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High-fidelity pfu DNA polymerase mutants, their encoding DNA and their application in NGS

A polymerase and mutant technology, applied in the biological field, can solve the problems affecting the application and promotion of Pfu enzyme and the low amplification efficiency of Pfu enzyme, and achieve the effects of improving library sequencing quality, reducing base mismatch rate, and ultra-high fidelity

Active Publication Date: 2021-12-07
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the amplification efficiency of Pfu enzyme is low, which affects the application and promotion of Pfu enzyme.

Method used

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  • High-fidelity pfu DNA polymerase mutants, their encoding DNA and their application in NGS
  • High-fidelity pfu DNA polymerase mutants, their encoding DNA and their application in NGS
  • High-fidelity pfu DNA polymerase mutants, their encoding DNA and their application in NGS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 High-fidelity enzyme six-site mutant activity test

[0026] Select calf thymus gDNA (Yeasen, Cat#60612ES03) ultrasound fragment, 250-500bp as a template, use a conventional DNA library construction kit (Yeasen, Cat#12200) to build a library, prepare a PCR free library, and use 1×dsDNA HSAsay Kit for Qubit ® (Cat#12642) for concentration detection of PCR free library. Use different volumes (0.3 / 0.5 / 1 / 1.5 / 2 μL) of high-fidelity Pfu DNA polymerase six-site mutants (referred to as high-fidelity enzyme six-site mutants) to perform amplification tests on 10ngPCR free libraries, and compare the amplification of purified products Yield, select the optimal enzyme amount.

[0027] Table 1 PCR system

[0028] name Dosage Calf thymus gDNA PCR free library 10ng 10×PCR buffer 5 μL NGS Primer 5 μL high fidelity enzyme mutants 0.3 / 0.5 / 1 / 1.5 / 2μL ddH2O to 50 μL X μL

[0029] Table 2 PCR program

[0030]

[0031] Table 3...

Embodiment 2

[0034] Example 2 High-fidelity enzyme mutants 1-13, PCR free library amplification efficiency test

[0035] Use 50ng calf thymus DNA PCR free library for amplification test, the most suitable amount of high-fidelity enzyme mutants 1-13, and wild-type Pfu enzyme are 1.5μL, 2μL, 1μL, 1.2μL, 1μL, 1.5μL, 1μL , 1 μL, 2 μL, 1.5 μL, 1.5 μL, 1 μL, 1 μL, 1 μL, 10×PCR buffer 5 μL, water up to 50 μL, PCR amplification for 7 cycles, and compare the amplification efficiency of different mutants.

[0036] Table 4 Library yield

[0037]

[0038] Result analysis: Among the 13 high-fidelity enzyme mutants, the amplification efficiency of single mutants 2, 3, and triple mutant 10 was slightly improved, and single mutants 1, 4, 5, 6, and triple mutants 7, 8, and 9 The amplification efficiency of , 11, and 12 is significantly improved, and the amplification yield is about 1.5 times that of the wild-type pfu enzyme; the amplification efficiency of the six mutant 13 is the most obvious, and the...

Embodiment 3

[0039] Example 3 Test of PCR free library amplification efficiency of high-fidelity enzyme six-site mutants with different DNA inputs

[0040] 500pg-500ng calf thymus DNA PCR free library was used for amplification test, the amount of high-fidelity enzyme mutant was 1 μL, 10×PCR buffer 5 μL, water was added to 50 μL, PCR amplification was performed, and the amplification effect of different DNA input amounts was compared.

[0041] Table 5 Library Yield

[0042] group 1 2 3 Calf thymus gDNA PCR free library 500pg 50ng 500ng Number of Amplification Cycles (Cycles) 14 7 4 Library Yield (ng) 1530 1690 1550

[0043] Result analysis: The six-site mutant of the high-fidelity enzyme is compatible with the amplification of DNA PCR free libraries with different input amounts, and the corresponding cycle number of 500pg-500ng amplification can obtain considerable library yields.

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Abstract

The present invention provides a high-fidelity Pfu DNA polymerase mutant, which is characterized in that, on the basis of wild-type Pfu DNA polymerase, mutations of 1, 3 or 6 amino acid sites are carried out, wherein the mutation sites are as follows: Loci selected: Y403A, P411H, V438K, A741S, R757Q, S768K. And disclosed its coding DNA, and its application in NGS. The high-fidelity Pfu DNA polymerase mutant of the present invention has ultra-high fidelity and high amplification efficiency, can be stably and efficiently applied to the library enrichment process in next-generation sequencing, effectively improves the quality of library sequencing, and reduces the base mismatch rate. It can be used for precise analysis and interpretation of genetic information.

Description

technical field [0001] The patent of the present invention relates to a high-fidelity Pfu DNA polymerase mutant and its coding DNA, as well as its application in NGS, belonging to the field of biotechnology. Background technique [0002] High-Throughput Sequencing (High-Throughput Sequencing), also known as NextGeneration Sequencing (NGS), has a large sequencing throughput and can determine tens to millions of DNA molecular sequences at a time. It is a revolutionary technology for traditional sequencing technologies. change. At present, NGS technology has been applied to all aspects of life sciences, solving more and more biological problems. With the widespread popularization and application of sequencing technology, the market demand for library construction kits is also increasing. At the same time, people have put forward higher requirements for the quality standards of library construction kits. First, more people began to pay attention to quality, and among many qual...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54C12Q1/6869
CPCC12N9/1252C12Q1/6869C12Y207/07007
Inventor 秦雪梅曹振宋东亮曹欣茹任静
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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