Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis

A kit and prognostic technology, applied in the field of genetic engineering, can solve the problems that cannot really meet the clinical diagnosis and detection, complicated operation and high cost of fluorescence in situ hybridization technology, and achieve the effects of intuitive detection, low cost and strong primer specificity.

Inactive Publication Date: 2016-08-17
SHANGHAI TISSUEBANK BIOTECH +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Among the currently widely used molecular biology methods for detecting leukemia gene mutations at home and abroad, fluorescence in situ hybridization (FISH) can only perform qualitative detection, and the operation is complicated; fluorescent quantitative PCR has limitations in

Method used

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  • Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis
  • Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis
  • Primers, kit and method for detecting gene mutation related to AML (acute myeloid leukemia) prognosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Primers designed to detect gene mutations associated with AML prognosis

[0033]According to the sequence of each mutated gene related to AML prognosis published by GenBank, primers were designed using Primer Premier5.0 primer design software, and the designed primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. At the same time, in order to reduce the bottom peak of the sequencing peak map, the purity of the sequencing primers should be guaranteed to the greatest extent. The specific primers designed in the present invention are shown in Table 1.

[0034] Table 1, the present invention detects the primer situation table of the gene mutation relevant with AML prognosis

[0035]

[0036]

[0037] *The CEBPA gene has only one exon, and the numbers here represent the number of primers and not the number of amplified exons.

[0038] The primers designed in the present invention are not only aimed at the hotspot mutation regions of FLT3 a...

Embodiment 2

[0041] Example 2: Detection of gene mutations associated with AML prognosis in samples

[0042] (1) Reagents and materials

[0043] 1. Detection system PCR reaction solution: 10×PCR Buffer, dNTPs (2.5mM), LA Taq DNA Polymerase, ddH 2 O et al.

[0044] 2. Sequencing system:

[0045] (1) PCR product digestion reaction solution: shrimp alkaline phosphatase (Alkaline Phosphatas (Shrimp)) and exonuclease (Exonuclease I), 1:1 mixed;

[0046] (2) Purification solution for sequencing: EDTA (125 mmol), 85% absolute ethanol, 75% absolute ethanol, HIDI (highly deionized formamide);

[0047] (3) Sequencing reaction solution: Terminator V3.1 cycle sequencing Kit.

[0048] (2) Workflow

[0049] 1. PCR amplification

[0050] The preparation of the detection system PCR reaction solution is shown in Table 2.

[0051] Table 2 Preparation table of detection system PCR reaction solution

[0052] Element

Volume (ul)

10×PCR buffer

2.5

dNTPs (2.5mM)

3.0

...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses a primer combination for detecting gene mutation related to AML (acute myeloid leukemia) prognosis, a kit containing the primer combination and a method for detecting gene mutation related to AML prognosis. The primer combination covers all hotspot mutant sites of FLT3, NPM1, DNMT3A and CEBPA genes, and has the advantages of high specificity and wide covering range. The annealing temperatures of all the primers are close, so that the amplification stage can be completed at one time by one procedure, and the hotspot mutation can be subjected to combined parallel detection; and the detection efficiency is high. Besides, the forward and reverse amplification primers are adopted to directly carry out sequencing, and software is utilized to directly carry out mutation analysis, so that the method has the advantages of visual detection and low cost, thereby providing important references for therapeutic scheme determination and prognosis judgment of AML.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, kits and methods for detecting gene mutations related to AML prognosis. Background technique [0002] Acute myeloid leukemia (AML) is a group of malignant blood diseases originating from hematopoietic stem cells with high heterogeneity. At present, among the many factors for evaluating the risk of prognosis in AML, cytogenetics is considered as an important independent prognostic factor, and some specific karyotype abnormalities are often considered as markers for guiding treatment and judging prognosis. However, there are still a considerable number of patients with undetectable karyotype abnormalities, and these patients have large variations in both treatment response and prognosis evaluation. Due to the rapid development of molecular biology techniques, multiple molecular markers related to AML have been identified one after another, which fully proves t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156
Inventor 郑仲征杜金伟安雪茹郁晓晨徐郁尚
Owner SHANGHAI TISSUEBANK BIOTECH
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