Direct cloning of PCR amplified nucleic acids

• US 5,487,993 A
• Filed: 09/09/1993
• Issued: 01/30/1996
• Est. Priority Date: 09/27/1990
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Chimer, Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Fernandez, Joseph M., Mead, David A., Smith, Lloyd, Herrnstadt, Corinna
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): HOUTTEMAN, SCOTT W
• Application Number:
  US08/119,313
• Time in Patent Office: 873 Days
• Field of Search: 435/6, 435/69.1, 435/91.2, 435/172.3, 435/320.1, 435/91.5, 435/91.4, 435/91.41, 436/63
• US Class Current: 435/488
• CPC Class Codes: 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
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/131 Terminal transferase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine

Abstract

Methods are described for producing recombinant DNA molecules from suitable host vectors and nucleic acids subjected to 3'"'"'-terminal transferase activity. In one embodiment, the method takes advantage of the single 3'"'"'-deoxy-adenosine monophosphate (dAMP) residues attached to the 3'"'"' termini of PCR generated nucleic acids. Vectors are prepared with recognition sequences that afford single 3'"'"'-terminal deoxy-thymidine monophosphate (dTMP) residues upon reaction with a suitable restriction enzyme. Thus, PCR generated copies of genes can be directly cloned into the vectors without need for preparing primers having suitable restriction sites therein. The invention also contemplates associated plasmid vectors and kits for implementing the methods.

Claims

1. A method of direct cloning of polymerase chain reaction amplified nucleic acids containing a target DNA segment, which method comprises:

(a) subjecting said target DNA segment to polymerase chain reaction amplification to produce a plurality of double-stranded nucleic acids including said DNA segment, each of said nucleic acids having a single, overhanging dAMP residue located at each 3'"'"' terminus;

(b) forming a ligation reaction mixture by admixing the plurality of double-stranded nucleic acids with linear double-stranded DNA molecules, said linear DNA molecules each having a single, overhanging (unpaired) dTMP residue at each 3'"'"' terminus;

(c) maintaining said mixture under predetermined reaction conditions for a time period sufficient to effect ligation of the nucleic acids to the linear DNA molecules to produce recombinant DNA nucleic acid molecules; and

(d) transforming a compatible host cell line with said recombinant DNA molecules.

2. The method of claim 1, wherein said DNA segment includes a selectable marker gene.

3. A method of direct cloning of polymerase chain reaction amplified nucleic acids containing a target DNA segment, which method comprises:

(a) subjecting said target DNA segment to polymerase chain reaction amplification using a polymerase having 3'"'"'-terminal adenosine transferase activity to produce a plurality of double-stranded nucleic acids including the DNA segment, each of the nucleic acids having a single, overhanging 3'"'"'-dAMP residue at each 3'"'"'terminus;

(b) reacting a DNA plasmid with at least one restriction enzyme to form a linear DNA molecule having a single overhanging 3'"'"'-dTMP residue at each DNA 3'"'"'terminus;

(c) forming a ligation reaction mixture by combining the double-stranded nucleic acids and the linear plasmid molecules with a preselected ligase enzyme;

(d) maintaining said admixture under predetermined reaction conditions for a time period sufficient to effect ligation of the nucleic acids to the linear plasmid molecules to produce recombinant DNA molecules; and

(e) transforming a compatible host cell line with said recombinant DNA molecules.

4. The method of claim 3 wherein said polymerase having 3'"'"'-terminal adenosine transferase activity is thermophilic.

5. The method of claim 3, wherein the DNA segment includes a selectable marker gene.

6. The method of claim 3, further comprising separating said linear DNA molecules in step (b) from smaller restriction fragments prior to performing step (c).

7. The method of claim 3, wherein said plasmid contains a first nucleotide sequence recognized by a first restriction enzyme and a second nucleotide sequence recognized by a second restriction enzyme, wherein each enzyme generates a single overhanging 3'"'"'-dTMP at each 3'"'"' terminal upon reaction with the first and second nucleotide sequences.

8. The method of claim 7, wherein said first and second restriction enzymes are XcmI or HphI.

9. The method of claim 7, wherein said first and second nucleotide sequences are identical.

10. A kit for direct cloning of PCR amplified nucleic acids, which kit comprises, in separate containers:

an aliquot of plasmids, said plasmids each having at least two nucleotide sequences recognized by at least one restriction enzyme capable of generating a single, overhanging 3'"'"'-dTMP group; and

an aliquot of said restriction enzyme sufficient to cleave at least a portion of said plasmids.

11. The kit of claim 10, wherein said enzyme is selected from the group consisting of XcmI, MboII or HphI.

12. The kit of claim 10, wherein said plasmids are lyophilized.

13. The kit of claim 10, further comprising in a separate container an aliquot of a ligase capable of ligating a nucleic acid sequence containing a 3'"'"'-dAMP overhang into said plasmids at said overhanging 3'"'"'-dTMP groups.

14. The kit of claim 10, further comprising in a separate container an aliquot of primer molecules for use in PCR amplification of a target DNA sequence.

15. The kit of claim 10, further comprising in a separate container an aliquot of a thermophilic polymerase having 3'"'"'-terminal transferase activity.

16. The kit of claim 10, further comprising an aliquot of competent host cells capable of replicating said plasmids upon transformation therewith.

17. A kit for direct cloning of PCR amplified nucleic acids, which kit comprises, in separate containers:

an aliquot of lyophilized cloning plasmids, said plasmids each having at least two nucleotide sequences recognized by a restriction enzyme capable of generating a single, overhanging 3'"'"'-dTMP; and

an aliquot of competent host cells capable of replicating said plasmids upon transformation therewith.

18. A kit for direct cloning of PCR amplified nucleic acids, which kit comprises, in separate containers:

an aliquot of linear plasmid DNA molecules having single, overhanging 3'"'"'-dTMP termini;

an aliquot of control plasmids;

an aliquot of each of forward and reverse PCR primers suitable for PCR amplification;

an aliquot of a DNA ligase, said DNA ligase being capable of ligating a nucleic acid sequence containing 3'"'"'-dAMP overhangs into said linear plasmid DNA at said overhanging 3'"'"'-dTMP sites.

19. The kit of claim 18 wherein said linear plasmid DNA is pTA112-L.

20. The kit of claim 18 wherein said linear plasmid DNA is pTA12-L.