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Nucleic acid amplification method

A nucleic acid and amplification product technology, applied in the field of nucleic acid amplification, can solve problems such as false positives, and achieve the effect of suppressing false positives

Active Publication Date: 2018-09-07
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Since the nucleic acid amplification device is repeatedly used, there is a problem that false positives may occur by mixing the amplified nucleic acid amplification product into the subsequent nucleic acid amplification reaction solution

Method used

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Embodiment approach

[0025] In the present embodiment, there is provided a nucleic acid amplification product that suppresses the influence of carryover by obtaining a target nucleic acid amplification product (first nucleic acid amplification product) in excess of the remaining nucleic acid and performing second nucleic acid amplification using it as a template. increase method. In the present embodiment, at least two nucleic acid amplification steps of the "first nucleic acid amplification step" and the "second nucleic acid amplification step" are performed. In the second nucleic acid amplification step, a new amplification reagent (polymerase or dNTPs) is added to part or all of the reaction solution containing the amplified product of the first nucleic acid amplification step to perform a nucleic acid amplification reaction. Usually, the second nucleic acid amplification step is performed in a container different from the container used in the first nucleic acid amplification step. Embodiment...

Embodiment 1

[0090] [Example 1: Preparation of DNA fragments containing uracil bases]

[0091] (1) Preparation of PCR reaction solution

[0092] Prepare a PCR reaction solution with the following composition: 12.5μl 10×PCR buffer, 12.5μl 100mM MgCl 2 , 2, 5 μl, 1 μl of primers 1 and 2 (10 μM), 0.5 μl template (7 pg / μl), 89.5 μl distilled water, 2.5 μl dNTP (10 mM each: dUTP, dGTP, dATP, dCTP), 2.5 μl Taq DNA polymerase (Peqlab: cat01-1020). The template is 500 copies of mutated DNA. The base sequence of one strand of the template is represented by SEQ ID NO: 1, and the other strand that forms a double strand (KRAS DNA fragment containing dUTP) together with the strand of SEQ ID NO: 1 is represented by SEQ ID NO: 2. Primers 1 and 2 are represented by SEQ ID NO:3 and 4.

[0093] (2) PCR cycle

[0094] The PCR reaction was carried out with the following cycles: 94°C for 2 minutes, 94°C for 10 seconds → 68°C for 1 minute → 70°C for 10 seconds three times, 94°C for 10 seconds → 59°C for 1 ...

Embodiment 2

[0123] In the same manner as in Example 1, BEAMing was carried out using a KRAS DNA sample prepared so that the variation rate was 0% or 1%, or a sample obtained by adding uracil-containing DNA to the same sample.

[0124] DNA containing dUTP was prepared by the same method as in Example. Pre-PCR was carried out in 50 μl of PCR reaction solution. The composition of each reaction solution is as follows: Phusion U: 5×Phusin high-fidelity buffer, 0.8 μl of Phusion U (both NEB), 0.2 μM of primer 1, 0.2 μM of primer 2, 0.25 mM of each dNTP, and 0.5mM MgCl 2 ; KOD Plus neo: 10× buffer, 0.8 μl KOD Plus neo, 0.2 μM primer 1, 0.2 μM primer 2, 0.2 mM each dNTP, 1.5 mM MgSO 4 .

[0125] The PCR conditions were the same as in Example 1. The conditions from emulsion PCR to analysis by flow cytometry were also the same as in Example 1.

[0126] As a result, similar to the results of Example 1, when Phusion U was used in the pre-PCR, the mutation rate was abnormally higher than 1% under...

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Abstract

An aim of the invention is to provide a nucleic acid amplification method capable of simply suppress false positive caused by leftover. Disclosed is a nucleic acid amplification method used for solving the above problem. The nucleic acid amplification method is characterized by comprising the following steps: performing a first nucleic acid amplification using a target nucleic acid contained in asample as a template; and performing a second nucleic acid amplification using the amplification product produced in the first nucleic acid amplification step as a template. In the first nucleic acidamplification, a DNA polymerase that selectively amplifies a nucleic acid not comprising a uracil base is used to produce an amplification product that does not comprise a uracil base. In the second nucleic acid amplification, (i) dUTP and / or a primer comprising a uracil base, and (ii) a DNA polymerase capable of amplifying a nucleic acid comprising a uracil base are used to produce an amplification product that comprises a uracil base.

Description

【Technical field】 [0001] The present invention relates to nucleic acid amplification methods. 【Background technique】 [0002] Since the nucleic acid amplification device is repeatedly used, there is a problem that a false positive may occur due to the amplified product after nucleic acid amplification being mixed into the subsequent nucleic acid amplification reaction solution. This is generally called carryover, and the method described in Patent Document 1 is known as a technique for suppressing it. [0003] Patent Document 1 describes that an amplification product containing a uracil base is generated in a nucleic acid amplification step, and before the subsequent nucleic acid amplification, treatment with an enzyme that decomposes a nucleic acid containing a uracil base is performed, and the pre-decomposed nucleic acid A process method for amplifying an amplification product containing a uracil base. When DNA is used as a template, this method decomposes the uracil bas...

Claims

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Application Information

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IPC IPC(8): C12P19/34
CPCC12P19/34C12Q1/6844C12Q1/6858C12Q2521/101C12Q1/6848C12Q2525/101C12Q2537/149C12Q1/686
Inventor 能登谷伦崇山下秀治吉村美香前田丰
Owner SYSMEX CORP
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