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

• US 20020192769A1
• Filed: 05/07/2001
• Published: 12/19/2002
• Est. Priority Date: 05/07/2001
• Status: Active Grant

• Current Assignee: Bioneer Corporation
• Original Assignee: Bioneer Corporation
• Inventors: Weon, Se-Yeon, Park, Han Oh, Rhee, Joo-Won, Jeon, Jin-Tae, Joung, In-Suk, Song, Su-Nam
• Granted Patent: US 6,849,404 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• 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; and

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

2. A series of hairpin loop adapters which have single-stand cohesive end, which comprises hairpin loop adapters of which single-stand cohesive ends comprising all sorts of single-strand DNAs which can be obtained by a random combination of four (4) nucleotides.

3. 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; and

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

4. The process according to claim 3, which further comprises eliminating hairpin loops from the DNA fragments on which hairpin loop adapter are ligated in step iii).

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

6. The process according to claim 3, wherein the restriction enzyme is type IIip restriction enzyme.

7. The process according to claim 3, wherein the DNA ligase in step iii) is T4 DNA ligase.

8. The process according to claim 3, wherein the exonuclease in step iv) is exonuclease III.

9. The process according to claim 4, wherein the hairpin loop is eliminated by using alkaline solution.

10. The process according to claim 4, wherein the hairpin loop is eliminated by using RNase.

11. The process according to claim 4, wherein the hairpin loop is eliminated by using single strand specific exonuclease.

12. The process according to claim 3, wherein the DNA Polymerase is Taq DNA polymerase.