Unlock instant, AI-driven research and patent intelligence for your innovation.

PCR (polymerase chain reaction) system and kit for high-GC segment amplification

The technology of a reaction system and a kit, which is applied in the biological field, can solve the problems of unsuccessful amplification, high price and high use cost, and achieve the effects of cost reduction and convenient purification.

Active Publication Date: 2017-08-11
GUANGZHOU SUPBIO BIO TECH & SCI
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The above method can amplify DNA fragments with a GC content of 80%, but cannot successfully amplify DNA fragments with a GC content greater than 80%.
In addition, because the price of 7-deaza-dGTP is very expensive, the use cost of the above method is very high, so the general applicability and market prospect of the above method are relatively poor

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • PCR (polymerase chain reaction) system and kit for high-GC segment amplification
  • PCR (polymerase chain reaction) system and kit for high-GC segment amplification
  • PCR (polymerase chain reaction) system and kit for high-GC segment amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 A kind of PCR kit for amplification of DNA fragments with high GC content

[0060] Described PCR kit comprises:

[0061] (1) dNTPs, (2) Tris-HCl at pH 8.7, (3) MgSO 4 , (4) (NH 4 ) 2 SO 4 、 (5) BeCl 2 , (6) DMSO, (7) betaine, (8) DTT and (9) DNA polymerase.

Embodiment 2

[0062] Embodiment 2 A kind of PCR reaction system and its application for high GC content DNA fragment amplification

[0063] The INSR gene (NCBI No. AH002851.2) was used as the target amplified fragment and corresponding PCR primers were designed. The nucleic acid sequence of the target amplified product is:

[0064] CGCGGCCCCCAGCGCCTCTTGGGTGGCCGCCTCGGAGCATGACCCCCGCGGGCCAGCGCCGCGCGCTCTGATCCGAGGAGACCCCGCGCTCCCGCAGCCATGGGCACCGGGGGCCGGCGGGGAGCGGCGGCCGCGCCGCTGGTGGCGGTGGCCGCGCTGCTACTGGGCGCCGCGGGC

[0065] The fragment length of the target amplification product is 180bp, and the GC content of the fragment is 82.8%.

[0066] (1) PCR primers are:

[0067] Upstream primer: 5'-CGCGGCCCCCAGCGCCTCTT-3'

[0068] Downstream primer: 5'-GCCCGCGGCGCCCAGTAGCA-3'.

[0069] (2) The PCR template is: the amplified sample is human whole blood.

[0070] (3) The PCR reaction system is:

[0071]

[0072] After the above reagents were added to the PCR tube, Taq DNA polymerase was added, and the...

Embodiment 3

[0078] Embodiment 3 A kind of PCR reaction system and its application for high GC content DNA fragment amplification

[0079] Target amplified fragment: same as Example 2.

[0080] (1) PCR primers: same as Example 2.

[0081] (2) PCR template: same as Example 2.

[0082] (3) PCR reaction system:

[0083]

[0084] After the above reagents were added to the PCR tube, Taq DNA polymerase was added, and the system was made up to 50 μL.

[0085] (4) Carry out PCR reaction according to the following PCR procedure:

[0086] 95℃, 10min;

[0087] 95°C, 15sec; 62°C, 15sec; 72°C, 30sec; 40 cycles;

[0088] 72°C, 45sec.

[0089] (5) Perform agarose gel electrophoresis on the PCR amplification products. Electrophoresis results such as figure 2 As shown, lanes 3 and 4 are the amplification products of this example. It can be clearly seen that the target DNA fragment with a GC content as high as 82.8% can be amplified using the amplification system of this example.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of biology, and particularly relates to the technical field of PCR (polymerase chain reaction), more particular to a PCR (polymerase chain reaction) system and kit for high-GC segment amplification. The PCR system and kit comprises dNTPs, Tris-HCl, MgSO4, (NH4)2SO4 and PCR auxiliary, wherein the PCR auxiliary is one or multiple of BeCl2, dimethyl sulfoxide (DMSO), betaine and dithiothreitol (DTT). Compared with methods for amplifying high segments through 7-deaza-dGTP, the PCR system and kit has the advantage that cost is lowered greatly. Compared with existing high-GC-content amplification methods, the PCR system can amplify DNA segments higher in GC content, can amplify DNA segments higher than 80% in GC content and even can amplify DNA segments up to 90% in GC content.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically relates to the field of PCR technology, and more specifically relates to a PCR reaction system and a kit for amplifying high GC fragments. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction, PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as special DNA replication outside the body. The biggest feature of PCR is that it can Trace amounts of DNA increased dramatically. Therefore, whether it is ancient organisms in fossils, the remains of historical figures, or the hair, skin or blood left by the murderer in a murder case decades ago, as long as a little bit of DNA can be isolated, it can be amplified by PCR for comparison. right. This is where the power of "trace evidence" lies. In 1983, Mullis of the United States first proposed the idea, and in 1985, he invented the polymerase chain reaction, that is, a ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/10
CPCC12Q1/686C12Q2527/125
Inventor 张晓玮彭春梅张嘉邓可基李海茵谢丽娟李家导乐小炎林志豪罗园香张新陈观芝陈凤英林敏深石壮壮林若琳王星王法余培煜莫静嫣
Owner GUANGZHOU SUPBIO BIO TECH & SCI