DNA polymerase with increased gene mutation specificity and PCR buffer composition for increasing activity thereof

A gene mutation and polymerase technology, applied in the field of DNA polymerase, can solve problems such as inability to obtain clear information and poor synthesis of polymerase, and achieve improved amplification efficiency, high matching extension selectivity, and reliable gene mutation-specific amplification Effect

Active Publication Date: 2019-01-22
GENECAST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason for the shortcomings of these PCR-based methods is the poor synthesis of the polymerases used in the methods used to adequately discriminate between mismatched bases
Therefore, it has not been possible to directly obtain clear information on the presence or absence of mutations by PCR.
To date, definitive diagnosis of mutations has required additional time and costly purification and analysis methods

Method used

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  • DNA polymerase with increased gene mutation specificity and PCR buffer composition for increasing activity thereof
  • DNA polymerase with increased gene mutation specificity and PCR buffer composition for increasing activity thereof
  • DNA polymerase with increased gene mutation specificity and PCR buffer composition for increasing activity thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1 Inducing Mutation of Taq Polymerase

[0134] 1-1. Fragment PCR

[0135] In this example, in the amino acid sequence with SEQ ID NO: 1, the arginine at the 536th amino acid residue (in the amino acid sequence of SEQ ID NO: 1 was prepared according to the following method) was substituted by lysine for Taq DNA polymerization enzyme (hereinafter referred to as "R536K"), Taq DNA polymerase in which the arginine at the 660th amino acid residue was replaced with valine (hereinafter referred to as "R660V"), and the arginine at the 536th amino acid residue was replaced with Taq DNA polymerase in which lysine was substituted and arginine at the 660th amino acid residue was substituted by valine (hereinafter referred to as "R536K / R660V") was prepared as follows.

[0136] First, Taq DNA polymerase fragments (F1 to F5) were amplified by PCR using the mutation-specific primers shown in Table 1, as figure 1 (a) shown. The reaction conditions are shown in Table 2.

[013...

Embodiment 2

[0163] Example 2 Introduction of E507K mutation

[0164] 2-1. Fragment PCR

[0165] The activity of the Taq polymerase of "R536K", "R660V" and "R536K / R660V" prepared in Example 1 was tested, and the results confirmed that the activity decreased (data not shown), respectively for "R536K", "R660V " and "R536K / R660V" introduced the E507K mutation (in the amino acid sequence of SEQ ID NO: 1, the glutamic acid at the 507th amino acid residue was replaced by lysine), as a control group, in the wild-type (WT) Taq DNA polymer The E507K mutation was also introduced into the enzyme. The preparation method of the TaqDNA polymerase with the E507K mutation introduced is the same as in Example 1.

[0166] Use the mutation-specific primers shown in Table 9, such as image 3 Taq DNA polymerase fragments (F6 to F7) were amplified by PCR as indicated. The reaction conditions are shown in Table 10.

[0167] Table 9

[0168]

[0169] Table 10

[0170]

[0171] *Template plasmid: pUC1...

Embodiment 3

[0184] Example 3 Using the DNA polymerase of the present invention to carry out qPCR

[0185] Using Taq polymerases each containing the "E507K / R536K", "E507K / R660V" and "E507K / R536K / R660V" mutations obtained in Example 2 above, it was confirmed that it extends the mismatched primer for the template containing the SNP. whether the ability is reduced. As a control, "E507K" Taq polymerase containing the E507K mutation was used.

[0186] The templates containing SNPs used in this example are rs1408799, rs1015362 and rs4911414. The genotypes of each template and the sequence information of their corresponding specific primers (IDT, USA) are shown in Table 12 and Table 13.

[0187] Table 12

[0188]

[0189] Table 13

[0190]

[0191] The qPCR conditions (Applied Biosystems 7500 Fast) are shown in Table 14 below.

[0192] Table 14

[0193]

[0194] The probes were double labeled as shown in Table 15 below.

[0195] Table 15

[0196]

[0197] Oral epithelial cells ...

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Abstract

The present invention relates to a DNA polymerase having increased gene mutation specificity and a PCR buffer composition for increasing activity of the DNA polymerase. More specifically, provided, inthe present invention, are a DNA polymerase in which a mutation is induced at a specific amino acid position to increase gene mutation specificity, a nucleic acid sequence encoding the polymerase, avector comprising the nucleic acid sequence, and a host cell transformed with the vector. In addition, provided is a method for in vitro detecting one or more gene mutations or SNPs in one or more templates by using a DNA polymerase having increased gene mutation specificity, a composition for detecting a gene mutation or SNP comprising the DNA polymerase, and a PCR kit comprising said composition. Furthermore, provided are a PCR buffer composition for increasing the activity of a DNA polymerase having increased gene mutation specificity, a PCR kit for detecting a gene mutation or SNP comprising the PCR buffer composition and/or the DNA polymerase having increased gene mutation specificity, and a method for in vitro detecting one or more gene mutations or SNPs in one or more templates by using the kit.

Description

technical field [0001] The present invention relates to a DNA polymerase with improved gene mutation-specific amplification efficiency and its application, specifically, a DNA polymerase with improved gene mutation-specific amplification efficiency induced by amino acid sites, encoding the polymerase A nucleic acid sequence, a vector comprising the nucleic acid sequence and providing the vector with a transgenic host cell. The present invention also provides a method for detecting more than one gene variation or SNP in more than one template in vitro (invitro) by using a DNA polymerase with improved gene mutation-specific amplification efficiency, comprising the DNA polymerase A composition for detecting gene variation or SNP and a PCR kit comprising the composition. Background technique [0002] Since the discovery of the first human gene sequence, researchers have focused on discovering genetic differences among individuals such as single nucleotide mutations (single nucl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12Q1/6827
CPCC12N9/1252C12Q1/6827C12Y207/07007C12Q2521/101C12Q2531/113
Inventor 李炳哲朴日铉李辉皓
Owner GENECAST CO LTD
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