CN104762301B - A kind of kit and method for detecting liver cancer risk genes TSPYL5 methylation levels - Google Patents

Abstract

The invention discloses a kit and a method for detecting the methylation level of liver cancer risk gene TSPYL5 , belonging to the field of biotechnology. The kit for detecting the methylation level of the TSPYL5 gene includes primer pair A for the MSRE-qPCR method or primer pair B for the BSP method, and the sequences of these primers are shown in SEQ ID NO.1-22. The present invention discovers the correlation between the methylation of TSPYL5 gene and the occurrence of liver cancer, and identifies the methylation site of TSPYL5 . The present invention adopts specific methylation-sensitive restriction endonuclease combined with specific primers to establish a method for detecting methylation of TSPYL5 by fluorescent quantitative PCR. The MSRE-qPCR-based kit and method of the present invention detect the methylation level of TSPYL5 , with a sensitivity as high as 83.9% and a specificity as high as 93.25%.

Description

A kit and method for detecting methylation level of liver cancer risk gene TSPYL5

technical field

The invention relates to the field of biotechnology, in particular to a kit and method for detecting the methylation level of liver cancer risk gene TSPYL5 .

Background technique

DNA methylation is one of the main contents of epigenetics research. It studies the reversible genetic changes that occur without changing the base sequence of the gene. It is a covalent modification method after DNA molecule replication. In eukaryotes, DNA methylation refers to the transfer of methyl groups to specific bases in DNA molecules using S-adenosylmethionine as a methyl donor under the action of DNA methyltransferases (DNMTs) process, which mainly occurs on cytosines in CpG dinucleotides. In the genome, the regions where the distribution of CpG dinucleotides is relatively concentrated are called CpG islands (CpG islands, CGIs), ranging in size from 100 to 1000 bp, mainly located in the promoter region and the first exon region of genes; the abnormality of CpG islands Methylation can directly lead to silencing of related gene expression. DNA-specific methylation patterns are of great significance for maintaining genome stability and correct gene expression in time and space. Abnormal changes in methylation patterns will directly participate in the occurrence of human diseases and even cancer.

Methylation-sensitivity restriction enzyme digestion and PCR (MSRE-PCR) is based on the property that methylation-sensitivity restriction enzymes do not cut methylated sites. , DNA was digested into fragments of different sizes and then PCR amplified, and the methylation status was analyzed according to the difference of electrophoresis products. See Figure 1 for the specific principle. After digestion with a restriction endonuclease sensitive to the methylation sequence of a specific DNA fragment, PCR is performed with the sequence outside the methylation site to be tested as the starting point of amplification. If the DNA fragment is methylated, there will be amplification products; if there is no methylation, no fragment amplification will appear. When a methylation-insensitive endonuclease digestion product is used as a PCR template, no fragments should amplify regardless of whether the site is methylated or not.

Bisulfite sequencing PCR (BSP) method, after the extracted DNA is modified by bisulfite, the unmethylated cytosine is deaminated and converted into uracil, while the methylated cytosine remains constant. All uracils were converted into thymines by PCR. PCR products were gel-cut and purified after agarose electrophoresis, then connected to the pMD18-T vector, transformed into Escherichia coli, and after overnight culture, single positive clones were picked to extract plasmids for sequencing, and the distribution map of methylation sites in specific regions of each DNA molecule was obtained. Thus, the methylation status of each CpG site in the region can be obtained. This method is a highly reliable and accurate method for detecting the methylation status, and can clarify the methylation status of each CpG site in the target fragment. In searching for meaningful key CpG sites, there are advantages unmatched by other methods. However, this method consumes time, money, and energy. At least 10 or more clones must be sequenced to obtain reliable data. A large number of clones and plasmid extraction and sequencing are required, and the process is cumbersome and expensive. Therefore, it is difficult to achieve high-throughput automated detection in the clinic, but it can be used as a comparison method for new methods of methylation detection.

This kit intends to establish a method based on methylation-sensitive restriction endonuclease combined with fluorescent quantitative PCR (MSRE-qPCR), and change the subsequent detection of MSRE-PCR method to fluorescent quantitative PCR, which can quantitatively detect specific genes in trace DNA samples The methylation level of the site. The method is easy to use, does not require bisulfite treatment, and has good compatibility with paraffin sections.

Contents of the invention

Based on this, the purpose of the present invention is to provide a kit for detecting the methylation level of the liver cancer risk gene TSPYL5 with high sensitivity and good specificity.

The object of the present invention is achieved through the following technical solutions:

A fluorescent quantitative PCR method for detecting TSPYL5 gene methylation, comprising the following steps: ① extracting the genomic DNA of a sample to be tested; ② using a methylation-sensitive restriction endonuclease to digest the genomic DNA; ③ using Primer pair A was used to perform fluorescent quantitative PCR amplification on the digested product; ④ Analyze the Ct value of the PCR product; ⑤ Determine the methylation level of the TSPYL5 gene in the sample based on the analysis results.

Among them, the primer pair A in step ③ is preferably one of the following four pairs of primers (the length of the amplified fragment of the primer pair is indicated in parentheses):

AF1: 5'-CCCCGCGAGCGCATATCAGAG-3' (176bp),

AR1: 5'-GCAACCGCCGACGTCACGAAC-3';

AF2: 5'-ATATCAGAGAAACTCGCCGAG-3' (150bp),

AR2: 5'-CACGAACGTACAACTGTACCG-3';

AF3: 5'-CAGAGAAACTCGCCGAGACCTA-3' (231bp),

AR3: 5'-TTCAAAGACACGCTGTGACCCT-3';

AF4: 5'-AGCGCATATCAGAGAAACT-3' (140bp),

AR4: 5'-GTACCGTCGCGAGAGGACGTGA-3'.

The above primers were designed by the following method: Obtain the CpG island sequence of the TSPYL5 gene from the UCSC database, analyze the restriction site in the sequence, and use the online primer design software (http://simgene.com/Primer3) primerpremier 3.0 to design including 2 -PCR primers with 6 restriction sites. After repeated tests, screening and verification, primers with high amplification efficiency, good specificity, and the ability to detect the methylation level of the TSPYL5 gene in samples were obtained.

A kit A for detecting the methylation level of TSPYL5 gene based on methylation-sensitive restriction endonuclease combined with fluorescent quantitative PCR, comprising the above-mentioned primer pair A.

The kit A also includes methylation-sensitive restriction endonucleases.

The kit A also includes a methylation negative control and a positive control. Preferably, negative controls include normal human peripheral blood DNA, commercial unmethylated human genomic DNA controls; positive controls include commercial fully methylated human genomic DNA, confirmed target gene fragments fully methylated HCC cell line DNA.

The kit A also includes SYBR Green fluorescent dye for fluorescent quantitative PCR and the like.

The method for using the above kit A includes the following steps: ① extracting the genomic DNA of the sample to be tested; ② digesting the genomic DNA with a methylation-sensitive restriction endonuclease; Quantitative PCR amplification; ④ Analyze the Ct value of PCR products.

The enzyme digestion system is preferably: 300ng of DNA, 5 μL of Buffer (10×), 30 U of methylation-sensitive restriction endonuclease, and supplemented with ddH ₂ O to 50 μL.

The reaction system for PCR amplification is preferably: 10 μL of SYBR Mix (2×), 0.5 μL of upstream primer (F), 0.5 μL of downstream primer (R), 7 μL of ddH ₂ O, and 2 μL of digested product.

The reaction conditions of the PCR amplification are preferably: 95° C. for 5 minutes; 95° C. for 30 s, 61° C. for 30 s, 72° C. for 30 s, and 35-40 cycles.

The kit A of the present invention based on methylation-sensitive restriction endonuclease combined with fluorescent quantitative PCR can be divided into two parts: a detection system and a monitoring system. The detection system includes the above-mentioned primer pair A, which can be formed by any combination. The monitoring system includes: ① Negative controls include two types, the first is a normal human genomic DNA template that has been confirmed to be unmethylated in the region to be tested; the second is a commercial unmethylated human genomic DNA control . Under normal circumstances, the methylation level of all negative controls should be close to 0, otherwise it indicates that the sample addition process is contaminated or the enzyme digestion is incomplete. ②Positive controls are commercialized fully methylated human genomic DNA, and verified DNA of liver cancer cell line with fully methylated target gene fragments; under normal reaction, the methylation level of the positive control should approach 1, otherwise the experimental fail.

Another BSP method for detecting TSPYL5 gene methylation comprises the following steps: ① extracting the genomic DNA of the sample to be tested; ② using sodium bisulfite to modify the genomic DNA; ③ using primer pair B to modify the DNA Perform PCR amplification; ④PCR product cloning and sequencing to analyze the methylation status of each CG site.

Wherein, the primer pair B in step ③ includes an outer primer (W) and an inner primer (N), wherein the outer primer is one of the following 3 pairs:

BWF1: 5'-TAAGAGATAATTGGAGGA-3' (465bp),

BWR1: 5'-ACCTTTACCCCCGATTTTTA-3';

BWF2: 5'-ACGTTCGAGTATTTTTTTTA-3' (498bp),

BWR2: 5'-GACCTTTACCCCAATTTTTA-3';

BWF3: 5'-AGATAATTGGAGGAGTTGAAGA-3' (526bp),

BWR3: 5'-TACTATAAAAAATCCGAATCGC-3';

The inner primer is one of the following 4 pairs of primers:

BNF1: 5'-AATAGGTGATGGGGGATAGGT-3' (376bp),

BNR1: 5'-CCGCTCATAATAACGACGAAA-3';

BNF2: 5'-AGAAAATAGGTGATGGGGGA-3' (383bp),

BNR2: 5'-CGACCGCTCATAATAACGAC-3';

BNF3: 5'-TTAGAAAATAGGTGATGGGGGATAG-3' (373bp),

BNR3: 5'-ATAACGACGAAAACAACTTCAAAAA-3';

BNF4: 5'-AATAGGTGATGGGGGATAG-3' (378bp),

BNR4: 5'-GACCGCTCATAATAACGAC-3'.

The above primers were designed by the following method: the CpG island sequence of the TSPYL5 gene was obtained from the UCSC database, and the primers used in the BSP method were designed using the online methylation primer design software (http://www.urogene.org/methprimer/) MethPrimer. After repeated tests, screening and verification, BSP primers with high amplification efficiency, good specificity, and the ability to detect the methylation level of the TSPYL5 gene in samples were obtained.

A kit B for detecting the methylation level of the TSPYL5 gene based on sodium bisulfite modified PCR, comprising the above-mentioned primer pair B.

The kit B also includes a methylation negative control and a positive control. Preferably, the negative control includes normal human peripheral blood DNA, commercial non-methylated human genomic DNA control; the positive control includes commercial methylated human genomic DNA, confirmed target gene fragments fully methylated liver cancer cells Department of DNA.

The kit B also contains PCR reagents (dNTPs, DNA polymerase, DNA polymerase buffer, etc.) and reagents required for the sodium bisulfite modification process (sodium bisulfite, hydroquinone, sodium hydroxide, ammonium acetate, glycogen, etc.).

The method for using the above kit B includes the following steps: ① extract the genomic DNA of the sample to be tested; ② use sodium bisulfite to modify the genomic DNA; ③ use the primer pair B to perform PCR amplification on the modified DNA (using the outer Primers and internal primers for nested PCR amplification); ④PCR product cloning and sequencing to analyze the methylation status of each CG site.

The reaction system for PCR amplification is preferably: 5 μL of Buffer (10×), 1 μL of dNTPs (10 mM), 4 μL of MgCl ₂ (25 mM), 2 μL of Taq (1U/μL), 1 μL of upstream primer (F), 1 μL of downstream primer ( R) 1 μL, ddH ₂ O 32 μL, DNA template 4 μL.

The reaction conditions of the outside and inside of the nested PCR amplification are preferably: 95°C, 5min; 95°C, 30s, 68°C-56°C, 2cycles/2°C, 45s, 72°C, 45s, the annealing temperature is reduced to 25cycles at 56°C; 10min at 72°C.

The present invention discovers the correlation between the methylation of TSPYL5 gene and the occurrence of liver cancer, and identifies the methylation site of TSPYL5 , and establishes a fluorescent quantitative PCR detection method in combination with methylation-sensitive restriction endonucleases and specific primers . And then adopt the method of BSP to verify this conclusion.

The sensitivity of the TSPYL5 gene methylation detection kit of the present invention is as high as 80.37%, and the specificity is as high as 93.25%.

The method utilizes the fluorescence quantitative PCR method to detect the DNA methylation, which greatly improves the detection efficiency and reduces the detection cost. The test results are simple, intuitive, and high-throughput, allowing many samples to be tested at one time.

Description of drawings

Figure 1 is the schematic diagram of MSRE-PCR; in the figure, CH3 represents methylation, and enz represents methylation-sensitive restriction endonuclease; the DNA on the left is not methylated, the DNA is cut, and the PCR product cannot be obtained; the right figure DNA is methylated, DNA remains intact, and PCR products can be obtained.

Fig. 2 is the amplification curve (a) and the melting curve (b) figure of negative control in embodiment 1, and wherein gray and black line represent respectively add enzyme and do not add enzyme system, two duplicate wells of each sample; RFU represents expansion The fluorescence value of the increase reaction; -d(RFU)/dt represents the negative first derivative of the change of the fluorescence signal.

Fig. 3 is positive control amplification curve (a) and melting curve (b) figure in embodiment 1, and wherein gray line and black line represent adding enzyme and not adding enzyme system respectively, each sample has two duplicate wells; RFU represents amplification The fluorescence value of the reaction; -d(RFU)/dt represents the negative first derivative of the change in the fluorescence signal.

Fig. 4 is a scatter diagram of detection of TSPYL5 gene methylation levels in liver cancer and para-cancerous tissues by MSRE-qPCR in Example 1.

FIG. 5 is a ROC curve for distinguishing liver cancer and paracancerous tissues by TSPYL5 gene methylation level in Example 1. FIG.

Fig. 6 is a box diagram of detection of TSPYL5 gene methylation levels in liver cancer and para-cancerous tissues by BSP cloning sequencing method in Example 2.

7 is a bar graph showing the methylation of each CpG site of the TSPYL5 gene in liver cancer and paracancerous tissues in Example 2.

detailed description

In order to understand the technical content of the present invention more clearly, the following examples are given in detail. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

For the experimental methods not specified in the following examples, generally follow conventional conditions, such as Sambrook et al., Molecular Cloning: The conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacture conditions recommended by the manufacturer. Various commonly used chemical reagents used in the examples are all commercially available products. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the technical field of the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments, and are not used to limit the present invention.

Example 1 TSPYL5 gene methylation fluorescent PCR detection of 163 pairs of liver cancer and paracancerous tissue samples

Genomic DNA of paraffin samples and frozen tissue samples were extracted using kits from Qiagen (QIAamp DNAFFPE Tissue kit; QIAamp DNA Mini Kit). The concentration and purity of DNA samples were detected by NanoDrop-2000c.

300ng DNA sample was digested with methylation-sensitive restriction endonuclease (HinP1I). The digestion system was: DNA 300ng, Buffer (10×) 5μL, methylation-sensitive restriction endonuclease 30U, ddH ₂ O supplemented to 50μL, this is the enzyme group. The treatment of the non-enzyme group was the same as that of the enzyme-added group except that ddH ₂ O was used to replace the restriction endonuclease. Negative control (commercial unmethylated human genomic DNA control) and positive control (commercial fully methylated human genomic DNA) were also treated in the same way.

Design PCR primers for the restriction sites, and use fluorescent quantitative PCR to amplify the DNA samples that have been digested (enzyme-added group) and undigested (unenzyme-added group), negative control and positive control (two for each sample). multiple wells). The amplification system is: SYBR Mix (2×) 10 μL, upstream primer (F) 0.5 μL, downstream primer (R) 0.5 μL, ddH ₂ O 7 μL, DNA 2 μL; the upstream and downstream primers are as follows:

Upstream primer AF2: 5'-ATATCAGAGAAACTCGCCGAG-3',

Downstream primer AR2: 5'-CACGAACGTACAACTGTACCG-3';

The amplification conditions are: 95°C for 5min; 95°C for 30s, 61°C for 30s, 72°C for 30s, and 35 cycles.

The level of methylation was calculated using the formula of 100%×2 ^{△Cq (no enzyme added group-enzyme added group)} , where △Cq (no enzyme added group-enzyme added group) was expressed as the Cq value of two duplicate wells in the enzyme added group Mean value - the mean value of Cq values of two duplicate wells in the group without enzyme.

It was found that the methylation level of the negative control was 0.00% (Figure 2), and the methylation level of the positive control was 100.00% (Figure 3). The methylation level of TSPYL5 gene in cancer tissue was significantly higher than that in non-cancerous tissue ( P <0.0001, Figure 4 ), and ROC curve analysis can well distinguish cancer from non-cancerous tissue, and the area under the curve can reach 91.4% (88.0 %-94.8%) (Figure 5), when the methylation level was 7.015% as the cut off value, the sensitivity and specificity were 80.37% and 93.25%, respectively.

Example 2 Detection of TSPYL5 gene methylation level in liver cancer and paracancerous tissues by BSP cloning sequencing method

Genomic DNA of paraffin samples and frozen tissue samples were extracted using kits from Qiagen (QIAamp DNAFFPE Tissue kit; QIAamp DNA Mini Kit). The concentration and purity of DNA samples were detected by NanoDrop-2000c.

2 μg of DNA samples were modified with sodium bisulfite, and the negative control (commercial unmethylated human genomic DNA control) and positive control (commercial fully methylated human genomic DNA) were also treated in the same way. The specific process is as follows :

(1) Take 2 μg of DNA and place it in a 2 mL EP tube, add appropriate amount of autoclaved water to make up the volume to 50 μL;

(2) Add 5.5 μL of freshly prepared 3mol/L NaOH, and bathe in water at 55°C for 20 minutes;

(3) Add 30 μL of freshly prepared hydroquinone solution (0.04 mol/L) and 520 μL of sodium bisulfite solution (3.6 mol/L), add 200 μL of paraffin oil, and bathe in water at 55°C for 16 hours in the dark;

(4) Wizard DNA Clean-up System purification (according to the operating instructions);

(5) Add 4 μL of 3mol/L NaOH to the above-mentioned purified DNA, and bathe in water at 37°C for 15 minutes;

(6) Add 22 μL 10M ammonium acetate, 4 μL 20g/L glycogen, 250 μL ice-free ethanol, and place at -20°C for overnight precipitation;

(7) The precipitated DNA was recovered, washed twice with 70% ethanol, dried at room temperature, dissolved in 50 μL TE, and stored at -20°C.

Design PCR primers for the transformed sequence, and use nested PCR (using the outer primer and inner primer in turn, the sequence is as follows) to amplify the modified DNA. The primer sequence is as follows:

Outer primers:

Upstream BWF2: 5'-ACGTTCGAGTATTTTTTTTA-3',

Downstream BWR2: 5'-GACCTTTACCCCAATTTTTA-3';

Inside primer:

Upstream BNF2: 5'-AGAAAATAGGTGATGGGGGA-3',

Downstream BNR2: 5'-CGACCGCTCATAATAACGAC-3'.

Both inner and outer PCR amplification conditions are: 95°C, 5min; 95°C, 30s, 68°C-56°C, 2cycles/2°C, 45s, 72°C, 45s, 25cycles when the annealing temperature drops to 56°C; 72°C, 10min.

The PCR product was gel-cut and purified, connected to the pMD18-T vector, and then transferred to a DH5α competent medium. After overnight culture on the Amp(+) plate, 10 positive clones were picked to extract the plasmid and sequenced. The sequencing results were analyzed, and the methylation level of each site and each sample was calculated using 100×(the number of methylated cytosines/the number of all cytosines).

In this example, the methylation level of the TSPYL5 gene in some liver cancers and paracancerous tissues in Example 1 was detected by using the BSP cloning sequencing method. The results showed that the methylation level of CpG island in the promoter region of TSPYL5 gene in cancer tissue was higher than that in paracancerous tissue ( P <0.0001, Figure 6), and the methylation level of each CpG site in the amplified fragment of TSPYL5 gene in cancer tissue The levels were higher than those in paracancerous tissues (Figure 7).

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims (6)

1. one kind is horizontal based on methylation sensitive restriction enzyme combination fluorescence quantitative PCR detection TSPYL5 gene methylations Kit, it is characterised in that：Include following primer pair：

AF2：5 '-ATATCAGAGAAACTCGCCGAG-3 ',

AR2：5’-CACGAACGTACAACTGTACCG-3’；

Described methylation sensitive restriction enzyme is HinP1I.

2. kit according to claim 1, it is characterised in that：Also include methylation sensitive restriction enzyme.

3. kit according to claim 1, it is characterised in that：Also include quantitative fluorescent PCR reagent.

4. according to the kit described in claim any one of 1-3, it is characterised in that：Also comprising methylate negative control and methyl Change positive control.

5. kit according to claim 4, it is characterised in that：The described negative control that methylates includes Normal human peripheral Blood DNA, the non-human genome DNA to methylate.

6. kit according to claim 4, it is characterised in that：Described methylation positive control includes the people to methylate The liver cancer cell lines DNA that type genomic dna, target fragment methylate.