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SFRP1 gene promoter methylation detection primers and detection method thereof

A methylation and promoter technology, applied in the fields of life science and biology, can solve the problems of high instrument requirements, high probe cost, cumbersome operation, etc., and achieve cost saving, good detection stability, and high detection rate Effect

Inactive Publication Date: 2016-10-05
杭州艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the MSP method uses PCR amplification detection to determine whether there is methylation in the sample. This method is practical and common, but it has high false positives and poor stability; bisulfite pyrosequencing is not suitable because of its cumbersome operation. Mass detection; MS-HRM is to judge whether there is methylation by converting the difference of single base sequence into the difference of melting curve. This method has high requirements on the instrument, and a fluorescent quantitative PCR instrument with HRM module is required , and at the same time, this method can only analyze the overall methylation status of the detection fragment, but cannot clarify the methylation status of each CG site; MS-PCR is a technology developed based on PCR, mainly because TaqMan probes are added to the detection. This ensures high sensitivity and accuracy, but it can only do overall analysis for the detection of multiple methylation sites. At the same time, the cost of probes is high, so this method is more suitable for a small number of sites and a large number of sites. sample testing

Method used

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  • SFRP1 gene promoter methylation detection primers and detection method thereof
  • SFRP1 gene promoter methylation detection primers and detection method thereof
  • SFRP1 gene promoter methylation detection primers and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0034] The SFRP1 gene promoter region CG island methylation detection kit includes a pair of specific degenerate primers SEQ NO 1 and SEQ NO 2. At the same time, this primer is a sequencing primer, and its base sequence is as follows:

[0035] SEQ NO 1: 5'-CGGTGACGGACGTGGTAAYGAG-3';

[0036] SEQ NO 2: 5'-CGACTCCCGAAAATACGACRAACA-3';

[0037] SFRP1 gene promoter region CG island methylation detection kit also includes the following reagents:

[0038] (1) Sulfite treatment reagents: Bisulfite Mix, RNase free water, DNA Protect Buffer;

[0039] (2) PCR amplification reagents: 10*PCR Buffer, 2mM dNTP, 5*Q Solution Buffer, 5U / ul Taq enzyme, 10uM amplification primers (SEQ NO1, SEQ NO 2) and sterilized water;

[0040] (3) Enzyme purification reagents: EXOI, CIP;

[0041] (4) Sequencing reagents: Bigdye Terminator V3.1, absolute ethanol, EDTA, and sequencing primers (SEQ NO 1, SEQ NO 2).

[0042] (5) Positive quality control product: the methylated DNA of the CG island in the promo...

Embodiment 2

[0044] DNA extraction reagents and methods.

[0045] Take 5-8 paraffin sections (5-10μm thick, 1×1cm2 size), collect the paraffin-embedded tissue into a 1.5mL centrifuge tube, and centrifuge briefly; add 1mL tissue clear solution, shake fully, centrifuge at 13200rpm for 1min, remove the upper Supernatant solution: add 0.5mL tissue clear solution, shake fully, centrifuge at 13200rpm for 1min, remove supernatant; add 1mL ethanol (96-100%), shake vigorously, centrifuge at room temperature for 2min at 13200rpm, remove supernatant; open the lid, Incubate at room temperature (15-25°C) or in a metal bath at 37°C until the remaining ethanol is completely evaporated; take 180 μL Buffer ATL to resuspend the tissue in the tube, then add 20 μL proteinase K, and shake again; place the centrifuge tube containing the mixture in On a metal bath at 56°C, incubate for 1 hour, invert and mix 3 times until the sample is completely dissolved; transfer the centrifuge tube to a metal bath at 90°C, a...

Embodiment 3

[0047] The sulfite treatment reagents and methods are as follows.

[0048] Operation steps: dilute the genomic DNA extracted in Example 2 and 1.5ml EP tube to 50ul with double distilled water, add 5.5ul freshly prepared 3M NaOH solution, and bathe in water at 42°C for 30min; prepare the following solution during the water bath: 10mM hydroquinone solution (Hydryquinone) : Dissolve 55mg hydroquinone in 50ml water; 3.6mol / LNaHSO 3 : Take 18.8g NaHSO 3 Add 40ml of water, adjust the pH of the solution to 5.0 with 3M NaOH solution, and finally dilute to 50ml. Add 30ul 10mM hydroquinone solution and 520ul3.6mol / LNaHSO 3 Put the EP tube into the solution after the water bath, wrap the EP tube with aluminum foil to avoid light, gently invert the solution, add 100ul paraffin oil to the tube, and centrifuge briefly to cover the liquid surface with paraffin oil to prevent water evaporation during the water bath and limit oxidation by 50 ℃ Protect from light and water bath for 16 hours;...

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Abstract

The invention discloses SFRP1 gene promoter multi-CG-locus methylation detection primers and a detection method thereof. The method comprises the following steps: (1) forward and reverse degenerate primers SEQ NO 1 and SEQ NO 2 in the SFRP1 gene promoter region are amplified; (2) the target sequence amplified by the primers comprise at least 7 CG loci; and (3) as for the problems of severe paraffin section DNA fragmentation and low effective template concentration in the bisulfite modification saturated state, Touch-down PCR amplification and Sanger sequencing are combined to obviously enhance the detection sensitivity. The primers and method have the advantages of high specificity, high accuracy, low risk of contamination and the like and can detect multi-CG-locus methylation in the SFRP1 gene promoter region of the tumor patient surgery paraffin section; and the result can instruct the doctors to perform prognosis evaluation on related diseases.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and relates to a primer and a detection method for detecting the methylation state of the SFRP1 gene promoter. Background technique [0002] The SFRP1 gene is located on chromosome 8p12-11.1 and encodes a protein of about 30KDa. It is involved in the formation and cell differentiation of the eye, brain, and vascular system during embryonic development. It is also mainly expressed in the choroid of the brain, retina of the eye, lens, and ciliary Body, vascular endothelium and other tissues, participate in the metabolism and function of these tissues. As a secreted upstream inhibitor of Wm signaling, SFRP1 is located outside the cell and contains a CRD (cysteine-rich domain) domain, whose amino-terminal sequence is similar to that of Frz, and its mechanism of action may be to compete with Frz for binding Wnt Protein or combined with Frz into a non-functional complex, thereby blocking ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/118C12Q2600/154C12Q2523/125C12Q2535/101C12Q2531/113
Inventor 林有升单战王淑一
Owner 杭州艾迪康医学检验中心有限公司
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