PCR primer capable of reducing non-specific amplification and PCR method using the PCR primer

• US 20090258353A1
• Filed: 08/12/2008
• Published: 10/15/2009
• Est. Priority Date: 04/15/2008
• Status: Active Grant

• Current Assignee: iNtRON Biotechnology, Inc.
• Original Assignee: iNtRON Biotechnology, Inc.
• Inventors: Lee, Hyunyoung, Yu, Jinseok, Yoon, Seongjun, Kang, Sanghyeon, Jun, Sooyoun
• Granted Patent: US 10,077,468 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2549/101 Hot start

Abstract

The present invention relates to a PCR primer facilitating hot-start PCR by suppressing non-specific amplification at room temperature and at the same time capable of reducing significantly non-specific amplification by dominating the amplification of the PCR product rather than the amplification of the original template from the third PCR cycle, more precisely a PCR primer prepared by additionally inserting the reverse-complementary sequence to a certain region starting from the 5′-start site of the 5′-terminus of the original primer which is composed of priming sequence to anneal to a PCR template into the 5′-terminus of the original primer and a PCR method using the same. The primer of the present invention has a original primer sequence composed of priming sequence to anneal to a PCR template and an additional reverse-complementary sequence, which inserted into the 5′-terminus of the original primer, to a certain region starting from the 5′-start site of the 5′-terminus of the original primer sequence, suggesting that a template-specific sequence and its reverse-complementary sequence are included in the same primer. The present invention can improve PCR specificity by reducing non-specific amplification.

Claims

1. A PCR primer comprising a first original primer sequence composed of priming sequence to anneal to a PCR template and a second additional reverse-complementary sequence to a certain region starting from the 5′

-start site of the 5′

-terminus of the original primer sequence, which inserted into the 5′

-terminus of the original primer.

2. The PCR primer according to claim 1, wherein the PCR is the DNA polymerase-mediated PCR and RT-PCR.

3. The PCR primer according to claim 1, wherein the original primer is composed of 10-50 nucleotides.

4. The PCR primer according to claim 1, wherein the additional reverse-complementary sequence added to the 5′

-terminus of the original primer is composed of 1-15 nucleotides.

5. The PCR primer according to claim 1, wherein the additional reverse-complementary sequence added to the 5′

-terminus of the original primer exhibits GC ratio of 20-100% by the total base of the primer.

6. The PCR primer according to claim 1, wherein the linker sequence is additionally inserted in between the additional reverse-complementary sequence added to the 5′

-terminus of the original primer and the original primer sequence.

7. The PCR primer according to claim 1, wherein the primer has the structure of (A) or (B) shown in FIG. 1.

8. A PCR method using the primer of claim 1.

9. A PCR kit containing the primers of claim 1.

10. The PCR kit according to claim 9, wherein the kit is characteristically a diagnostic kit using PCR and RT-PCR for diagnosis.