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