CN112941162A - Method for detecting correlation between DNA telomere length and gene methylation degree - Google Patents
Method for detecting correlation between DNA telomere length and gene methylation degree Download PDFInfo
- Publication number
- CN112941162A CN112941162A CN202011600874.2A CN202011600874A CN112941162A CN 112941162 A CN112941162 A CN 112941162A CN 202011600874 A CN202011600874 A CN 202011600874A CN 112941162 A CN112941162 A CN 112941162A
- Authority
- CN
- China
- Prior art keywords
- dna
- methylation
- adsorption column
- telomere length
- apc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000011987 methylation Effects 0.000 title claims abstract description 39
- 238000007069 methylation reaction Methods 0.000 title claims abstract description 39
- 108091035539 telomere Proteins 0.000 title claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 title claims description 18
- 238000000034 method Methods 0.000 title claims description 7
- 108700001666 APC Genes Proteins 0.000 claims abstract description 22
- 238000001179 sorption measurement Methods 0.000 claims description 33
- 108020004414 DNA Proteins 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 16
- 210000003411 telomere Anatomy 0.000 claims description 16
- 102000055501 telomere Human genes 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 12
- 239000002699 waste material Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000010219 correlation analysis Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 238000010818 SYBR green PCR Master Mix Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 238000003753 real-time PCR Methods 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 4
- 230000007067 DNA methylation Effects 0.000 claims description 3
- 108010067770 Endopeptidase K Proteins 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000012937 correction Methods 0.000 claims description 3
- 239000012149 elution buffer Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000013207 serial dilution Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 16
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 abstract description 12
- 238000011160 research Methods 0.000 abstract description 3
- 238000005070 sampling Methods 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 230000000391 smoking effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000004199 lung function Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 206010006458 Bronchitis chronic Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 201000006306 Cor pulmonale Diseases 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000004186 Pulmonary Heart Disease Diseases 0.000 description 1
- 208000004756 Respiratory Insufficiency Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 229940124630 bronchodilator Drugs 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 208000007451 chronic bronchitis Diseases 0.000 description 1
- 230000012085 chronic inflammatory response Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000007933 dermal patch Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 201000004193 respiratory failure Diseases 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The detection method can quickly and efficiently detect the correlation between the DNA telomere length and the APC gene methylation degree in the sample, one sample can be analyzed, repeated sampling is not needed, and the detection can be efficiently and quickly carried out. The detection efficiency and the detection accuracy can be effectively improved, and the related research on the chronic obstructive pulmonary disease can be promoted.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a method for detecting correlation between DNA telomere length and gene methylation degree.
Background
Chronic obstructive pulmonary disease is a chronic bronchitis and/or emphysema characterized by airflow obstruction that can further progress to the common chronic diseases of pulmonary heart disease and respiratory failure. Related to abnormal inflammatory reaction of harmful gas and harmful particles, the disability rate and the fatality rate are high, and the worldwide incidence rate of over 40 years old is up to 9-10%. Chronic Obstructive Pulmonary Disease (COPD) is a common disease that can be prevented and treated characterized by persistent airflow limitation, which progresses in a progressive manner, associated with an enhanced chronic inflammatory response of the airways and lungs to toxic particles or gases.
The national guideline for diagnosis and treatment of chronic obstructive pulmonary diseases defines the standard for diagnosing the lung function of COPD as 'forced expiratory volume/forced vital capacity (FEV1/FVC) < 0.70) at 1 second after the bronchodilator is inhaled'. It is noted that "0.70" in the diagnostic criteria is a fixed value that does not necessarily distinguish between variables such as gender, age, height, weight, race, etc., whereas the measure of lung function is related to the subject's factors just described. There is increasing evidence that FEV1/FVC < 0.70 as a diagnostic criterion for COPD may lead to a large number of clinical misdiagnoses.
Disclosure of Invention
The invention discovers for the first time that the correlation between the telomere length of DNA and the methylation of APC gene in patients with chronic obstructive pulmonary disease has obvious correlation after excluding age, sex, smoking and the like. Therefore, the diagnosis of chronic obstructive pulmonary disease can be assisted by detecting the correlation between the length of DNA telomeres and the methylation degree of APC genes.
However, the existing detection method only aims at single DNA telomere length detection or APC gene methylation degree detection, one sample can only be detected once, the detection time is long, and DNA telomere length detection and APC gene methylation degree detection cannot be simultaneously carried out on one sample.
Aiming at the technical problems, the invention provides a method for detecting the correlation between DNA telomere length and APC (active cell death) based on methylation degree.
The technical scheme of the invention is as follows:
a method for detecting the correlation between DNA telomere length and APC gene methylation degree comprises the following steps:
and (3) extracting leucocyte DNA:
step 1-1: adding 20 mul of protease K solution into 200 mul of fresh, frozen or anticoagulant-added blood, and mixing uniformly;
step 1-2: adding 200 μ l buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10 min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;
step 1-3: adding 200 μ l of anhydrous ethanol, shaking thoroughly, mixing for 15 sec to obtain flocculent precipitate, and centrifuging briefly to remove water drop on the inner wall of the tube cover;
step 1-4: adding the solution and flocculent precipitate obtained in the step 1-3 into an adsorption column CB3, centrifuging at 12,000rpm for 30 sec, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe;
step 1-5: adding 500 μ l buffer solution GD12 into adsorption column CB3, centrifuging at 000rpm for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
step 1-6: adding 600 μ l of rinsing liquid PW into adsorption column CB3, centrifuging at 12,000rpm for 30 sec, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube; repeating the operation twice;
step 1-7: placing adsorption column CB3 back into the collecting tube, centrifuging at 12,000rpm for 2 min, and pouring off waste liquid; placing the adsorption column CB3 at room temperature, and completely airing the residual rinsing liquid in the adsorption material;
step 1-8: transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5 min, centrifuging at 12,000rpm for 2 min, and collecting the solution into the centrifugal tube to obtain sample DNA;
telomere length measurement:
step 2-1: 36B4 is selected as a monoclonal gene, and primers for synthesizing telomere and 36B4 genes are obtained. The following were used:
Telo u, 5’-GGTTTTT- GAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3’;
Telo d, 5’-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3’;
36B4u, 5’-CAGCAAGTGGGAAGGTGTAATCC-3’;
36B4d, 5’-CCCATTCTATCATCAACGGGTACAA-3’；
step 2-2: preparing a premixing system: containing 5 ml SYBR Green PCR Master Mix, 0.4. mu.l primer Mix, 0.2. mu.l dye and 3.4. mu.l water; wherein the primers are TELO and 36B4, and two pre-mixed reagents are respectively added into two 96-well plates;
step 2-3: preparation of a standard curve: standard Curve DNA five concentrations of 0.25 ng/. mu.l, 0.74 ng/. mu.l, 2.22 ng/. mu.l, 6.67 ng/. mu.l and 20 ng/. mu.l were generated by serial dilution of Haila DNA, and 3 wells of each concentration were added to a 96-well plate;
step 2-4: adding the sample DNA holes obtained in the steps 1-8 into a 96-well plate;
step 2-5: running on a fluorescence quantitative PCR instrument in an absolute quantitative PCR mode;
step 2-6: dividing the final TELO copy number by the copy number of 36B4 to obtain the relative telomere length of the corresponding sample, and correcting by using the correction sample to obtain the final relative telomere length;
APC gene methylation measurement:
step 3-1: primers for DNA methylation were synthesized, which were classified into methylated (M) and unmethylated (U) as follows:
RUNX3-F(M) ： CGAGGTTTCGTTGGTTCGA
RUNX3-R(M) ：GCCGCGACCCAAACAA
RUNX3-F(U) ：TGAGGTTTCGTTGGTTTGA
RUNX3-R(U) ：GCCACAACCCAAACAA
APC-F(M) ：GAACCAAAACGCTCCCCAT
APC-R(M) ：TTATATGTCGGTTACGTGCGTTTATAT
APC-F(U) ：GAACCAAAACACTCCCCAT
APC-R(U) ：TTATATGTTGGTTATGTGTGTTTATAT
PCDH20-F(M) ：GAAGAGTTAGTGATTATTGGTTGAAG
PCDH20-R(M) ：CAAATTCCTATAACTAATACTACTCCT
PCDH20-F(U) ：TTAGCAATTTGTATGAATCAAGA
PCDH20-R(U) ：TGTGAATATAAATTGTTGAATATT；
3-2: preparing whole gene sequence plasmid of corresponding gene by using competent cell;
step 3-3: plasmid 10 fold diluted to 5 gradients;
step 3-4: preparing a premixing system: containing 10 ml SYBR Green PCR Master Mix, 4. mu.l primer Mix, and 4. mu.l water;
step 3-5: mu.l of sample DNA and 18. mu.l of the system were added to a 96-well plate and mixed well. Each sample DNA and plasmid DNA occupies 2-3 replicate wells;
step 3-6: obtaining the copy number of the corresponding gene according to the concentration gradient of the plasmid, and dividing methylation (M) by the sum of methylation (M) and non-methylation (U) to obtain the methylation degree of the DNA;
DNA telomere length and APC gene methylation correlation analysis:
step 4-1: and (3) carrying out correlation analysis on the DNA telomere length and the methylation degree of the APC gene.
The detection method can quickly and efficiently detect the correlation between the DNA telomere length and the APC gene methylation degree in the sample, one sample can be analyzed, repeated sampling is not needed, and the detection can be efficiently and quickly carried out. The detection efficiency and the detection accuracy can be effectively improved, and the related research on the chronic obstructive pulmonary disease can be promoted.
Drawings
FIG. 1 is a structural table of the DNA telomere length and APC gene methylation degree correlation analysis according to the present invention;
FIG. 2 is a scattergram showing the correlation between DNA telomere length and APC gene methylation degree according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, for which specific conditions are not noted in the examples, are generally carried out according to conventional conditions, laboratory manuals or according to conditions recommended by the manufacturer.
Example (b): and verifying the difference between patients with chronic obstructive pulmonary disease and normal persons in the correlation between DNA telomere length and APC gene methylation degree.
Clinical study subjects:
1. clinical study subjects:
a research sample is selected, and the sample source is the first subsidiary hospital of Kunming technology university. The sample ensemble information is as follows:
all subjects signed informed consent.
The extraction method of leucocyte DNA comprises the following steps:
1. 200. mu.l of fresh, frozen or blood added with various anticoagulants is used, 20. mu.l of protease K solution is added, and mixed well.
2. Adding 200 μ l buffer GB, mixing thoroughly, standing at 70 deg.C for 10 min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover.
3. Add 200. mu.l of absolute ethanol, mix well for 15 sec with shaking, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover.
4. The solution and flocculent precipitate obtained in the previous step are added into an adsorption column CB3 to be centrifuged for 30 sec at 12,000rpm (13,400 Xg), waste liquid is poured off, and the adsorption column CB3 is put back into the collecting pipe.
5. 500. mu.l of buffer GD12 (GD12, 000rpm) (-13, 400 Xg) is added into the adsorption column CB3, the mixture is centrifuged for 30 s, waste liquid is poured off, and the adsorption column CB3 is placed into a collection tube.
6. 600. mu.l of a rinsing solution PW was added to the adsorption column CB3, centrifuged at 12,000rpm (-13,400 Xg) for 30 sec, the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube. The operation was repeated twice.
7. The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm (-13,400 Xg) for 2 min and the waste liquid was decanted. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.
8. Transferring the adsorption column CB3 into a clean centrifuge tube, hanging and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5 min, centrifuging at 12,000rpm (13,400 Xg) for 2 min, and collecting the solution into the centrifuge tube.
The telomere length measuring method comprises the following steps:
1. 36B4 was selected as the monoclonal gene.
2. Synthesizing telomere and 36B4 gene to obtain primer. The following were used:
Telo u, 5’-GGTTTTT- GAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3’;
Telo d, 5’-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3’;
36B4u, 5’-CAGCAAGTGGGAAGGTGTAATCC-3’;
36B4d, 5’-CCCATTCTATCATCAACGGGTACAA-3’。
3. preparing a premixing system: this contained 5 ml of SYBR Green PCR Master Mix (Universal SYBR Rapid quantitation; KaPa, Tokyo, Japan), 0.4. mu.l of primer Mix, 0.2. mu.l of dye (Universal SYBR Rapid quantitation; KaPa, Tokyo, Japan), and 3.4. mu.l of water. The primers were TELO and 36B4 and two pre-mixed reagents were added to two 96-well plates, respectively.
4. Preparation of a standard curve: standard Curve DNA five concentrations (0.25, 0.74, 2.22, 6.67 and 20 ng/. mu.l) were generated by serial dilution of Haila DNA and 3 wells of each concentration were added to a 96-well plate.
5. Three wells per sample were added to a 96-well plate.
6. Runs were performed on an Applied Biosystem 7500 (Sammer Feishell technology, Shanghai, China) by means of absolute quantitative PCR.
7. And dividing the final TELO copy number by the copy number of 36B4 to obtain the relative telomere length of the corresponding sample, and correcting by using the correction sample to obtain the final relative telomere length.
Methylation degree measurement method:
1. primers for DNA methylation were synthesized, which were classified into methylated (M) and unmethylated (U) as follows:
RUNX3-F(M) ： CGAGGTTTCGTTGGTTCGA
RUNX3-R(M) ：GCCGCGACCCAAACAA
RUNX3-F(U) ：TGAGGTTTCGTTGGTTTGA
RUNX3-R(U) ：GCCACAACCCAAACAA
APC-F(M) ：GAACCAAAACGCTCCCCAT
APC-R(M) ：TTATATGTCGGTTACGTGCGTTTATAT
APC-F(U) ：GAACCAAAACACTCCCCAT
APC-R(U) ：TTATATGTTGGTTATGTGTGTTTATAT
PCDH20-F(M) ：GAAGAGTTAGTGATTATTGGTTGAAG
PCDH20-R(M) ：CAAATTCCTATAACTAATACTACTCCT
PCDH20-F(U) ：TTAGCAATTTGTATGAATCAAGA
PCDH20-R(U) ：TGTGAATATAAATTGTTGAATATT
2. preparing whole gene sequence plasmid of corresponding gene by using competent cell.
3. Plasmid was 10-fold diluted into 5 gradients.
4. Preparing a premixing system: this contained 10 ml of SYBR Green PCR Master Mix (Universal SYBR Rapid quantitation; KaPa, Tokyo, Japan), 4. mu.l of primer Mix, and 4. mu.l of water.
5. Mu.l of sample DNA and 18. mu.l of the system were added to a 96-well plate and mixed well. Each sample DNA and plasmid DNA occupies 2-3 replicate wells.
6. The copy number of the corresponding gene is obtained from the concentration gradient of the plasmid, and the methylation degree of the DNA is obtained by dividing the methylation (M) by the sum of the methylation (M) and the non-methylation (U).
And (3) correlation analysis:
the obtained DNA telomere length and APC gene methylation degree are subjected to correlation analysis, and age, age and sex, age, sex and smoking history are sequentially excluded, and the results are shown in FIGS. 1-2.
And performing correlation analysis between the relative telomere length of the COPD patient and the methylation degree of the APC gene, and performing bias analysis on the correlation analysis to remove the influence of age, sex and smoking history, wherein the telomere length and the methylation degree of the APC gene have a negative correlation in the COPD patient, and the difference has statistical significance, namely when the telomere length increases, the methylation degree of the APC gene correspondingly decreases.
SEQUENCE LISTING
<110> Yuyu Zhen Shu; huangzhen Ming; pink-skin patch; stretching cloudy glow; liu Rui; paiyu (jade)
<120> detection method for correlation between DNA telomere length and APC gene methylation degree
<130> 2020-10-23
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 37
<212> DNA
<213> Artificial Synthesis
<400> 1
ggtttttgag ggtgagggtg agggtgaggg tgagggt 37
<210> 2
<211> 39
<212> DNA
<213> Artificial Synthesis
<400> 2
tcccgactat ccctatccct atccctatcc ctatcccta 39
<210> 3
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 3
cagcaagtgg gaaggtgtaa tcc 23
<210> 4
<211> 25
<212> DNA
<213> Artificial Synthesis
<400> 4
cccattctat catcaacggg tacaa 25
<210> 5
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 5
gaaccaaaac gctccccat 19
<210> 6
<211> 27
<212> DNA
<213> Artificial Synthesis
<400> 6
ttatatgtcg gttacgtgcg tttatat 27
<210> 7
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 7
gaaccaaaac actccccat 19
<210> 8
<211> 27
<212> DNA
<213> Artificial Synthesis
<400> 8
ttatatgttg gttatgtgtg tttatat 27
Claims (1)
1. A method for detecting the correlation between DNA telomere length and APC gene methylation degree is characterized by comprising the following steps:
and (3) extracting leucocyte DNA:
step 1-1: adding 20 mul of protease K solution into 200 mul of fresh, frozen or anticoagulant-added blood, and mixing uniformly;
step 1-2: adding 200 μ l buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10 min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;
step 1-3: adding 200 μ l of anhydrous ethanol, shaking thoroughly, mixing for 15 sec to obtain flocculent precipitate, and centrifuging briefly to remove water drop on the inner wall of the tube cover;
step 1-4: adding the solution and flocculent precipitate obtained in the step 1-3 into an adsorption column CB3, centrifuging at 12,000rpm for 30 sec, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe;
step 1-5: adding 500 μ l buffer solution GD12 into adsorption column CB3, centrifuging at 000rpm for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
step 1-6: adding 600 μ l of rinsing liquid PW into adsorption column CB3, centrifuging at 12,000rpm for 30 sec, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube; repeating the operation twice;
step 1-7: placing adsorption column CB3 back into the collecting tube, centrifuging at 12,000rpm for 2 min, and pouring off waste liquid; placing the adsorption column CB3 at room temperature, and completely airing the residual rinsing liquid in the adsorption material;
step 1-8: transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50-200 mu l of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5 min, centrifuging at 12,000rpm for 2 min, and collecting the solution into the centrifugal tube to obtain sample DNA;
telomere length measurement:
step 2-1: 36B4 is selected as a monoclonal gene, and primers for synthesizing telomere and 36B4 genes are obtained. The following were used:
Telo u, 5’-GGTTTTT- GAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3’;
Telo d, 5’-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3’;
36B4u, 5’-CAGCAAGTGGGAAGGTGTAATCC-3’;
36B4d, 5’-CCCATTCTATCATCAACGGGTACAA-3’；
step 2-2: preparing a premixing system: containing 5 ml SYBR Green PCR Master Mix, 0.4. mu.l primer Mix, 0.2. mu.l dye and 3.4. mu.l water; wherein the primers are TELO and 36B4, and two pre-mixed reagents are respectively added into two 96-well plates;
step 2-3: preparation of a standard curve: standard Curve DNA five concentrations of 0.25 ng/. mu.l, 0.74 ng/. mu.l, 2.22 ng/. mu.l, 6.67 ng/. mu.l and 20 ng/. mu.l were generated by serial dilution of Haila DNA, and 3 wells of each concentration were added to a 96-well plate;
step 2-4: adding the sample DNA holes obtained in the steps 1-8 into a 96-well plate;
step 2-5: running on a fluorescence quantitative PCR instrument in an absolute quantitative PCR mode;
step 2-6: dividing the final TELO copy number by the copy number of 36B4 to obtain the relative telomere length of the corresponding sample, and correcting by using the correction sample to obtain the final relative telomere length;
APC gene methylation measurement:
step 3-1: primers for DNA methylation were synthesized, which were classified into methylated (M) and unmethylated (U) as follows:
RUNX3-F(M) ： CGAGGTTTCGTTGGTTCGA
RUNX3-R(M) ：GCCGCGACCCAAACAA
RUNX3-F(U) ：TGAGGTTTCGTTGGTTTGA
RUNX3-R(U) ：GCCACAACCCAAACAA
APC-F(M) ：GAACCAAAACGCTCCCCAT
APC-R(M) ：TTATATGTCGGTTACGTGCGTTTATAT
APC-F(U) ：GAACCAAAACACTCCCCAT
APC-R(U) ：TTATATGTTGGTTATGTGTGTTTATAT
PCDH20-F(M) ：GAAGAGTTAGTGATTATTGGTTGAAG
PCDH20-R(M) ：CAAATTCCTATAACTAATACTACTCCT
PCDH20-F(U) ：TTAGCAATTTGTATGAATCAAGA
PCDH20-R(U) ：TGTGAATATAAATTGTTGAATATT；
3-2: preparing whole gene sequence plasmid of corresponding gene by using competent cell;
step 3-3: plasmid 10 fold diluted to 5 gradients;
step 3-4: preparing a premixing system: containing 10 ml SYBR Green PCR Master Mix, 4. mu.l primer Mix, and 4. mu.l water;
step 3-5: mu.l of sample DNA and 18. mu.l of the system were added to a 96-well plate and mixed well. Each sample DNA and plasmid DNA occupies 2-3 replicate wells;
step 3-6: obtaining the copy number of the corresponding gene according to the concentration gradient of the plasmid, and dividing methylation (M) by the sum of methylation (M) and non-methylation (U) to obtain the methylation degree of the DNA;
DNA telomere length and APC gene methylation correlation analysis:
step 4-1: and (3) carrying out correlation analysis on the DNA telomere length and the methylation degree of the APC gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011600874.2A CN112941162A (en) | 2020-12-30 | 2020-12-30 | Method for detecting correlation between DNA telomere length and gene methylation degree |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011600874.2A CN112941162A (en) | 2020-12-30 | 2020-12-30 | Method for detecting correlation between DNA telomere length and gene methylation degree |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112941162A true CN112941162A (en) | 2021-06-11 |
Family
ID=76234936
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011600874.2A Pending CN112941162A (en) | 2020-12-30 | 2020-12-30 | Method for detecting correlation between DNA telomere length and gene methylation degree |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112941162A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101680025A (en) * | 2007-01-09 | 2010-03-24 | 癌甲基化组科学公司 | Epigenetic change in selected genes and cancer |
| WO2012018613A2 (en) * | 2010-07-26 | 2012-02-09 | The Johns Hopkins University | Genetic make-up modifies cancer outcome |
| US20120122088A1 (en) * | 2010-11-15 | 2012-05-17 | Hongzhi Zou | Methylation assay |
| CN105986026A (en) * | 2015-03-04 | 2016-10-05 | 李南方 | Application of L102Q mutational site of KCNJ5 gene |
| CN106755477A (en) * | 2017-01-19 | 2017-05-31 | 中国科学院生物物理研究所 | A kind of method for detecting cell and body aging level |
-
2020
- 2020-12-30 CN CN202011600874.2A patent/CN112941162A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101680025A (en) * | 2007-01-09 | 2010-03-24 | 癌甲基化组科学公司 | Epigenetic change in selected genes and cancer |
| WO2012018613A2 (en) * | 2010-07-26 | 2012-02-09 | The Johns Hopkins University | Genetic make-up modifies cancer outcome |
| US20120122088A1 (en) * | 2010-11-15 | 2012-05-17 | Hongzhi Zou | Methylation assay |
| CN105986026A (en) * | 2015-03-04 | 2016-10-05 | 李南方 | Application of L102Q mutational site of KCNJ5 gene |
| CN106755477A (en) * | 2017-01-19 | 2017-05-31 | 中国科学院生物物理研究所 | A kind of method for detecting cell and body aging level |
Non-Patent Citations (12)
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104450901B (en) | The nucleic acid markers of quick diagnosis mucocutaneous lymphnode syndrome and test kit thereof | |
| CN110484624B (en) | Gastric cancer biomarker based on peripheral blood and detection method and application thereof | |
| CN109576370B (en) | Biomarker and detection kit for bladder cancer diagnosis and recurrence monitoring | |
| CN114457192B (en) | Application of reagent for methylation typing of EB virus CpG sites in saliva in preparation of nasopharyngeal carcinoma diagnostic kit | |
| CN111172287A (en) | Application of exosome lncRNA RN7SL5P as internal reference gene in gastric cancer lncRNA detection | |
| JP2023113881A (en) | Method for assisting in detection of breast cancer | |
| Wan et al. | Targeted sequencing of genomic repeat regions detects circulating cell-free echinococcus DNA | |
| JP2023113877A (en) | Method for assisting in detection of pancreatic cancer | |
| JPWO2008090930A1 (en) | How to diagnose cancer | |
| CN111004849B (en) | Primer, method and kit for detecting multiple site mutations of CDH1 gene | |
| CN108977533B (en) | MiRNA composition for predicting chronic hepatitis B injury | |
| CN105671179B (en) | application of serum microRNA in liver cancer diagnosis and diagnosis kit | |
| CN112941162A (en) | Method for detecting correlation between DNA telomere length and gene methylation degree | |
| WO2019117270A1 (en) | Method for assisting detection of head and neck cancer | |
| CN116121376A (en) | Application of NMRK2 as diagnostic gene translocation renal cell carcinoma marker | |
| CN111500733B (en) | Molecular marker for early diagnosis of non-small cell lung cancer in peripheral blood mononuclear cells | |
| CN102021169A (en) | Serum/plasma miRNA composition and use thereof | |
| CN104962612B (en) | G.41256139delT, BRCA1 gene frameshift mutation and its is preparing the application in Computer-aided Diagnosis of Breast Cancer kit | |
| CN113322318A (en) | Application of LINC00485 as molecular marker in preparation of product for diagnosis and/or prognosis of hepatocellular carcinoma | |
| CN115803448A (en) | Micronucleus DNA from peripheral red blood cells and uses thereof | |
| CN114150072A (en) | Biomarker for brain glioma diagnosis, detection primer set and kit thereof | |
| CN109207595A (en) | A kind of Human epidermal growth factor receptor gene T790M mutation detection kit and its detection method | |
| CN116732176B (en) | Application of gene translocation type renal cell carcinoma marker group | |
| CN113930493B (en) | Application, kit and using method of molecular marker hsa_circ_0003665 for stroke-related infection | |
| CN114107484B (en) | Ulcerative colitis marker gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210611 |