Primers for PCR amplification comprising a basic parts within the primer sequences

• US 8,513,399 B2
• Filed: 10/02/2008
• Issued: 08/20/2013
• Est. Priority Date: 10/05/2007
• Status: Active Grant

• Current Assignee: Bioneer Corporation
• Original Assignee: Bioneer Corporation
• Inventors: Kim, Hyun Bae, Kim, Seong Youl, Gil, Jun Mo, Park, Hae Joon, Park, Han Oh
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/681,754
• Publication Number:
  US 20110008845A1
• Time in Patent Office: 1,783 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 536/26.6, 435/6.1, 435/91.1
• US Class Current: 536/24.33
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites

Abstract

The present invention relates to primers for PCR amplification comprising abasic parts within the primer sequences and a method for PCR amplification using the same. More precisely, the present invention relates to primers capable of amplifying different templates and having abasic parts complementary to mutated site or polymorphic site of template DNA and a method for PCR amplification comprising the steps of mixing the composition for PCR amplification comprising the primers with nucleic acid template; and performing PCR with the mixture. The primers for PCR amplification of the present invention contain abasic parts not having specific coding information in their nucleotide sequences, so that they can amplify different templates having mutated sites at the same time.

Claims

1. A primer for PCR amplification capable of commonly amplifying different nucleic acid templates having mutated or polymorphic sites in their nucleotide sequences, said primer comprising abasic part in the region corresponding to the mutated or polymorphic sites of said different nucleic acid templates to be hybridized, wherein said primer commonly amplifies different nucleic acid templates having mutated or polymorphic sites in their nucleotide sequences at the same time.

2. The primer according to claim 1, wherein the abasic part is selected from the group consisting of dSpacer (5′

-O-dimethoxytrityl-1′

,2′

-dideoxyribose-3′

-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), 1′

-OMe-dSpacer (5-O-dimethoxytrityl-1′

-methoxy-2′

-dideoxyribose-3′

-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), PC Spacer Phosphoamidite ([4-(4,4′

-Dimethoxytrityloxy)butyramidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite), rSpacer CE Phosphoamidite (5′

-O-Dimethoxytrityl -1′

-Deoxyribose-2′

-O-Triisopropylsilyloxymethyl-3′

-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), Spacer C12 CE Phosphoamidite (12-(4,4′

-Dimethoxytrityloxy)-dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), Spacer Phosphoamidite 18 (18-O-Dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), Spacer Phosphoamidite 9 (9-0-Dimethoxytrityl-triethyleneglycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite), and Spacer Phosphoamidite C3 (3-(4,4′

-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite).

3. The primer according to claim 1, wherein the number of the abasic part in the primer is 1-10.

4. The primer according to claim 3, wherein the number of the abasic part in the primer is 1-3.

5. The primer according to claim 1, wherein a nucleotide complementary to template is added to a terminus of the primer to compensate the lowered Tm by abasic part.

6. A composition for PCR amplification comprising reaction buffer, 4 kinds of dNTPs, DNA polymerase and any primer for PCR amplification according to claim 1.

7. The composition according to claim 6, wherein the composition further comprises a dye and/or a stabilizer.

8. The composition according to claim 7, wherein the dye is one or more compounds selected from the group consisting of Bromophenol blue, Xylene cyanole, Bromocresol red and Cresol red.

9. The composition according to claim 7, wherein the stabilizer is one or more compounds selected from the group consisting of gelatin, bovine serum albumin, Thesit, PEG-8000 and polyol.

10. A method for PCR amplification comprising the steps of mixing the composition for PCR amplification comprising the primer according to claim 1 with nucleic acid template;

and performing PCR with the mixture.

11. The method for PCR amplification according to claim 10, wherein the method is to commonly amplify different nucleic acid templates having mutated sites in their nucleotide sequences.

12. The method for PCR amplification according to claim 10, wherein the method is to commonly amplify different nucleic acid templates having polymorphic sites in their nucleotide sequences.

13. The method for PCR amplification according to claim 10, wherein one primer of the primer set for PCR amplification is a primer for PCR amplification comprising abasic part and the other primer of the primer set is a normal primer not comprising abasic parts.

14. The primer of claim 2, wherein a nucleotide complementary to template is added to a terminus of the primer to compensate the lowered Tm by abasic part.

15. The primer of claim 3, wherein a nucleotide complementary to template is added to a terminus of the primer to compensate the lowered Tm by abasic part.

16. The primer of claim 4, wherein a nucleotide complementary to template is added to a terminus of the primer to compensate the lowered Tm by abasic part.