CN104762301A - Kit and method for detecting methylation level of liver cancer risk gene TSPYL5 - Google Patents
Kit and method for detecting methylation level of liver cancer risk gene TSPYL5 Download PDFInfo
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Abstract
The invention discloses a kit and a method for detecting the methylation level of liver cancer risk gene TSPYL5, and belongs to the field of biotechnologies. The kit for detecting the methylation level of the TSPYL5 comprises a primer pair A used for an MSRE-qPCR method or a primer pair B used for a BSP method, wherein the primer sequence is shown in SEQ ID NO. 1-22. According to the kit and the method disclosed by the invention, the relevance between the methylation of the TSPYL5 gene and liver cancer occurrence is found, and the methylation loci of the TSPYL5 are authenticated; the methylation method for the TSPYL5 by virtue of fluorescent quantitative PCR is established by combining specific methylation sensitive restriction endonuclease with the specific primers; the methylation level of the TSPYL5 is detected through the kit and the method based on MSRE-qPCR, the sensitivity is up to 83.9%, and the specificity is up to 93.25%.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of detection liver cancer risk genes
tSPYL5the test kit of methylation level and method.
Background technology
DNA methylation is one of main contents of epigenetics research, research be the reversible hereditary change that gene base sequence occurs when not changing, be that a kind of DNA molecular copies rear covalent modification mode.In eukaryote, DNA methylation refers under the effect of dnmt rna (DNMTs), with S-adenosylmethionine be methyl donor by Methyl transporters to the process in the particular bases in DNA molecular, it mainly occurs on the cytosine(Cyt) in CpG dinucleotides.In genome, the region of CpG dinucleotides distribution Relatively centralized is called CpG island (CpG islands, CGIs), and size, at 100 ~ 1000bp, is mainly positioned at promoter region and the First Exon district of gene; The abnormal methylation on CpG island directly can cause the expression silencing of genes involved.The specific methylation patterns of DNA is for maintaining Genome stability and the correct spatial and temporal expression of gene is significant, and the abnormal change of methylation patterns will participate in the human diseases even generation of cancer directly.
Methylation sensitive restriction enzyme is in conjunction with method (the Methylation-sensitive restriction enzyme digestion and PCR of PCR, MSRE-PCR) be the characteristic of methylation sites not being cut based on methylation sensitive restriction enzyme, be carry out pcr amplification again, according to electrophoresis product variance analysis methylation state after the fragment of different size by DNA digestion.Concrete principle, see Fig. 1, is selected and is carried out after enzyme cuts, with sequence outside methylation sites to be measured for amplification starting point carries out PCR to the restriction enzyme of specific DNA fragment methylated DNA fragments sensitivity.Methylate if this DNA fragmentation exists, amplified production will be had and occur; If without methylating, then do not have any fragment amplification and occur.When with insensitive endonuclease digestion product that methylates as pcr template time, no matter whether this position methylates, the fragment that all do not have expands.
The method of bisulfite cloning and sequencing (bisulfite sequencing PCR, BSP), the DNA of extraction, after bisulphite modified, methylated cytosine deamination does not occur and is transformed into uracil, and methylated cytosine(Cyt) remain unchanged.All thymus pyrimidine is changed into through PCR uracil.Glue purification is cut after PCR primer agarose electrophoresis, be connected to pMD18-T carrier subsequently, transformation of E. coli, choose single positive colony extraction plasmid after incubated overnight to check order, obtain the distribution plan of each DNA molecular specific region methylation sites, thus obtain the methylation state in each CpG site, this region.The method is the method for the detection methylation state that a kind of reliability and tolerance range are very high, the methylation state in each CpG site in the fragment that can have a definite purpose.On the significant key CpG site of searching, there is the advantage that additive method is incomparable.But the method expends time in, fund and energy, the clone of more than 10 of at least will checking order could obtain authentic data, and need a large amount of clones and plasmid extraction order-checking, process is comparatively loaded down with trivial details, expensive.So be difficult to realize high-throughout Aulomatizeted Detect clinical, but can as the control methods of DNA methylation assay novel method.
This test kit plans to build the method for the methylation sensitive restriction enzyme combined with fluorescent quantitative PCR (MSRE-qPCR) that is based on, change the subsequent detection of MSRE-PCR method into quantitative fluorescent PCR, can the methylation level in detection by quantitative minim DNA sample specific gene site.The method is easy to use, without the need to carrying out bisulf iotate-treated, and has good compatibility to paraffin section.
Summary of the invention
Based on this, the object of the present invention is to provide a kind of highly sensitive, detection liver cancer risk genes that specificity is good
tSPYL5the test kit of methylation level
.
Object of the present invention is achieved through the following technical solutions:
A kind of being used for is detected
tSPYL5the method of the quantitative fluorescent PCR of gene methylation, comprises the steps: 1. to extract sample to be tested genomic dna; 2. use methylation sensitive restriction enzyme to carry out enzyme to genomic dna to cut; 3. primer pair A is used to carry out fluorescent quantitative PCR to digestion products; 4. the Ct value of PCR primer is analyzed; 5. according in analytical results judgement sample
tSPYL5the methylation level of gene.
Wherein, step 3. in primer pair A be preferably a pair (in bracket, representing the length of primer pair amplifies fragment) in following four pairs of primers:
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’。
Above-mentioned primer designs by the following method and obtains: obtain from UCSC database
tSPYL5cpG island sequence, by analyzing the restriction enzyme site in this sequence, adopts online primer-design software (http://simgene.com/Primer3) primer premier 3.0 design packet containing the PCR primer of 2-6 restriction enzyme site.Through repetition test screening and checking, obtain that amplification efficiency is high, specificity good, can sample be detected
tSPYL5the primer of gene methylation level.
One detects based on methylation sensitive restriction enzyme combined with fluorescent quantitative PCR
tSPYL5the test kit A of gene methylation level, comprises above-mentioned primer pair A.
Described test kit A also comprises methylation sensitive restriction enzyme.
Described test kit A also comprises methylate negative control and positive control.Preferably, negative control comprises normal human peripheral blood DNA, the unmethylated human genome DNA's contrast of commercialization; Positive control comprises the human genome DNA of business-like exhaustive methylation, the hepatoma cell line DNA of certified target fragment exhaustive methylation.
Described test kit A also comprises the SYBR Green fluorescence dye etc. of quantitative fluorescent PCR.
The using method of mentioned reagent box A, comprises the steps: 1. to extract sample to be tested genomic dna; 2. use methylation sensitive restriction enzyme to carry out enzyme to genomic dna to cut; 3. primer pair A is used to carry out fluorescent quantitative PCR to digestion products; 4. the analysis of Ct value is carried out to PCR primer.
The system preference that described enzyme is cut is: DNA 300ng, Buffer(10 ×) 5 μ L, methylation sensitive restriction enzyme 30U, ddH
2o complements to 50 μ L.
The reaction system of described pcr amplification is preferably: SYBR Mix(2 ×) 10 μ L, upstream primer (F) 0.5 μ L, downstream primer (R) 0.5 μ L, ddH
2o 7 μ L, digestion products 2 μ L.
The reaction conditions of described pcr amplification is preferably: 95 DEG C of 5min; 95 DEG C of 30s, 61 DEG C of 30s, 72 DEG C of 30s, 35-40 cycles.
The test kit A that the present invention is based on methylation sensitive restriction enzyme combined with fluorescent quantitative PCR can be divided into detection system and supervisory system two parts.Detection system comprises above-mentioned primer pair A, can be combined by wherein any one.Supervisory system then comprises: 1. negative control comprises 2 kinds, and the 1st kind is attested region to be measured is non-methylated normal people's genomic DNA template; 2nd kind is the business-like non-human genome DNA's contrast that methylates.The methylation level of all negative controls answers convergence 0 under normal circumstances, otherwise shows that application of sample process has pollution or enzyme to cut not exclusively.2. positive control is the human genome DNA of business-like exhaustive methylation, the hepatoma cell line DNA of certified target fragment exhaustive methylation; Under normal reaction, the methylation level of positive control answers convergence 1, otherwise illustrative experiment failure.
Another kind of being used for is detected
tSPYL5the method of the BSP of gene methylation, comprises the steps: 1. to extract sample to be tested genomic dna; 2. sodium bisulfite is used to modify genomic dna; 3. primer pair B is used to carry out pcr amplification to the DNA after modification; 4. PCR primer cloning and sequencing analyzes the methylation status in each CG site.
Wherein, step 3. in primer pair B comprise Outside primer (W) and inner primer (N), wherein Outside primer is a pair of following 3 centerings:
BWF1：5’-TAAGAGATAATTGGAGGA-3’（465bp），
BWR1：5’-ACCTTTACCCCGATTTTTA-3’；
BWF2：5’-ACGTTCGAGTATTTTTTTTA-3’（498bp），
BWR2：5’-GACCTTTACCCCAATTTTTA-3’；
BWF3：5’-AGATAATTGGAGGAGTTGAAGA-3’（526bp），
BWR3：5’-TACTATAAAAAATCCGAATCGC-3’；
Inner primer is a pair in 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’。
Above-mentioned primer designs by the following method and obtains: obtain from UCSC database
tSPYL5cpG island sequence, the primer for BSP method adopts online methylated primers design software (http://www.urogene.org/methprimer/) MethPrimer to design.Through repetition test screening and checking, obtain that amplification efficiency is high, specificity good, can sample be detected
tSPYL5the BSP primer of gene methylation level.
A kind of PCR modified based on sodium bisulfite detects
tSPYL5the test kit B of gene methylation level, comprises above-mentioned primer pair B.
Described test kit B also comprises methylate negative control and positive control.Preferably, negative control comprises normal human peripheral blood DNA, the non-methylated human genome DNA's contrast of commercialization; Positive control comprises the hepatoma cell line DNA of the business-like human genome DNA that methylates, certified target fragment exhaustive methylation.
Described test kit B also comprises PCR reagent (dNTPs, archaeal dna polymerase, archaeal dna polymerase buffer etc.) and the reagent (sodium bisulfite, quinhydrones, sodium hydroxide, ammonium acetate, glycogen etc.) needed for sodium bisulfite modification.
The using method of mentioned reagent box B, comprises the steps: 1. to extract sample to be tested genomic dna; 2. sodium bisulfite is used to modify genomic dna; 3. primer pair B is used to carry out pcr amplification (using Outside primer and inner primer to carry out Chao Shi pcr amplification successively) to the DNA after modification; 4. PCR primer cloning and sequencing analyzes the methylation status in each CG site.
The reaction system of described pcr amplification is preferably: Buffer(10 ×) 5 μ L, dNTPs(10mM) 1 μ L, MgCl
2(25mM) 4 μ L, Taq(1U/ μ L) 2 μ L, upstream primer (F) 1 μ L, downstream primer (R) 1 μ L, ddH
2o 32 μ L, DNA profiling 4 μ L.
Outside described Chao Shi pcr amplification, the reaction conditions of inner side is all preferably: 95 DEG C, 5min; 95 DEG C, 30s, 68 DEG C-56 DEG C, 2cycles/2 DEG C, 45s, 72 DEG C, 45s, carries out 25cycles when annealing temperature is down to 56 DEG C; 72 DEG C, 10min.
Present invention finds
tSPYL5the cognation that gene methylation and liver cancer occur, and identify
tSPYL5methylation sites, in conjunction with methylation sensitive restriction enzyme and specific primer, set up the detection method of quantitative fluorescent PCR.And adopt the method for BSP to verify this conclusion subsequently.
Of the present invention
tSPYL5the sensitivity of gene methylation detection kit is up to 80.37%, and specific degree is up to 93.25%.
Present method utilizes fluorescence quantifying PCR method to detect DNA methylation, substantially increases detection efficiency, reduces testing cost.Detected result is simple, intuitively, and high-throughput, once can detect many samples.
Accompanying drawing explanation
Fig. 1 is MSRE-PCR schematic diagram; In figure, CH3 represents and methylates, and enz represents methylation sensitive restriction enzyme; Left figure DNA is without methylating, and DNA is cut off, and cannot obtain PCR primer; Right figure DNA methylates, and DNA keeps complete, can obtain PCR primer.
Fig. 2 is the amplification curve (a) of negative control in embodiment 1 and melting curve (b) figure, wherein grey and black line represent respectively enzyme-added with do not add enzyme system, the multiple hole of two, each sample; RFU represents the fluorescent value of amplified reaction;-d (RFU)/dt represents the negative first order derivative that fluorescent signal changes.
Fig. 3 is positives contrast amplification curve (a) of embodiment 1 and melting curve (b) figure, wherein grey and black line represent respectively enzyme-added with do not add enzyme system, the multiple hole of two, each sample; RFU represents the fluorescent value of amplified reaction;-d (RFU)/dt represents the negative first order derivative that fluorescent signal changes.
Fig. 4 is that embodiment 1 adopts MSRE-qPCR to detect in liver cancer and cancer beside organism
tSPYL5the horizontal scatter diagram of gene methylation.
Fig. 5 is in embodiment 1
tSPYL5gene methylation level distinguishes liver cancer and cancer beside organism ROC graphic representation.
Fig. 6 is that embodiment 2 adopts BSP clone sequencing to detect in liver cancer and cancer beside organism
tSPYL5gene methylation filter box figure.
Fig. 7 is liver cancer and cancer beside organism in embodiment 2
tSPYL5the each CpG site of gene methylates bar graph.
Embodiment
In order to more clearly understand technology contents of the present invention, describe in detail especially exemplified by following examples.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Various conventional chemical reagent used in embodiment, is commercially available prod.Unless otherwise defined, all technology used in the present invention and scientific terminology are identical with belonging to the implication that those skilled in the art of the present invention understand usually.The object of the term used in specification sheets of the present invention just in order to describe specific embodiment, is not used in restriction the present invention.
embodiment 1
tSPYL5gene methylation fluorescent PCR detects 163 pairs of liver cancer and cancer beside organism's sample
Paraffin sample and frozen tissue sample genomic dna all adopt test kit (the QIAamp DNA FFPE Tissue kit of Qiagen company; QIAamp DNA Mini Kit) extract.The concentration of DNA sample and purity adopt NanoDrop-2000c to detect.
300ng DNA sample adopts methylation sensitive restriction enzyme to carry out enzyme and cuts (HinP1I), and the enzyme system of cutting is: DNA 300ng, Buffer(10 ×) 5 μ L, methylation sensitive restriction enzyme 30U, ddH
2o complements to 50 μ L, and this is enzyme-added group.The process of not enzyme-added group is except adopting ddH
2o substitutes outside restriction enzyme, and all the other are all identical with enzyme-added group.Negative control (the non-methylated human genome DNA's contrast of commercialization), positive control (human genome DNA of business-like exhaustive methylation) also do same process.
For restriction enzyme site design PCR primer, quantitative fluorescent PCR is adopted to increase (hole answered by two, each sample) to DNA sample, negative control and the positive control of cutting process (enzyme-added group) through enzyme and non-enzyme cuts (not enzyme-added group).Amplification system is: SYBR Mix(2 ×) 10 μ L, upstream primer (F) 0.5 μ L, downstream primer (R) 0.5 μ L, ddH
2o 7 μ L, DNA 2 μ L; Wherein upstream and downstream primer is as follows:
Upstream primer AF2:5 '-ATATCAGAGAAACTCGCCGAG-3 ',
Downstream primer AR2:5 '-CACGAACGTACAACTGTACCG-3 ';
Amplification condition is: 95 DEG C of 5min; 95 DEG C of 30s, 61 DEG C of 30s, 72 DEG C of 30s, 35 cycles.
Adopt 100% × 2
△ Cq (not enzyme-added group-enzyme-added group)the methylated level of formulae discovery, wherein △ Cq (not enzyme-added group-enzyme-added group) is expressed as the mean value of mean value-not enzyme-added group two the multiple hole Cq value of enzyme-added group two multiple hole Cq values.
Found that the methylation level of negative control is 0.00%(Fig. 2), the methylation level of positive control is 100.00%(Fig. 3).In cancerous tissue
tSPYL5gene methylation level be significantly higher than cancer beside organism (
p<0.0001, Fig. 4), and ROC tracing analysis its can well distinguish cancer and cancer beside organism, its area under curve can reach 91.4%(88.0%-94.8%) (Fig. 5), when methylation level with 7.015% for cut off value time, its sensitivity and specific degree are respectively 80.37% and 93.25%.
embodiment 2 BSP clone sequencing detects in liver cancer and cancer beside organism
tSPYL5gene methylation level
Paraffin sample and frozen tissue sample genomic dna all adopt test kit (the QIAamp DNA FFPE Tissue kit of Qiagen company; QIAamp DNA Mini Kit) extract.The concentration of DNA sample and purity adopt NanoDrop-2000c to detect.
2 μ g DNA samples adopt sodium bisulfite to modify, and negative control (the non-methylated human genome DNA's contrast of commercialization), positive control (human genome DNA of business-like exhaustive methylation) also do same process, and detailed process is as follows:
(1) get 2 μ g DNA and be placed in 2mL EP pipe, add appropriate autoclaving water and supply volume to 50 μ L;
(2) 5.5 μ L Fresh 3mol/L NaOH are added, 55 DEG C of water-bath 20min;
(3) add freshly prepared hydroquinone solution (0.04mol/L) 30 μ L and sodium sulfite solution (3.6 mol/L) 520 μ L, add 200 μ L paraffin oils, 55 DEG C of lucifuge water-baths 16 hours;
(4) Wizard DNA Clean-up System purifying (being undertaken by process specifications);
(5) in the DNA after above-mentioned purifying, 3mol/L NaOH 4 μ L is added, 37 DEG C of water-bath 15min;
(6) add 22 μ L 10M ammonium acetates, 4 μ L 20g/L glycogens, 250 μ L ice dehydrated alcohols, are placed in-20 DEG C of precipitates overnight;
(7) reclaim the DNA of precipitation, 70% ethanol washes twice, is dissolved in 50 μ L TE, is placed in-20 DEG C of preservations after drying at room temperature.
For the sequences Design PCR primer after conversion, adopt Chao Shi PCR(to use Outside primer successively, inner primer, sequence is as follows) DNA after modifying is increased, primer sequence is specific as follows:
Outside primer:
Upstream BWF2:5 '-ACGTTCGAGTATTTTTTTTA-3 ',
Downstream BWR2:5 '-GACCTTTACCCCAATTTTTA-3 ';
Inner primer:
Upstream BNF2:5 '-AGAAAATAGGTGATGGGGGA-3 ',
Downstream BNR2:5 '-CGACCGCTCATAATAACGAC-3 '.
Interior outside pcr amplification condition is: 95 DEG C, 5min; 95 DEG C, 30s, 68 DEG C-56 DEG C, 2cycles/2 DEG C, 45s, 72 DEG C, 45s, carries out 25cycles when annealing temperature is down to 56 DEG C; 72 DEG C, 10min.
Be connected with pMD18-T carrier after PCR primer cuts glue purification, proceed in DH5 α competence subsequently, in Amp (+) flat board, after incubated overnight, picking 10 positive colonies extract plasmid order-checking.Sequencing result is analyzed, adopts 100 × (quantity of methylated cytosine(Cyt) quantity/all cytosine(Cyt)s) to calculate the methylation level of each site and each sample.
The present embodiment is by adopting BSP clone sequencing in the part liver cancer in embodiment 1 and cancer beside organism
tSPYL5gene methylation level detects.Found that cancerous tissue
tSPYL5the methylation level on CpG island, gene promoter area all higher than cancer beside organism (
p<0.0001, Fig. 6), and in cancerous tissue
tSPYL5in gene amplification fragment, the methylation level in each CpG site is all higher than cancer beside organism (Fig. 7).
Each technical characteristic of the above embodiment can combine arbitrarily, for making description succinct, the all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics does not exist contradiction, be all considered to be the scope that this specification sheets is recorded.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.
Claims (9)
1. one kind is detected based on methylation sensitive restriction enzyme combined with fluorescent quantitative PCR
tSPYL5the test kit of gene methylation level, is characterized in that: comprise primer pair A, and described primer pair A is a pair in following four pairs of primers:
AF1：5’-CCCCGCGAGCGCATATCAGAG -3’，
AR1：5’-GCAACCGCCGACGTCACGAAC-3’；
AF2：5’-ATATCAGAGAAACTCGCCGAG-3’，
AR2：5’-CACGAACGTACAACTGTACCG-3’；
AF3：5’-CAGAGAAACTCGCCGAGACCTA-3’，
AR3：5’-TTCAAAGACACGCTGTGACCCT-3’；
AF4：5’-AGCGCATATCAGAGAAACT-3’，
AR4：5’-GTACCGTCGCGAGAGGACGTGA-3’。
2. test kit according to claim 1, is characterized in that: also comprise methylation sensitive restriction enzyme.
3. test kit according to claim 1, is characterized in that: also comprise quantitative fluorescent PCR reagent.
4. the PCR modified based on sodium bisulfite detects
tSPYL5the test kit of gene methylation level, is characterized in that: comprise primer pair B, and described primer pair B comprises Outside primer and inner primer;
Wherein, Outside primer is a pair in following 3 pairs of primers:
BWF1：5’-TAAGAGATAATTGGAGGA-3’，
BWR1：5’-ACCTTTACCCCGATTTTTA-3’；
BWF2：5’-ACGTTCGAGTATTTTTTTTA-3’，
BWR2：5’-GACCTTTACCCCAATTTTTA-3’；
BWF3：5’-AGATAATTGGAGGAGTTGAAGA-3’，
BWR3：5’-TACTATAAAAAATCCGAATCGC-3’；
Inner primer is a pair in following 4 pairs of primers:
BNF1：5’-AATAGGTGATGGGGGATAGGT-3’，
BNR1：5’-CCGCTCATAATAACGACGAAA-3’；
BNF2：5’-AGAAAATAGGTGATGGGGGA-3’，
BNR2：5’-CGACCGCTCATAATAACGAC-3’；
BNF3：5’-TTAGAAAATAGGTGATGGGGGATAG-3’，
BNR3：5’-ATAACGACGAAAACAACTTCAAAAA-3’；
BNF4：5’-AATAGGTGATGGGGGATAG-3’，
BNR4：5’-GACCGCTCATAATAACGAC-3’。
5. test kit according to claim 4, is characterized in that: also comprise bisulphite modified required reagent.
6. test kit according to claim 4, is characterized in that: also comprise PCR reagent.
7. the test kit according to any one of claim 1-6, is characterized in that: also comprise methylate negative control and methylation positive contrast.
8. test kit according to claim 7, is characterized in that: the described negative control that methylates comprises normal human peripheral blood DNA, non-methylated human genome DNA.
9. test kit according to claim 7, is characterized in that: described methylation positive contrasts methylated human genome DNA, the methylated hepatoma cell line DNA of target fragment.
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