METHOD FOR PREPARING DNA FRAGMENT HAVING STICKY END

• US 20110009607A1
• Filed: 03/10/2009
• Published: 01/13/2011
• Est. Priority Date: 03/11/2008
• Status: Abandoned Application

• Current Assignee: University of Tokyo
• Original Assignee: University of Tokyo
• Inventors: Kuzuya, Akinori, Komiyama, Makoto, Tanaka, Keita
• Application Number:
  US12/921,930
• Publication Number:
  US 20110009607A1
• Time in Patent Office: Days
• US Class Current: 536/24.33
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• 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: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

The present invention provides a method for preparing a DNA fragment, in which a desired double-stranded DNA fragment having a sticky end is directly and easily obtained from an amplification product (an amplified fragment) after PCR without a restriction enzyme digestion. The method for preparing a DNA fragment having a sticky end of the present invention comprises: (i) a step of performing a PCR reaction using a template DNA and specific primers to obtain an amplified DNA fragment; and (ii) a step of performing a prescribed treatment on the amplified DNA fragment to dissociate a protecting group from the fragment. Herein, the above-mentioned specific primers are composed of a complementary DNA portion consisting of a nucleotide sequence complementarily binding to an amplification target region in a template DNA and a non-complementary DNA portion consisting of a nucleotide sequence that links to the 5′ end of the complementary DNA portion but does not complementarily bind to the amplification target sequence, and at least a base corresponding to the 3′ end in the nucleotide sequence of the non-complementary DNA portion is modified with a protecting group capable of terminating the progression of DNA replication catalyzed by a DNA polymerase.

Claims

1. A primer used for PCR, which is composed of a complementary DNA portion consisting of a nucleotide sequence complementarily binding to an amplification target region in a template DNA and a non-complementary DNA portion consisting of a nucleotide sequence that links to the 5′

end of the complementary DNA portion but does not complementarily bind to the amplification target sequence,

wherein at least a base corresponding to the 3′

end in the nucleotide sequence of the non-complementary DNA portion is modified with a protecting group capable of terminating the progression of DNA replication catalyzed by a DNA polymerase.

2. The primer according to claim 1, wherein the protecting group can be dissociated from the modified base by a light irradiation treatment, an alkali treatment, an acid treatment, an oxidation treatment, a reduction treatment, a desilylation treatment, a heat treatment, an esterase treatment or a phosphatase treatment.

3. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the light irradiation treatment is a 2-(2-nitrophenyl)propyl group, a 2-(2-nitrophenyl)propyloxymethyl group, a 1-(2-nitrophenyl)ethyl group or a 6-nitropiperonyloxymethyl group.

4. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the alkali treatment is an isobutyryl group, a benzoyl group or an acetoxymethyl group.

5. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the acid treatment is a trityl group or a methoxy derivative thereof.

6. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the oxidation treatment is an allyloxymethyl group, a dimethoxybenzyloxymethyl group or a trimethoxybenzyloxymethyl group.

7. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the reduction treatment is a benzyloxymethyl group, or a benzyloxymethyl group substituted with any given substituent.

8. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the desilylation treatment is a t-butyldimethoxysilyloxymethyl group or a t-butyldiphenylsilyloxymethyl group.

9. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the heat treatment is an isocyanate group.

10. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the esterase treatment is an acetoxymethyl group.

11. The primer according to claim 2, wherein the protecting group that can be dissociated from the modified base by the phosphatase treatment is a methyl phosphate group.

12. The primer according to any one of claims 3 to 11, wherein at least a base corresponding to the 3′

end in the nucleotide sequence of the non-complementary DNA portion is thymine or guanine.

13. The primer according to claim 1, wherein the non-complementary DNA portion is 1 to 100 bases in length.

14. A method for preparing a DNA fragment having a sticky end, which comprises:

(i) a step of performing a PCR reaction using a template DNA and the primer according to claim 1 to obtain an amplified DNA fragment; and

(ii) a step of performing a prescribed treatment on the amplified DNA fragment to dissociate a protecting group from the fragment.

15. The method according to claim 14, wherein the prescribed treatment for dissociating the protecting group from the modified base is a light irradiation treatment, an alkali treatment, an acid treatment, an oxidation treatment, a reduction treatment, a desilylation treatment, a heat treatment, an esterase treatment or a phosphatase treatment.

16. The method according to claim 14, wherein the sticky end has the same nucleotide sequence as that of a sticky end obtained by a restriction enzyme digestion.

17. The method according to claim 14, wherein the sticky end has a nucleotide sequence different from that of a sticky end obtained by a restriction enzyme digestion.

18. A genetic recombination method, which comprises a step of binding the DNA fragment obtained by the method according to any one of claims 14 to 17 to a host DNA.

19. The method according to claim 18, wherein the binding operation is carried out without using a DNA ligase.

20. A substituent-introducing agent comprising a compound represented by the following formula (I′

);

21. A biomolecule having a 2-(2-nitrophenyl)propyloxymethyl group.

22. The biomolecule according to claim 21, which is represented by the following formula (I):

23. The biomolecule according to claim 21 or 22, which is a base.

24. The biomolecule according to claim 23, wherein the base is thymine or guanine.

25. The biomolecule according to claim 24, which is represented by the following formula (II):