Primers, method and kit for detecting secreted frizzled-related protein (SFRP)2 gene CpG island methylation

A technology of SFRP2-F and SFRP2-R, which is applied in the fields of life science and biology, can solve the problem of high probe ordering costs and achieve high accuracy and good specificity

Active Publication Date: 2014-06-25
郑州艾迪康医学检验所(普通合伙)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ordering cost of the probe is relatively high, and it is su

Method used

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  • Primers, method and kit for detecting secreted frizzled-related protein (SFRP)2 gene CpG island methylation
  • Primers, method and kit for detecting secreted frizzled-related protein (SFRP)2 gene CpG island methylation
  • Primers, method and kit for detecting secreted frizzled-related protein (SFRP)2 gene CpG island methylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Primers and kits for detection of methylation of SFRP2 gene CpG island

[0044] Primers for detecting methylation of SFRP2 gene CpG islands, including specific primers for amplifying SFRP2 gene fragments from sample nucleic acid and primers for pyrosequencing the obtained nucleic acid fragments, wherein,

[0045] Specific primers for amplifying the CpG island DNA fragment of the gene SFRP2 gene from the sample nucleic acid, the base sequence of which is:

[0046] SFRP2-F:Biotin-TGGGTTAAGAGATTATGAAGGAGGTGTTG

[0047] SFRP2-R:CTCAACCCAACAAAAAATAAAAAAAACAA

[0048] Wherein SFRP2-F is the 5' end biotin-labeled primer

[0049] The obtained nucleic acid amplification fragment is subjected to pyrosequencing primers, and its base sequence is:

[0050] SFRP2-S: AATATCCCAATAACACTACTTCAT (reverse).

[0051] Using pyrosequencing technology, through specific amplification primers SFRP2-F and SFRP2-R and pyrosequencing primers, the CG methylation of CpG islands related...

Embodiment 2

[0062] Example 2: DNA Extraction

[0063] DNA extraction reagents: stool suspension, stool DNA extraction kit (TIANGEN company).

[0064]Operation steps: Weigh 180-220mg of stool sample into a 2ml centrifuge tube, place the tube on ice and add 0.5ml of stool suspension, then add 1.4ml buffer GSL to the sample, shake intermittently for 1 minute until the sample is well mixed , incubate at 70°C for 5 minutes, vortex for 15 seconds, and centrifuge at 13,000 rpm for 1 minute. Transfer 1.2ml of the supernatant to a new 2ml centrifuge tube, add an inhibitor adsorption sheet InhibitEX, oscillate until the adsorption sheet is completely opened and resuspended, incubate at room temperature for 1 minute, so that the adsorption sheet can fully function, then centrifuge at 13000rpm for 3 minutes, and the obtained Transfer the supernatant to a new 1.5ml centrifuge tube, centrifuge at 13000rpm for 3 minutes, transfer the obtained supernatant 200μl to a new 1.5ml centrifuge tube, add 15μl p...

Embodiment 3

[0065] Embodiment 3: DNA sodium bisulfite treatment

[0066] After DNA was treated with sodium sulfite, the unmethylated cytosine in the CpG dinucleotide pair was converted to thymine (C→T), while the methylated 5'methylcytosine was not affected by sodium sulfite treatment, Still 5'methylcytosine.

[0067] 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 30ul10mM 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...

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Abstract

The invention discloses primers, method and kit for detecting secreted frizzled-related protein (SFRP)2 gene CpG island methylation. The primers include specific primers for amplifying deoxyribonucleic acid (DNA) segments of three CG loci (related to intestinal cancer) on an SFRP2 gene CpG island from a sample nucleic acid, and primers for pyrosequencing the obtained nucleic acid segments. By the primers, the method and the kit disclosed by the invention, the frequency of the SFRP2 gene methylation related to colorectal cancer susceptibility can be detected by adopting a pyrosequencing technique, so as to screen out susceptible people with early colorectal cancer. The primers, the method and the kit are good in specificity, and high in accuracy, and can detect samples at high flux.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to a method, a primer and a kit for detecting the methylation site of the SFRP2 gene. Pyrosequencing technology can be used to detect the methylation frequency of the SFRP2 gene, and the specificity is good. , with high accuracy, and can perform high-throughput screening of methylated samples. Background technique [0002] Colorectal cancer is the third most common tumor in the world, with nearly 1 million new cases and about 492,000 deaths from colorectal cancer every year. But colorectal cancer incidence and mortality have declined over the past decade, possibly due to effective screening and surveillance. It takes an average of 10 years for colonic epithelial cells to develop from abnormal hyperplasia through adenoma to cancer, which provides sufficient time for early screening and prevention. Colorectal cancer is the easiest to treat in the early stage...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 王淑一李文静周晓犊
Owner 郑州艾迪康医学检验所(普通合伙)
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