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Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method

A polymerase and activity technology, applied in the field of determination of Phi29DNA polymerase activity, can solve problems such as difficult to achieve high throughput, automation, radioactive pollution, long cycle, etc.

Inactive Publication Date: 2017-01-11
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is prone to radioactive contamination, and requires multiple steps such as precipitation and filtration. The cycle is long, and it is difficult to achieve high-throughput and automation, which brings difficulties to screening.

Method used

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  • Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method
  • Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method
  • Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Establishment of a method for measuring the polymerization activity of phi29 DNA polymerase by fluorescence method

[0033] 1. Standard curve establishment

[0034] (1) Rolling circle amplification (RCA) reaction

[0035] a) The materials required for the RCA reaction are as follows:

[0036] Template: M13 single-stranded DNA (ssDNA);

[0037] Amplification primer: 5'-aagccatccgcaaaaatgacctct-3' (SEQ ID NO: 1);

[0038] Polymerase: Phi29DNA polymerase (Enzymatics).

[0039] b) RCA reaction steps:

[0040] i. Prepare the primer-template hybridization reaction mixture, the reaction system is shown in Table 1 (can be adjusted according to the actual situation), and the reaction mixture is placed in a PCR instrument for primer-template hybridization at 95°C for 3-5 minutes.

[0041] Table 1 Primer template hybridization reaction mixture reference system

[0042] components Dosage M13 ssDNA 1.5μL wxya 2 O water

83.3μL 10×phi2...

Embodiment 2

[0066] Example 2 Application of RCA products based on nanoarray optical sequencing platform

[0067] 1. For the RCA products obtained in Example 1, after taking a certain amount of RCA products for Qubit detection concentration, the remaining RCA products can be added to the chip of the nano-array, and high-throughput optical sequencing platform BGI-1000 is used for high-throughput sequencing. Throughput sequencing.

[0068] 2. Sequencing primer: complementary to the amplification primer of the RCA reaction. If the non-M13 single-stranded DNA used in Example 1 is a circular single-stranded DNA with a specific sequence, the sequence corresponding to the known specific sequence can be used as a sequencing primer.

[0069] 3. Specific process: preprocessing of samples to be tested—loading of samples to be tested—data generation of samples to be tested—data processing of samples to be tested—downstream application analysis.

[0070] 4. Application: The data obtained by sequencin...

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Abstract

The invention discloses a method for measuring the activity of Phi29 DNA polymerase and a rolling circle sequencing method. The method for measuring the activity of Phi29 DNA polymerase comprises the following steps: taking a circular single-stranded DNA as a template; in the presence of an amplification primer, using Phi29 DNA polymerase to trigger rolling circle replication to generate a large amount of continuous single-stranded DNA copies; then doping fluorescent molecules into the amplification products, detecting the fluorescent signals of the amplification products to quantify the produced single-stranded DNA copy number, and thus calculating the activity of Phi29 DNA polymerase. The provided method has the advantages of no radioactive contamination, simpleness, rapidness, and high sensitivity, and can be applied to high-flux and automatic polymerase screening.

Description

technical field [0001] The invention relates to the technical field of measuring enzyme activity, in particular to a method for measuring the activity of Phi29 DNA polymerase and a rolling circle sequencing method. Background technique [0002] phi29 DNA polymerase is a mesophilic DNA polymerase cloned from Bacillus subtilis phage phi29. It has special multiple replacement and continuous synthesis characteristics, and has 3' to 5' exonuclease proofreading ability. Phi29 DNA polymerase is not resistant to high temperature, and can perform amplification reaction at 30°C, and its fidelity is equivalent to that of pfu polymerase, which is 10 times that of ordinary Taq enzyme 3 times. [0003] Phi29 DNA polymerase is an important molecular biology tool enzyme, which can be used for whole genome amplification, isothermal amplification, rolling circle replication, DNA sequencing, biochemical analysis, virus detection, nucleic acid detection, endonuclease activity determination, et...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/48
CPCC12Q1/6869C12Q1/48C12Q2531/125C12Q2563/107
Inventor 席凤郭苗苗韩鸿雁
Owner MGI TECH CO LTD
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