PCR primer pair and application thereof

Patent metadata

Field	Value
Publication number	US-11760995-B2
Application number	US-201716624791-A
Country	US
Kind code	B2
Filing date	Jun 20, 2017
Priority date	Jun 20, 2017
Publication date	Sep 19, 2023
Grant date	Sep 19, 2023

Abstract

Provided is 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; the first specific sequence is located on 3′ end of the first primer, and the first random sequence is located on 5′ end of the first primer; the second primer comprises a second specific sequence and a second random sequence, the second specific sequence is located on 3′ end of the second primer, and the second random sequence is located on 5′ end 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, and the first random sequence and the second random sequence are inverse complementary.

First claim

The invention claimed is:

1. A method for nucleic acid amplification, comprising:
performing amplification by using a 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 template sequence, and the first random sequence and the second random sequence are reversely complementary,
wherein the amplification comprises two rounds, wherein the first round of amplification is performed at a first annealing temperature, and the second round of amplification is performed at a second annealing temperature that is higher than the first annealing temperature so as to obtain a circular product.

2. The method according to claim 1 , wherein in the first round of amplification, the primer pair and the template sequence are subjected to amplification under an annealing temperature of 55-65° C., and in the second round of amplification, a product of the first round of amplification is subjected to amplification under an annealing temperature of 65-72° C.

3. The method according to claim 1 , wherein the two rounds of amplification are performed as the following amplification reaction procedure:

step 1
preheating for 2 minutes at 98° C.

step 2
denaturing for 10 seconds at 98° C.

step 3
annealing for 2 minutes at 55-65° C.

step 4
amplifying for 30 seconds at 72° C.

step 5
denaturing for 10 seconds at 98° C.

step 6
annealing for 1 minute at 65-72° C.

step 7
repeating steps 5 and 6 for 5-35 circles

step 8
extending for 5 minutes at 72° C.

4. A method for preparing a circular DNA library, comprising the steps of:
(1) subjecting a DNA sample comprising template sequences to the method of claim 1 , so as to obtain circular amplification products, wherein the circular amplification products are double-stranded, wherein each strand of each double stranded circular amplification product comprises a 5′ end and a 3′ end, wherein the 5′ end and the 3′ end of each strand of each double-stranded circular amplification product are not connected,
wherein at least one of the first primer and the second primer used in the amplification is phosphorylated at the 5′ end, wherein the 1-5th bases from each of the 5′ end and the 3′ end of the first primer respectively contain thio-modifications, wherein the 1-5th bases from each of the 5′ end and the 3′ end of the second primer respectively contain thio-modifications,

and
(2) subjecting the double-stranded circular amplification products to a ligation reaction by using a ligase, such that the 5′ end and the 3′ end of at least one strand of the double-stranded circular amplification products are ligated, thus forming ligated circular DNAs, the ligated circular DNAs constituting the circular DNA library,
wherein the circular DNA library is a single-stranded circular DNA library when only one of the first primer and the second primer is phosphorylated at the 5′ end; and the circular DNA library is a double stranded circular DNA library when both the first primer and the second primer are phosphorylated at the 5′ end.

5. The method according to claim 4 , further comprising: (3) removing unligated DNAs having 5′ and 3′ ends.

6. The method according to claim 5 , wherein the linear unligated DNAs having 5′ and 3′ ends are removed by digestion.

7. The method according to claim 4 , wherein in step (1), the DNA molecules of the sample to be tested are modified with universal sequences at the 5′ end and the 3′ end respectively.

8. The method according to claim 1 , wherein the first specific sequence and the second specific sequence each have a TM value of 55-65° C., and the first primer and the second primer each have a TM value of 65-75° C.

9. The method according to claim 1 , wherein the first round of amplification comprises: subjecting the primer pair and the template sequence to amplification under an annealing temperature of 55-65° C. so as to obtain a linear product; and
the second round of amplification comprises: subjecting the primer pair and the linear product to amplification under an annealing temperature of 65-72° C. so as to obtain the circular product.

10. The method according to claim 1 , wherein the first round of amplification further comprises:
subjecting the first specific sequence of the first primer to binding to the upstream of the template sequence, and subjecting the second specific sequence of the second primer to binding to the downstream of the template sequence, so as to obtain linear products; and the second round of amplification further comprises:
subjecting a second molecule of the first primer to binding to a linear product of the first round of amplification, wherein the first random sequence of the second molecule of the first primer binds to a 5′ end of the linear product, and the first specific sequence of the second molecule of the first primer binds to a 3′ end of the linear product, and
subjecting a second molecule of the second primer to binding to a linear product of the first round of amplification, wherein the second random sequence of the second molecule of the second primer binds to a 5′ end of the linear product, and the second specific sequence of the second molecule of the second primer binds to a 3′ end of the linear product, so as to obtain the circular products of the second round of amplification.

11. The method according to claim 1 , wherein in the second round of amplification, the first random sequence of a first primer and the second random sequence of a second primer bind to 5′ ends of products from the first round of amplification, and the first specific sequence of a first primer and the second specific sequence of a second primer bind to 3′ ends of products from the first round of amplification.

12. The method according to claim 1 , further comprising: subjecting the circular product to a ligation reaction by using a ligase, such that a 5′ end and a 3′ end of the circular product are ligated, thus forming a ligated circular product.

13. The method according to claim 1 , whe

Assignees

• Mgi Tech Co Ltd

Inventors

• Yang Lin
• Gao Ya
• Huang Guodong
• Wang Yicong
• Wang Yuqian
• Zhang Yanyan
• Chen Fang
• Zhong Na
• Jiang Hui
• Xu Xun

Classifications

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12N15/1093 Primary

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

• C12Q1/68

  involving nucleic acids · CPC title

• C12Q1/6806

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

• C12Q1/6853

  using modified primers or templates · CPC title