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

• US 10,077,468 B2
• Filed: 08/12/2008
• Issued: 09/18/2018
• Est. Priority Date: 04/15/2008
• Status: Active Grant

• Current Assignee: iNtRON Biotechnology, Inc.
• Original Assignee: iNtRON Biotechnology, Inc.
• Inventors: Yoon, Seongjun, Lee, Hyunyoung, Yu, Jinseok, Jun, Sooyoun, Kang, Sanghyeon
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US12/228,302
• Publication Number:
  US 20090258353A1
• Time in Patent Office: 3,689 Days
• Field of Search: None
• 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 method comprising amplifying via a polymerase chain reaction a selected target sequence with a primer, wherein the primer comprises a priming sequence and an additional sequence, wherein the priming sequence is complementary to 10-50 nucleotides of the 3′

end of the selected target sequence and the additional sequence is directly attached to the 5′

-terminal end of the priming sequence and is reverse-complementary to 3-15 nucleotides of the 5′

-terminal sequence of the priming sequence and does not hybridize to the selected target sequence.

2. A method for reducing non-specific amplification in PCR comprising amplifying a selected target sequence via a polymerase chain reaction with a primer prepared by steps a)-c):

a) determining a priming sequence of the primer based on the selected target sequence, wherein the priming sequence is complementary to 10-50 nucleotides of the 3′

end of the selected target sequence;

b) determining an additional sequence to be directly attached to the 5′

-terminal end of the priming sequence of the primer, wherein the additional sequence is reverse-complementary to 3-15 nucleotides of the 5′

-terminal sequence of the priming sequence and does not hybridize to the selected target sequence; and

c) synthesizing the primer comprising the priming sequence and the additional sequence, wherein the additional sequence is directly attached to the 5′

-terminal end of the priming sequence.