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Primer, kit and method for detecting APC gene mutation

A kit and reagent technology, applied in the field of primers and kits for detecting APC gene mutation, can solve the problems of tediousness, inability to do prenatal diagnosis, time-consuming, etc. Effect

Inactive Publication Date: 2018-10-16
上海浦东解码生命科学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These clinical detection methods are time-consuming and cumbersome, and are only applicable to babies who have already been born, and are powerless for prenatal diagnosis

Method used

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  • Primer, kit and method for detecting APC gene mutation
  • Primer, kit and method for detecting APC gene mutation
  • Primer, kit and method for detecting APC gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] A test kit for detecting APC gene mutation in familial adenomatous polyposis, comprising the following components:

[0079] (1) Perform PCR amplification on the 16 exon coding regions of the APC gene. Since the No. 16 exon is relatively large, 31 pairs of primers (nucleotide sequences shown in sequence as SEQ ID NO.1-62) were designed for Amplify.

[0080] (2) PCR amplification reagents: 10×PCR bμffer, dNTP, Takara LA Taq enzyme;

[0081] (3) PCR product purification reagent: ordinary agarose gel DNA recovery kit (spin column type) (Tiangen, DP209);

[0082] (4) Sequencing reagents: 2.2 μL BigDyemix (Bigdye, 5×seq), 2.5 μL EDTA, 30 μL ethanol solution (100%), 100 μL ethanol solution (70%), 8 μL Hi-Di.

[0083] This kit is stored at -20°C, and repeated freezing and thawing should be avoided as much as possible.

[0084] All of the above primer pairs can amplify the APC gene sequence shown in NCBI Reference Sequence: NM_000038.5.

Embodiment 2

[0086] A test kit for detecting the APC gene mutation of familial adenomatous polyposis, the steps for using it include the following steps:

[0087] (1) Sample genomic DNA extraction

[0088] The genomic DNA of the samples was extracted using a blood genomic DNA extraction kit (0.1-1 ml) (Tiangen, DP318).

[0089] (2) Different primer pairs are used for PCR amplification of APC gene

[0090] Primers (as shown in SEQ ID NO.1-62) were designed for the APC gene for PCR amplification. The 15 μL PCR reaction system includes: 1.5 μL of 10×PCR bμffer, 2.4 μL of dNTPs, 0.15 μL of Takara LA Taq enzyme, 0.3 μL of upper and lower primers (10 μM), 0.5 μL of genomic DNA (100 ng / μL), and ddH 2 O complements the reaction system.

[0091] The PCR reaction program was: 94°C pre-denaturation for 15 minutes; 94°C denaturation for 30 seconds, 62°C annealing for 30 seconds, 72°C extension for 45 seconds, 14 cycles; 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension...

Embodiment 3

[0103] APC gene amplification primer screening experiment, including the following primer sequences and reagents:

[0104] (1) There are 8 pairs of primers for the coding region of exons 5, 8 and 16 of the APC gene, and the nucleotide sequences thereof are shown in SEQ ID NO.63-78.

[0105] (2) PCR amplification reagents: 10×PCR bμffer, dNTP, Takara LA Taq enzyme;

[0106] (3) PCR product purification reagent: ordinary agarose gel DNA recovery kit (spin column type) (Tiangen, DP209);

[0107] (4) Sequencing reagents: 2.2 μL BigDyemix (Bigdye, 5×seq), 2.5 μL EDTA, 30 μL ethanol solution (100%), 100 μL ethanol solution (70%), 8 μL Hi-Di.

[0108] APC gene amplification primer screening experimental steps include:

[0109] (1) Sample genomic DNA extraction, same as in Example 2.

[0110] (2) APC gene PCR amplification, the same as in Example 2.

[0111] (3) Purification of the PCR product is the same as in Example 2.

[0112] (4) DNA sequencing reaction, same as in Example 2...

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Abstract

The invention relates to the field of detection of mutant genes, in particular to a primer, a kit and a method for detecting APC gene mutation. The primer for detecting the APC gene mutation comprisesany one pair of or multiple pairs of the following primer pairs, wherein the nucleotide sequences of the primer pairs 1 to 31 are as shown in SEQ ID NO. 1 to 62 in sequence. The primer for detectingthe APC gene mutation, which is provided by the invention, is obtained by screening through a design considering multiple aspects. During sequencing of a primer amplified product, the peak shape is tidy, the peak figure is intact, and the quality is high. By the APC gene detection for people (particularly newborns), populations having virulence genes can be found early, and a scientific basis is provided for diagnosis and treatment of late diseases, which contributes to taking measures in the early stage to prevent increase of polypus and avoid cancerization, so that the cancerization rate offamilial adenomatous polyposis is effectively decreased.

Description

technical field [0001] The invention relates to the field of mutation gene detection, in particular to a primer, a kit and a method for detecting APC gene mutation. Background technique [0002] Familial adenomatous polyposis (familial adenomatous polyposis, FAP) is called multiple polyposis, is an autosomal dominant genetic disease, the disease occurs in the colon adenomatous polyposis gene (adenomatoμspolyposis coli, APC) mutations are closely related, and more than 80% of FAP patients can detect APC gene mutations. The global incidence of FAP is 1 / 7000 to 1 / 10000 births. [0003] FAP has an equal impact on gender, the ratio of male to female is 1:1, and most cases show a high degree of autosomal dominant inheritance, which conforms to the law of Mendelian inheritance, with a penetrance rate of 70% to 95%, and sporadic cases account for about 1 / 3. Adult FAP patients will have a family history of their parents, and half of their children will suffer from FAP, involving s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 梅艳巧陈培华谢桂贞郭岳
Owner 上海浦东解码生命科学研究院
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