CN102888461A - PCR (polymerase chain reaction) method for quickly and efficiently detecting DNA (deoxyribonucleic acid) methylation modification after sulfite treatment - Google Patents
PCR (polymerase chain reaction) method for quickly and efficiently detecting DNA (deoxyribonucleic acid) methylation modification after sulfite treatment Download PDFInfo
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Abstract
The invention relates to a PCR (polymerase chain reaction) method for quickly and efficiently detecting DNA (deoxyribonucleic acid) methylation modification after sulfite treatment. The technical scheme is as follows: 1) a pair of inner primers A1 and A2 and a pair of outer primers B1 and B2 are designed; 2) PCR amplification: the PCR amplification comprises two PCR processes: the first PCR process is touchdown PCR, and uses the primers A1 and A2, or A1 and B2, or B1 and A2; the second PCR amplification process is common PCR, and uses the primers B1 and B2, the template is the first PCR process product 1-2ul, and the enzyme is Extaq or common rTaq; and 3) the amplification product is less than 700bp. The invention has the characteristics of high amplification efficiency, low experimental cost, short experimental period, quick and manageable detection and high experimental result stability, can be easily completed in the laboratory, and can be widely used in methylation modification detection of genome specific genes of humans, mammals and plants.
Description
Technical field
The invention belongs to biology field, relate in particular to the detection means of dna methylation modification mode in the epigenetics modification.
Background technology
Dna methylation (DNA methylation) is the modal a kind of DNA covalent modification form of eukaryotic gene group, is an importance of epigenetics.Dna methylation mainly forms 5-methylcytosine (5-mC) and a small amount of N6-methyladenine (N6-mA) and 7-methyl guanine (7-mG).In the eukaryote, dna methylation mainly is that the form with 5-methylcytosine exists.Particularly cytosine methylation is very general in the eukaryotic gene group owing to dna methylation, so receive the very big concern of life science circle.Studies show that in a large number dna methylation has participated in the various kinds of cell activity of higher organism.This wherein mainly comprises the expression of tissue-specific gene, heterochromatic formation, and developmental mechanism, the swivel base of transposon activates, the inactivation of X chromosome, the formation of genomic imprinting and oncogene etc.Dna methylation can also cause that chromosome structure, DNA conformation, dna stability and DNA and protein does the change of mode mutually, thereby controlling gene is expressed.Therefore, for methylate modification and the gene of eukaryote (the particularly mankind and plant) " time ", the relation between " sky " expression regulation; Very one of research of focus in the molecular biology.
Yet how utilizing fast simple method to detect methylated decorating site is a thorny problem, the more widely method of using at present is sulphite sequencing (bisulfite sequencing), the method can detect the situation that methylates of all cytosine(Cyt)s in the dna sequence dna in theory, the method mainly comprises three steps: sulfiting, pcr amplification purpose fragment, order-checking.(sulfiting has the test kit commodity although sulfiting and follow-up sequencing technologies all have forming technique, sequencing technologies has order-checking company), but the amplification of pcr amplification purpose fragment needs investigator oneself operation to finish, therefore in this step, remain problem be:
(1) owing to dna sequence dna behind the sulfiting can change, and the thymus pyrimidine T that forms forms hairpin structure easily, and general annealing temperature can't be opened two strands fully, for pcr amplification brings difficulty, need a lot of alternative primers of design, increase by the method for repeatedly attempting;
(2) if improve the primer annealing temperature, then because behind the sulfiting, genomic dna has been broken into the fragment about 800bp, therefore design primer with ordinary method, be not easy to increase or can not increase;
(3) Taq enzyme (gold medal rTaq, LATaq, the ExTaq etc.) expanding effect with high-fidelity does not improve, and success ratio is low, cost is high;
(4) need to design respectively primer to having methylated and non-methylated site, relatively very complicated.
Summary of the invention
In order to address the above problem, the invention provides a kind of PCR method that dna methylation is modified behind the sulfiting that rapidly and efficiently detects.
To achieve these goals, the technical solution used in the present invention is: a kind ofly rapidly and efficiently detect the PCR method that dna methylation is modified behind the sulfiting, comprise the steps:
At first, design pair of outer primer A1 and A2, a pair of inner primer B1 and B2; Its method of design is as follows:
1) according to the known primers;
2) the primer size is 28-35nt, avoid A, each continuous repetition more than 3 times of T;
3) the forward primer end can not be C;
4) forward primer is less than 5 C, and reverse primer is less than 5 G;
5) C in the forward primer replaces C/T with annexing base Y; G in the reverse primer replaces G/A with annexing base R;
6) according to two pairs of primers of mentioned above principle design, comprise the inside primer (B1 and B2) of design in a pair of outside primer (A1 and A2) according to purpose fragment and/or the design of its flanking sequence and a pair of externally primer amplification fragment.Generally, A1, B1 be as forward primer, A2, and B2 is as reverse primer.
Secondly, pcr amplification, it comprises two-wheeled PCR process,
The Amplification of first round PCR:
1) one takes turns Amplification: 95 ℃ or 94 ℃ of 45S or 30S, 62 ℃ of 45S or 30S, 72 ℃ of 60S;
2) every annealing temperature of taking turns is successively decreased 1 ℃ after, and amplification 9 is taken turns; Or annealing temperature successively decreases 0.7 ℃, and amplification 11 is taken turns;
3) then take turns by following parameter amplification 25: 95 ℃ or 94 ℃ of 45S or 30S, 52 ℃ of 45S or 30S, 72 ℃ of 60S;
4) first round PCR is landing-type PCR, and the primer is A1 and A2, or A1 and B2, or B1 and A2;
Second takes turns the Amplification of PCR:
1) takes turns according to following parameters amplification 35: 95 ℃ or 94 ℃ of 45S or 30S, 58 ℃ of 45S or 30S, 72 ℃ of 60S;
2) regular-PCR, the primer are B1 and B2, and template is first round PCR product 1-2ul, and used enzyme is ExTaq or common rTaq;
Three) amplified production is less than 700bp.
Design of primers thinking involved in the present invention and round pcr are to get through long practical experience, are applicable to the material of all sulphite order-checkings, regardless of species, are specially adapted to vegetable material.
Above-mentioned method is applied to detect animal-plant gene group dna methylation decorating site.
The invention has the beneficial effects as follows: the present invention combines two kinds of PCR programs and has solved following problem:
(1) reduced the cost of sulphite order-checking and improved conventional efficient, the present invention only need to design two pairs of primers, with ExTaq or common rTaq, template DNA behind the use 2ul sulfiting just can disposablely amplify the purpose fragment, and then connect and to be transformed in the competent cell, choose the bacterium order-checking;
(2) improve amplification efficiency, the present invention designs degenerate primer, only can be the common amplification in the site that methylates and do not methylate out by a PCR reaction;
(3) having solved dna profiling changes the conventional PCR primer amplification that causes and does not go out and the pcr amplification hard problem because processing rear sequence.Increase the length of primer, and utilize the amplification of uniting between two pairs of primers to solve this problem;
(4) adopt the again method of regular-PCR of first touchdown PCR, solved the regular-PCR program and be not easy the problem that increases or can not increase; And enlarged the combination range of primer, such as in the touchdown PCR circulation, both can use the A1/A2 combination, also can use the A1/B2 combination, also can use the B1/A2 combination, the purpose fragment expands out as long as have clearly, can carry out the second regular-PCR amplification of taking turns (primer is the B1/B2 combination) with this PCR product of taking turns again, easy to use
(5) the present invention is applicable to all higher organisms, the particularly mankind and plant.
In a word, the present invention has the amplification efficiency height, experimental cost is low, experimental period is short, detect and easily to grasp fast and experimental result good stability, the characteristics easily finished in the laboratory, can be widely applied to methylating of mankind's (particularly proto-oncogene and cancer suppressor gene), Mammals and Plant Genome specific gene and modify and detect.
Description of drawings
Fig. 1 is result after same template increases through different PCR programs among the embodiment 1;
M:100bpmarker; 1-3 uses the regular-PCR program, and annealing temperature is respectively 60 ℃, the amplification of 55 ℃ and 52 ℃; 4-6 uses the amplification of present patent application " I wheel pcr amplification " program, and per pass represents independent experiment one time, independently repeats for totally three times.
Fig. 2 is the rear result of II wheel amplification among the embodiment 1;
As shown be independent revision test twice.
Fig. 3 is that sulphite sequencing analysis result shows among the embodiment 1.
Fig. 4 is the pcr amplification result of embodiment 2-4;
M:marker; A-F amplified production size is followed successively by: 446bp, 332bp, 493bp, 425bp, 493bp, 402bp.
Fig. 5 is that sulphite sequencing analysis result shows among the embodiment 2-4.
Embodiment
One) design internal-external primer
1) downloads to the Tos17 sequence according to the clone AC087545 of Tos17 on Genebank in the NCBI website, its left side LTR sequence is 138bp, by setting out the sequence of left side each 600bp of LTR sequence upstream and downstream under the NCBI website, utilize primer-design software pimer5 to design as follows primer again;
2) the primer size is 28-35nt, avoid A, each continuous repetition more than 3 times of T;
3) the forward primer end can not be C;
4) forward primer is less than 5 C, and reverse primer is less than 5 G;
5) C in the forward primer replaces C/T with annexing base Y; G in the reverse primer replaces G/A with annexing base R;
6) two pairs of primers of design comprise the inside primer (B1 and B2) that designs in a pair of outside primer (A1 and A2) according to purpose fragment and/or the design of its flanking sequence and a pair of externally primer amplification fragment.Wherein, use primer A1/A2 amplification purpose clip size to be 626bp, primer B1/B2 combination amplification purpose clip size is 266bp, and the A1/A2 amplified fragments comprises the B1/B2 amplified fragments.Primer sequence is as follows:
A1: 5' GTTGGGATYAATGGGATTGGYAAGT 3'
A2: 5' CCTCRACCTRTRCARCAARCCARCAAC 3'
B1: 5' TGGAYAGATYAAGYYTAAYTTGGGAAG 3'
B2: 5' RACCATTRCTCTRATACCATCTTAACT 3'
Two) pcr amplification comprises two-wheeled PCR process,
Use primer A1/A2 to do I wheel pcr amplification
PCR reaction system 25ul, dosage adds routinely, and wherein ExTaq adds 0.1ul, and the sulfiting template adds 2ul, increases according to following parameters:
1) one takes turns Amplification: 95 ℃ of 45S, 62 ℃ of 45S, 72 ℃ of 60S;
2) later every 1 ℃ of lapse of temperature of withdrawing from a secret society or underworld gang of taking turns, amplification 9 is taken turns;
3) then take turns by following parameter amplification 25: 95 ℃ of 45S, 52 ℃ of 45S, 72 ℃ of 60S.
According to the said procedure amplification, obtain the purpose product, size is 626bp.Simultaneously, in order to verify the validity of present method, adopt same sulfiting template, same reaction system, but increase with common PCR program, the purpose that then can not increase fragment the results are shown in Figure 1.Be illustrated in figure 1 as same template through different PCR program amplifications after the result.M:100bpmarker; 1-3 uses the regular-PCR program, and annealing temperature is respectively 60 ℃, the amplification of 55 ℃ and 52 ℃, and only visible smear does not have the purpose product to increase; 4-6 uses the amplification of present patent application " I wheel pcr amplification " program, and per pass represents independent experiment one time, independently repeats for totally three times, and as seen clearly purpose fragment amplification of the same size is arranged.
Use primer B1/B2 to do II wheel pcr amplification
PCR reaction system 25ul, dosage adds routinely, gets 1ul as template take I wheel pcr amplification product, increases according to following parameters:
1) amplification 35 is taken turns: 95 ℃ of 45S, 58 ℃ of 45S, 72 ℃ of 60S;
2) regular-PCR, the primer are B1 and B2, and template is first round PCR product 2ul, and used enzyme is ExTaq 0.1ul.
According to the said procedure amplification, obtain the purpose product, size is seen Fig. 2 for 266bp().
Three) result verification after the order-checking
After will using conventional connection method for transformation through the PCR product behind the II wheel pcr amplification, choose the bacterium order-checking, sequencing result shows, after the PCR method of process two-wheeled optimization increases, the purpose fragment that the really experimental design of amplified fragments need to be increased, and comprise methylated and methylation fragment not, so the reliable (see figure 3) of real result.
One) design internal-external primer
1) downloads to this sequence according to its clone AP005292 on Genebank in the NCBI website, it is positioned at chromosomal 186486-190599 position No. seven, again by setting out the sequence of left side each 600bp of LTR sequence upstream and downstream under the NCBI website, namely be positioned at chromosomal 185886-187086 place No. seven, utilize primer-design software pimer5 to design as follows primer;
2) the primer size is 28-35nt, avoid A, each continuous repetition more than 3 times of T;
3) the forward primer end can not be C;
4) forward primer is less than 5 C, and reverse primer is less than 5 G;
5) C in the forward primer replaces C/T with annexing base Y; G in the reverse primer replaces G/A with annexing base R;
6) two pairs of primers of design comprise the inside primer (B1 and B2) that designs in a pair of outside primer (A1 and A2) according to purpose fragment and/or the design of its flanking sequence and a pair of externally primer amplification fragment.Wherein, use primer A1/A2 amplification purpose clip size to be 535bp, A1/B2 amplification purpose clip size is 446bp, and primer B1/B2 combination amplification purpose clip size is 332bp, and the A1/A2 amplified fragments comprises the B1/B2 amplified fragments.Primer sequence is as follows:
A1: 5' TGTGYATAGGATAYATTYTCGTTGAA 3'
A2: 5' ATAAATATRAATTRRARRARRTTRCTTA 3'
B1: 5' YATGTGTGGTTTYTATYAYTYGGATGT 3'
B2: 5' RTTCARACCATTRCTCTRATACCATCT 3'
Two) pcr amplification comprises two-wheeled PCR process
Use primer A1/B2 to do I wheel pcr amplification
PCR reaction system 25ul, dosage adds routinely, and wherein ExTaq adds 0.1ul, and the sulfiting template adds 2ul, increases according to following parameters:
1) one takes turns Amplification: 95 ℃ of 45S, 62 ℃ of 45S, 72 ℃ of 60S;
2) later every 1 ℃ of lapse of temperature of withdrawing from a secret society or underworld gang of taking turns, amplification 9 is taken turns;
3) then take turns by following parameter amplification 25: 95 ℃ of 45S, 52 ℃ of 45S, 72 ℃ of 60S.
According to the said procedure amplification, obtain the purpose product, size is 446bp.(seeing Fig. 4 A road).
Use primer B1/B2 to do II wheel pcr amplification
PCR reaction system 25ul, dosage adds routinely, gets 1ul as template take I wheel pcr amplification product, increases according to following parameters:
1) amplification 35 is taken turns: 95 ℃ of 45S, 58 ℃ of 45S, 72 ℃ of 60S;
2) regular-PCR, the primer are B1 and B2, and template is first round PCR product 2ul, and used enzyme is ExTaq 0.1ul.
According to the said procedure amplification, obtain the purpose product, size is seen Fig. 4 B road for 332bp().
Three) result verification after the order-checking
After will using conventional connection method for transformation through the PCR product behind the II wheel pcr amplification, choose the bacterium order-checking, sequencing result shows, after the PCR method of process two-wheeled optimization increases, the purpose fragment that the really experimental design of amplified fragments need to be increased, and comprise methylated and methylation fragment not, so real result reliable (seeing Fig. 5 A).
The PCR method that the dna methylation of sequence A P001551 among the GENE BANK behind the embodiment 3 detection sulfiting is modified
One) design internal-external primer
1) downloads to this sequence according to its clone AP001551 on Genebank in the NCBI website, it is positioned at chromosomal 59722-64876 position No. one, again by setting out the sequence of left side each 300bp of sequence upstream and downstream under the NCBI website, namely be positioned at chromosomal 59422-60022 place No. one, utilize primer-design software pimer5 to design as follows primer:
2) the primer size is 28-35nt, avoid A, each continuous repetition more than 3 times of T;
3) the forward primer end can not be C;
4) forward primer is less than 5 C, and reverse primer is less than 5 G;
5) utilize the merger method, the C in the forward primer replaces C/T with annexing base Y; G in the reverse primer replaces G/A with annexing base R;
6) two pairs of primers of design comprise the inside primer (B1 and B2) that designs in a pair of outside primer (A1 and A2) according to purpose fragment and/or the design of its flanking sequence and a pair of externally primer amplification fragment.Wherein, use primer A1/A2 amplification purpose clip size to be 493bp, primer B1/B2 combination amplification purpose clip size is 425bp, and the A1/A2 amplified fragments comprises the B1/B2 amplified fragments.Primer sequence is as follows:
A1: 5' TGTTTGTTAAGTGTTAAYGAAYTYYTGAAG 3'
A2: 5' TCTTCATTACCARCARATCATRCTACT 3'
B1: 5' YTGAAAAGAATTAGTGAYTAGTTAGGTG 3'
B2: 5' ACTATCATRTTRTRAARACTCRCTTTCCA 3'
Two) pcr amplification comprises two-wheeled PCR process
Use primer A1/A2 to do I wheel pcr amplification
PCR reaction system 25ul, dosage adds routinely, and wherein ExTaq adds 0.1ul, and the sulfiting template adds 2ul, increases according to following parameters:
1) one takes turns Amplification: 95 ℃ of 45S, 62 ℃ of 45S, 72 ℃ of 60S;
2) later every 1 ℃ of lapse of temperature of withdrawing from a secret society or underworld gang of taking turns, amplification 9 is taken turns;
3) then take turns by following parameter amplification 25: 95 ℃ of 45S, 52 ℃ of 45S, 72 ℃ of 60S.
According to the said procedure amplification, obtain the purpose product, size is 493bp.(seeing Fig. 4 C road).
Use primer B1/B2 to do II wheel pcr amplification
PCR reaction system 25ul, dosage adds routinely, gets 1ul as template take I wheel pcr amplification product, increases according to following parameters:
1) amplification 35 is taken turns: 95 ℃ of 45S, 58 ℃ of 45S, 72 ℃ of 60S;
2) regular-PCR, the primer are B1 and B2, and template is first round PCR product 2ul, and used enzyme is ExTaq 0.1ul.
According to the said procedure amplification, obtain the purpose product, size is seen Fig. 4 D road for 425bp().
Three) result verification after the order-checking
After will using conventional connection method for transformation through the PCR product behind the II wheel pcr amplification, choose the bacterium order-checking, sequencing result shows, after the PCR method of process two-wheeled optimization increases, the purpose fragment that the really experimental design of amplified fragments need to be increased, and comprise methylated and methylation fragment not, so real result reliable (seeing Fig. 5 B).
One section sequence that embodiment 4 chooses on the rice genome is carried out dna methylation modification detection
One) design internal-external primer
1) choose at random one section sequence on the NCBI website, it is positioned at chromosomal 63919-64519 position No. three, utilizes primer-design software pimer5 to design as follows primer:
2) the primer size is 28-35nt, avoid A, each continuous repetition more than 3 times of T;
3) the forward primer end can not be C;
4) forward primer is less than 5 C, and reverse primer is less than 5 G;
5) utilize the merger method, the C in the forward primer replaces C/T with annexing base Y; G in the reverse primer replaces G/A with annexing base R;
6) two pairs of primers of design comprise the inside primer (B1 and B2) that designs in a pair of outside primer (A1 and A2) according to purpose fragment and/or the design of its flanking sequence and a pair of externally primer amplification fragment.Wherein, use primer A1/A2 amplification purpose clip size to be 493bp, primer B1/B2 combination amplification purpose clip size is 402bp, and the A1/A2 amplified fragments comprises the B1/B2 amplified fragments.Primer sequence is as follows:
A1: 5' ATAAATGAAATYAAYATYAYAAAATTG 3'
A2: 5' ACAATCATRTTRTRAARACTCRCTTTCC 3'
B1: 5' YTGYATGGYAGYATAAGYAAAGTGTAA 3'
B2: 5' TRAARACTCRCTTTCCARATRCTCCA 3'
Two) treating with sulfurous acid
1) process genomic dna with EZ DNA Methylation-Gold Kit, the CT Conversion Reagent that interpolation 130uL prepares mixes in the 20uLDNA sample;
2) place according to following temperature: placed 10 minutes for 98 ℃, placed 2.5 hours for 64 ℃, then 4 ℃ of lower preservations;
3) then the M-Binding Buffer that adds 600uL adds the DNA sample in Zymo-Spin IC Column, close the lid and mix for several times by repeatedly putting upside down post;
4) (〉 10 at full speed 000*g) centrifugal 30 seconds, removed effluent liquid;
5) the M-Wash Buffer that adds 200uL in post, centrifugal 30 seconds;
6) the M-Desulphonation Buffer that adds 200uL is in post and placed centrifugal 30 seconds at full speed 15-20 minute;
7) the M-Wash Buffer that adds 200uL centrifugal 30 seconds, repeats this step for several times in post;
8) the M-Elution Buffer that directly adds 10uL is placed in the 1.5mL pipe in base for post matter, the centrifugal DNA that elutes.
Three) pcr amplification comprises the two-wheeled pcr amplification
Use primer A1/A2 to do I wheel pcr amplification
PCR reaction system 25ul, dosage adds routinely, increases according to following parameters:
1) one takes turns Amplification: 95 ℃ of 45S, 62 ℃ of 45S, 72 ℃ of 60S;
2) later every 1 ℃ of lapse of temperature of withdrawing from a secret society or underworld gang of taking turns, amplification 9 is taken turns;
3) then take turns by following parameter amplification 25: 95 ℃ of 45S, 52 ℃ of 45S, 72 ℃ of 60S;
According to the said procedure amplification, obtain the purpose product, size is 493bp.(seeing Fig. 4 E road).
Use primer B1/B2 to do II wheel pcr amplification
PCR reaction system 25ul, dosage adds routinely, gets 1ul as template take I wheel pcr amplification product, increases according to following parameters:
1) amplification 35 is taken turns: 95 ℃ of 45S, 58 ℃ of 45S, 72 ℃ of 60S;
2) regular-PCR, the primer are B1 and B2, and template is first round PCR product 2ul, and used enzyme is ExTaq 0.1ul.
According to the said procedure amplification, obtain the purpose product, size is seen Fig. 4 F road for 402bp().
Four) order-checking
After will using conventional connection method for transformation through the PCR product behind the II wheel pcr amplification, choose the bacterium order-checking, sequencing result carries out methylation analysis by http://www.gmi.oeaw.ac.at/CyMATE/, the result shows, after the PCR method of process two-wheeled optimization increases, the purpose fragment that the really experimental design of amplified fragments need to be increased, and comprise methylated and methylation fragment not, so real result reliable (seeing Fig. 5 C).
Claims (3)
1. one kind is rapidly and efficiently detected the PCR method that dna methylation is modified behind the sulfiting, it is characterized in that comprising the steps:
One) design pair of outer primer A1 and A2, a pair of inner primer B1 and B2;
Two) pcr amplification: comprise two-wheeled PCR process,
The Amplification of first round PCR:
1) one takes turns Amplification: 95 ℃ or 94 ℃ of 45S or 30S, 62 ℃ of 45S or 30S, 72 ℃ of 60S;
2) every annealing temperature of taking turns is successively decreased 1 ℃ after, and amplification 9 is taken turns; Or annealing temperature successively decreases 0.7 ℃, and amplification 11 is taken turns;
3) then take turns by following parameter amplification 25: 95 ℃ or 94 ℃ of 45S or 30S, 52 ℃ of 45S or 30S, 72 ℃ of 60S;
4) first round PCR is landing-type PCR, and the primer is A1 and A2, or A1 and B2, or B1 and A2;
Second takes turns the Amplification of PCR
1) takes turns according to following parameters amplification 35: 95 ℃ or 94 ℃ of 45S or 30S, 58 ℃ of 45S or 30S, 72 ℃ of 60S;
2) regular-PCR, the primer are B1 and B2, and template is first round PCR product 1-2ul, and used enzyme is ExTaq or common rTaq;
Three) amplified production is less than 700bp.
2. according to a kind of PCR method that dna methylation is modified behind the sulfiting that rapidly and efficiently detects claimed in claim 1, it is characterized in that described design pair of outer primer A1 and A2, the method for a pair of inner primer B1 and B2 is as follows:
1) according to the known primers;
2) the primer size is 28-35nt, avoid A, each continuous repetition more than 3 times of T;
3) the forward primer end can not be C;
4) forward primer is less than 5 C, and reverse primer is less than 5 G;
5) C in the forward primer replaces C/T with annexing base Y; G in the reverse primer replaces G/A with annexing base R;
6) according to the method described above, design outside primer A1 and A2 according to purpose fragment and/or its flanking sequence, and inside primer B1 and the B2 of design in a pair of externally primer amplification fragment.
3. claim 1 or the 2 described methods application in detecting animal-plant gene group dna methylation decorating site.
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| CN104046680B (en) * | 2013-03-11 | 2016-04-06 | 苏州承美生物科技有限公司 | Methylate DNA detection method |
| CN109706220A (en) * | 2019-03-11 | 2019-05-03 | 淮北师范大学 | A kind of Efficient amplification method of plant gene promoter BSP segment |
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