Method and primer for detection of polymorphic site of seventh exon of WT1 gene

A polymorphic site and exon technology, applied in the fields of life science and biology, can solve the problems of single, low abundance of non-specific amplification products, achieve simple operation, optimal amplification efficiency, and improve amplification specific effect

Active Publication Date: 2014-04-09
JILIN ADICON CLINICAL LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Touch-down PCR amplification can ensure that the combination of the forward and reverse amplification primers and the sample DNA template occurs between the most complementary sequences. When the annealing temperature is reduced to the level where non-specific amplification occurs, the specific amplification product is At this time, there is already a geometric number of initial advantages, and the abundance is high. In the remaining amplification reactions, the specific amplification products compete with the non-specific amplification products, but due to the low abundance of non-specific amplification products , the specific amplification product is always preferentially amplified, resulting in a single dominant specific amplification product

Method used

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  • Method and primer for detection of polymorphic site of seventh exon of WT1 gene
  • Method and primer for detection of polymorphic site of seventh exon of WT1 gene
  • Method and primer for detection of polymorphic site of seventh exon of WT1 gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Primers and kits for detecting the polymorphic site of exon 7 of WT1 gene:

[0040] Primers for detecting the polymorphic site of exon 7 of WT1 gene, said primers include: forward and reverse amplification primers for amplifying the polymorphic site of exon 7 of WT1 gene, and its base sequence for:

[0041] WT1-7-F: GGATCTGGAGTGTGAATGG

[0042] WT1-7-R: TTCTAACACCAACACGATTC.

[0043] Preferably, the primers also include a pair of sequencing primers, the base sequence of which is:

[0044] WT1-7-S-F: GGATCTGGAGTGTGAATGG

[0045] WT1-7-S-R: TTCTAACACCAACACGATTC.

[0046] A kit for detecting the polymorphic site of exon 7 of WT1 gene, the kit includes sample DNA extraction reagent, absolute ethanol, detection system PCR reaction solution, sequencing system reaction solution, positive control substance, negative Control substance and blank control substance, the detection system PCR reaction solution includes 2×PCR Buffer, dNTPs, KOD FX DNA Polymerase, ddH 2 O and a p...

Embodiment 2

[0063] Detection process:

[0064] (1) Use the blood DNA extraction kit (Tiangen Biology) to extract tissue DNA from blood:

[0065] 1) Take 500uL of blood and add 1000uL of red blood cell lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 3000rpm for 5min, suck off the supernatant, leave the white blood cell pellet, add 200uL buffer GA, shake until thoroughly mixed.

[0066] 2) Add 20 μl proteinase K solution and mix well.

[0067] 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0068] 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0069] 5) Add the solution and flocculent precipi...

Embodiment 3

[0096] The detection kit of the present invention is used to detect clinical samples.

[0097] Twenty cases of anticoagulated blood samples from patients with acute myeloid leukemia (AML) were taken for inspection, and the genomic DNA in the samples was extracted, reagents were prepared, electrophoresis and sequencing were performed according to the detection process described in Example 2.

[0098] Add 2 ul of the genomic DNA solution of each sample extracted according to the detection process described in Example 2 into the PCR reaction solution of the detection system. Do positive, negative and blank controls at the same time. A 96-well ordinary PCR machine can detect 46 samples at the same time. Each sample is repeated twice, one positive control, one negative control and one blank control. The detection time is 160 minutes.

[0099] After each sample has been sequenced twice, the mutation status will be compared, and a third sequence will be performed for samples with in...

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Abstract

The invention discloses a method and primers for detection of a polymorphic mutation site of a seventh exon of a WT1 gene. The primers comprise forward and reverse amplification primers for amplification of mutational sites of the seventh exon of the WT1 gene and a pair of sequencing primers. Touch-down PCR amplification and Sanger sequencing method are combined to accurately and specifically detect mutation of the seventh exon of the WT1 gene of patient with leukemia, especially acute myeloblastic leukemia.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and relates to a method and primers for detecting the polymorphic site of the seventh exon of WT1 gene. Background technique [0002] The WT1 gene is the first gene found to be associated with the occurrence and development of Wilms tumor. It is about 50,000 bp in length and has 10 exons in total. The loss of function of its allele plays an important role in the occurrence of Wilms tumor. The protein encoded by the gene is a zinc finger protein with a relative molecular mass of 52000-54000, and it is also a bidirectional transcriptional regulator. In recent years, it has been found that the WT1 gene is abnormally highly expressed in leukemia, especially in acute myeloid leukemia (AML), and the expression level is related to prognosis, and high expression may lead to poor prognosis. In addition, several studies have shown that WT1 mutations have a similar effect as its high expressio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2535/101C12Q2531/113
Inventor 王淑一宋坤孙翠莲
Owner JILIN ADICON CLINICAL LAB
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