in claim 1, wherein PCR is effected using two primers, one of which carries means for immobilization or is already immobilized.

6. A method as claimed claim 1, wherein site-specific mutagenesis is effected between the steps of PCR amplification and formation of the cyclic DNA product.

7. A method as claimed in claim 6, wherein amplified target DNA is immobilized prior to said site-specific mutagenesis.

8. A method as claimed in claim 1, further comprising the steps of recovering cloned DNA from said host organism having introduced therein said cyclic DNA and then determining in said cloned DNA a sequence in said target DNA.

9. A method as claimed in claim 1, wherein the single stranded linear vector DNA is produced by a method comprising immobilizing only one end of only one strand of a double-stranded linear vector DNA and separating the non-immobilized strand from the immobilized strand wherein said non-immobilized strand is said single stranded linear vector.

13. A method of cloning a target DNA according to claim 1, wherein said overlapping complementary regions are 20-250 base pairs in length.

14. A method of cloning a target DNA according to claim 1, wherein said overlapping complementary regions are about 20 base pairs in length.

10. A kit for cloning target DNA comprising:

(a) a linear vector

(i) in single stranded form having terminal regions that are complementary to respective terminal regions of a target DNA, whereby said complementary terminal regions overlap and hybridize to form a cyclic DNA product comprising single stranded target DNA, single stranded vector DNA, and two double stranded DNA regions wherein said double stranded regions are separated from each other by a region of single stranded target DNA, and wherein said cyclic DNA product is essentially single stranded apart from said double stranded complementary regions;

or

(ii) in double stranded form and immobilized by one end of one strand thereof, wherein the single stranded linear vector is produced by separation of the non-immobilized strand from the immobilized strand;

(b) a polymerase;

(c) two PCR primers for amplification of the target DNA, wherein said PCR primers correspond to the terminal regions of said vector;

(d) nucleoside triphosphates.

11. A kit as claimed in claim 10 additionally comprising means for sequencing target DNA including either labelled primer or labelled nucleoside triphosphates.

12. A kit as claimed in claim 10 additionally comprising means for site-specific mutagenesis including:

(a) a site specific mutagenic amplification primer;

(b) a second polymerase; and

(c) a ligase.