PCR primer pair and application thereof

Patent metadata

Field	Value
Publication number	US-11739319-B2
Application number	US-201716624779-A
Country	US
Kind code	B2
Filing date	Jun 20, 2017
Priority date	Jun 20, 2017
Publication date	Aug 29, 2023
Grant date	Aug 29, 2023

Abstract

Provided are a PCR primer pair and an application thereof. The PCR primer pair comprises: a first primer and a second primer, wherein the first primer comprises a first specific sequence and a first random sequence; the first specific sequence is located on end 3′ of the first primer, and the first random sequence is located on end 5′ of the first primer; the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is located on end 3′ of the second primer, and the second random sequence is located on end 5′ of the second primer; moreover, the first specific sequence and the second specific sequence are an upstream primer and a downstream primer directed to a target sequence, respectively; the first random sequence and the second random sequence are reverse complementary; a predetermined restriction enzyme cutting site is connected between the first specific sequence and the first random sequence; and a predetermined restriction enzyme cutting site is connected between the second specific sequence and the second random sequence.

First claim

What is claimed is:

1. A PCR primer pair, comprising a first primer and a second primer, wherein
the first primer comprises a first specific sequence and a first random sequence, and
the second primer comprises a second specific sequence and a second random sequence, wherein
the first specific sequence is located at the 3′ end of the first primer and the first random sequence is located at the 5′ end of the first primer,
the second specific sequence is located at the 3′ end of the second primer and the second random sequence is located at the 5′ end of the second primer,
the first specific sequence and the second specific sequence are respectively an upstream primer and a downstream primer for a target sequence,
the first random sequence and the second random sequence are reversely complementary,
the first specific sequence is connected to the first random sequence via a first predetermined restriction enzyme cutting site,
the second specific sequence is connected to the second random sequence via a second predetermined restriction enzyme cutting site, and
the target sequence of the PCR primer pair does not comprise any predetermined restriction enzyme cutting site,
wherein the first random sequence and the second random sequence each have a length of 15-45 bp, and
the first specific sequence and the second specific sequence each have a length of 15-30 bp,
wherein the first predetermined restriction enzyme cutting site and the second predetermined restriction enzyme cutting site each are a uracil (U) base site.

2. The PCR primer pair according to claim 1 , wherein the 1 st -5 th bases from each of the 5′ end and the 3′ end of the first primer respectively have thio-modification, and
the 1 st -5 th bases from each of the 5′ end and the 3′ end of the second primer respectively have thio-modification.

3. The PCR primer pair according to claim 2 , wherein the thio-modification is selected from phosphorothioate modification and methyl-sulfate modification.

4. The PCR primer pair according to claim 1 , wherein the first primer has a sequence as shown in SEQ ID NO.: 1-18, and the second primer has a sequence as shown in SEQ ID NO.: 19-36.

5. The PCR primer pair according to claim 1 , wherein the first primer and the second primer each have a length of 30-70 bp.

6. The PCR primer pair according to claim 1 , wherein the 1 st -5 th bases from each of the 5′ end and the 3′ end of the first primer respectively have a peptide nucleic acid modification.

Assignees

• Mgi Tech Co Ltd

Inventors

• Yang Lin
• Huang Guodong
• Gao Ya
• Zhang Yanyan
• Zhang Haiping
• Chen Fang
• Jiang Haojun
• Zhang Shaohong
• Wang Yuqian
• Xie Yifan
• Jiang Hui

Classifications

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title