Polymerase chain reaction of DNA of which base sequence is completely unidentified

• US 6,849,404 B2
• Filed: 05/07/2001
• Issued: 02/01/2005
• Est. Priority Date: 05/07/2001
• Status: Expired due to Fees

• Current Assignee: Bioneer Corporation
• Original Assignee: Bioneer Corporation
• Inventors: Park, Han Oh, Rhee, Joo-Won, Jeon, Jin-Tae, Weon, Se-Yeon, Joung, In-Suk, Song, Su-Nam
• Primary Examiner(s): Sitton, Jehanne
• Assistant Examiner(s): CHUNDURU, SURYAPRABHA
• Application Number:
  US09/849,597
• Publication Number:
  US 20020192769A1
• Time in Patent Office: 1,366 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 536/24.2, 536/23.1
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label

Abstract

The present invention relates to a process for amplifying DNA of an organism. More particularly, the present invention is directed to a process for amplifying DNA of an organism through Polymerase Chain Reaction(PCR) without any information regarding a primer needed for amplifying DNA of an organism.

Claims

1. A process for preparing a library of DNA fragments of which terminal sequences are known by using a DNA of which base sequence is completely unidentified, which comprises:

i) digesting a DNA into fragments which have single-strand cohesive ends by using a restriction enzyme,

ii) preparing a series of hairpin loop adapters which have single-strand cohesive ends of which base sequence is known;

iii) ligating the DNA fragments with the hairpin loop adapters prepared in the above step ii) by using a DNA ligase;

iv) removing DNA fragments and hairpin loop adapters which have not participated in the ligation reaction by using an exonuclease; and

(v) eliminating a hair pin loop structure only from the DNA fragments which contain the hairpin loop adapters, obtained in step iii), by using an alkaline solution, am RNase or a single strand specific exonuclease.

11. The method according to claim 1, wherein the series of hairpin loop adapters have a single-stand cohesive end comprising all sorts of single-strand DNAs which can be obtained by a random combination of four (4) nucleotides.

2. A process for selective amplifying DNA of which base sequence is completely unidentified, which comprises:

i) digesting a DNA into fragments which have a single-strand cohesive end group by using a restriction enzyme,

ii) preparing hairpin loop adaptors which have the single-strand cohesive end which can be complementarily combined to and ligated on the both ends of the DNA fragments obtained in step i);

iii) ligating the DNA fragments with the hairpin loop adapters thus prepared by using a DNA ligase;

iv) removing DNA fragments and hairpin loop adapters which have not participated in the ligation reaction by using an exonuclease;

v) eliminating a hairpin loop structure from the DNA fragments on which said hairpin loop adapters are ligated in step iii); and

vi) amplifying the DNA fragments by using a DNA polymerase and a primer which can combine complementarily to a residual sequence from the adapters.

3. The process according to claim 2, wherein the restriction enzyme is type IIs restriction enzyme.

4. The process according to claim 2, wherein the restriction enzyme is type hip restriction enzyme.

5. The process according to claim 2, wherein the DNA ligase in step iii) is T4 DNA ligase.

6. The process according to claim 2, wherein the exonuclease in step iv) is exonuclease III.

7. The process according to claim 2, wherein the hairpin loop is eliminated by using alkaline solution.

8. The process according to claim 2, wherein the hairpin loop is eliminated by using RNase.

9. The process according to claim 2, wherein the hairpin loop is eliminated by using single strand specific exonuclease.

10. The process according to claim 2, wherein the DNA Polymerase is Taq DNA polymerase.

12. The method according to claim 2, wherein the series of hairpin loop adapters have a single-stand cohesive end comprising all sorts of single-strand DNAs which can be obtained by a random combination of four (4) nucleotides.