Uracil-DNA glycosylase of psychrobacter sp. HJ147 and use thereof

• US 20080299609A1
• Filed: 06/26/2007
• Published: 12/04/2008
• Est. Priority Date: 03/12/2007
• Status: Active Grant

• Current Assignee: Sungkyunkwan University Foundation For Corporate Collaboration (SungKyunKwan University)
• Original Assignee: Sungkyunkwan University Foundation For Corporate Collaboration (SungKyunKwan University)
• Inventors: Lee, Jung-Hyun, Lee, Mi-Sun, Kwon, Suk-Tae, Kim, Gun-A.
• Granted Patent: US 7,723,093 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 9/2497 hydrolysing N- glycosyl compounds (3.2.2)
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Y 302/02027 Uracil-DNA glycosylase (3.2.2.27)

Abstract

The present invention provides uracil-DNA glycosylase (UDG) gene originating from Psychrobacter sp. HJ147, and amino acid sequences deduced from the gene; expression and purification of Psp HJ147 UDG gene in Escherichia coli; and characterization of UDG obtained therefrom, and the use thereof in a polymerase chain reaction (PCR). The UDG according to the present invention has a specific activity of excising uracil bases in a uracil-containing DNA substrates at a low temperature, and is easily heat-inactivated. It thus can effectively eliminate cross contamination and carry-over contamination of PCR templates often occurring after a PCR process using dUTP. Therefore, it is useful for increasing preciseness (elimination of false positives), purity and amplification efficiency of PCR.

Claims

1. A uracil-DNA glycosylase which comprises an amino acid sequence having at least 80% of sequence homology with the amino acid sequence set forth in SEQ ID NO:

2 or its functional equivalent.

2. The uracil-DNA glycosylase of claim 1, wherein the sequence homology is at least 90%.

3. The uracil-DNA glycosylase of claim 1, wherein the sequence homology is at least 95%.

4. The uracil-DNA glycosylase of claim 1, which is isolated from Psychrobacter sp. HJ147.

5. A polynucleotide having a nucleic acid sequence that encodes the UDG of claim 1.

6. The polynucleotide of claim 5, wherein the nucleic acid sequence comprises the sequence set forth in SEQ ID NO:

1.

7. The polynucleotide of claim 6, which is isolated from Psychrobacter sp. HJ147.

8. A recombinant vector comprising the polynucleotide of claim 5.

9. The recombinant vector of claim 8, which is pTPSUDG (KCCM10838P).

10. A host cell transformed by the vector of claim 8.

11. A method for producing a UDG, which comprises the steps of:

(a) inserting a polynucleotide encoding the UDG of claim 1 into a vector;

(b) introducing the vector into a host cell; and

(c) culturing the host cell under suitable medium and conditions so as to express the UDG.

12. A composition for PCR, which comprises the UDG according to claim 1.

13. The composition of claim 12, which comprises 0.5-10 units of the UDG of claim 1.

14. The composition of claim 12, which further comprises a polymerase, at least one nucleotide triphosphate, a primer and a buffer.

15. A method for removing cross contamination from PCR reaction products, comprising the steps of:

(a) reacting a uracil-containing DNA substrate with a composition comprising the UDG according to claim 1 at 50°

C. or lower; and

(b) conducting PCR reaction.

16. The method of claim 15, wherein the step (a) is conducted at 25-50°

C. for 0-5 minutes.