CN106591434A - Low-concentration PCR product concentrating method used for direct sequencing - Google Patents
Low-concentration PCR product concentrating method used for direct sequencing Download PDFInfo
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Abstract
The invention relates to a low-concentration PCR product concentrated perborate and 2ul nucleic acid settling agent (artificially synthesized DNA sequence), and mixing evenly; adding a 2-2 method. The 2-2 method comprises the specific steps of adding 3M sodium acetate with 1/10 volume and pH of 5.2, absolute ethyl alcohol with 0.5 volume and 300 ul 40% PEG8000 into a PCR product to be purified, placing the solution at -20 DEG C for 10-30 min; conducting centrifugation at 4 DEG C and 12000 rpm for 10-20 min, and abandoning the supernatant; adding 1 ml 70% ethyl alcohol to be subjected to precooling at 4 DEG C, reversing evenly, conducting centrifugation at 4 DEG C and 12000 rpm for 2-10 min, and abandoning the supernatant; placing a centrifuge tube with a cover opened at room temperature, and making the remaining liquid fully volatilize; adding water or a TE solution to dissolve precipitated DNA. By the adoption of the method, the recycled product can be directly used for sequencing, the success rate of the low-concentration PCR product is improved, the operation is simple and convenient, the method is economic and applicable, and the result is stable and reliable.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of low concentration PCR primer for direct Sequencing
Method for concentration.
Background technology
DNA sequencing method is the most reliable method of detection gene order, and the influence factor of PCR primer direct Sequencing most critical is
The concentration and purity of product after template and pcr amplification reaction, different method for concentration can affect to a great extent template and product
The concentration and purity of thing.At present, conventional nucleic acid condensation method mainly had column purification concentration, agarose gel electrophoresiies to reclaim dense
Contracting method, ethanol/sodium acetate concentration.
It is the centrifugation rod structure using special silicon matrix adsorbing material to cross column purification method for concentration, specific adsorption DNA, and
RNA is passed through with protein.Although the DNA purity that this method is reclaimed is high, sample is limited to the carrying capacity of pillar, extraction efficiency
It is relatively low, and need to be centrifuged repeatedly, complex operation, and it is relatively costly.
Agarose gel electrophoresiies are reclaimed after concentration method rubber tapping, reuse the DNA that test kit is reclaimed in blob of viscose, and whole process takes
It is long, and complex steps.
Ethanol/sodium acetate concentration of DNA, wherein ethanol can eliminate the hydrated sheath of nucleic acid, make negatively charged phosphate group
Come out.The ion balance of sodium ion etc can be combined with these charged groups, and in precipitation forming part many nucleoside are reduced
Repulsive interaction between sour chain.Ethanol just can occur when the amount only in cation be enough to neutralize the electric charge of exposed phosphate moiety
Precipitation.Low temperature can reduce the movement rate of DNA molecular, promote the precipitation of nucleic acid.But this method is when target DNA is precipitated,
By primer dimer and the primer coprecipitation not consumed, serious interference is caused to sequencing.
The present invention with the addition of PEG8000 and nucleic acid settling agent on the basis of ethanol/sodium acetate concentration of DNA.Wherein, core
Sour settling agent is the DNA sequence of one section of synthetic.PEG8000 and nucleic acid settling agent can not only promote the precipitation of target DNA, carry
The yield of high nucleic acid precipitation, can also remove primer dimer and the primer not consumed, and improve the success rate and accuracy rate of sequencing.
This method is simple to operation, and low cost, the suitability is wide.
The content of the invention
The purpose of the present invention is to propose to a kind of low concentration PCR primer method for concentration for direct Sequencing.Using the present invention
Method recovery product can be directly used for sequencing, improve the success rate of low concentration PCR primer sequencing, and its is easy to operate, and economy is suitable
With as a result reliable and stable.The technical scheme for being adopted is comprised the following steps：
1. take 100ulPCR products, add 1/10 volume pH to be 5.2 3M sodium acetates and the nucleic acid settling agent of 2ul, up and down
It is reverse to mix；
2. the dehydrated alcohol of 2-2.5 volumes, mixing of turning upside down are added；
3. the 40%PEG8000 of 300ul, mixing of turning upside down, -20 DEG C of placement 10-30min are added；
4.4 DEG C of 12000rpm are centrifuged 5-20min, are carefully removed from supernatant, suck all of drop on tube wall；
5. 1ml70%4 degree Celsius of pre-cooled ethanol is added, is gently mixed, 4 DEG C of 12000rpm are centrifuged 2-10min, are carefully removed from
Supernatant, sucks all of drop on tube wall；
6. the EP pipes uncapped are placed at room temperature, make evaporating to dry, macroscopic white precipitate change for residual
For transparence；
7. appropriate bulk hydrops or TE buffer solutions precipitation DNA are added.
Wherein, the first precedence technique scheme of the invention is, the volume of described addition dehydrated alcohol be PCR primer and
2-2.5 times of NaAC cumulative volumes, preferably 2 times.
The second precedence technique scheme of the present invention is, after described addition dehydrated alcohol, the time of -20 DEG C of placements is 10-
30min, preferred 15min.
The 3rd precedence technique scheme of the present invention is, after described addition dehydrated alcohol is placed, 4 DEG C of 12000rpm centrifugations
Time is 5-20min, preferred 10min.
The 4th precedence technique scheme of the present invention is that 4 DEG C of 12000rpm are centrifuged after described 4 degrees Celsius of pre-cooled ethanols of addition
Time is 2-10min, preferred 5min.
The technical advantage of the present invention：
1. it is economic and practical for easy to operate.Cross column purification concentration and agarose gel electrophoresiies recovery concentrating process step is equal
Comparatively laborious, time-consuming, and relatively costly.The present invention is added after dehydrated alcohol, is placed a period of time at -20 DEG C, and low temperature is reduced
The movement rate of nucleic acid molecules, contributes to the precipitation of DNA, improves concentrated effect.
2. PEG8000 and nucleic acid settling agent used in the present invention, can not only promote the precipitation of target DNA, improve settling nucleic acid
The yield in shallow lake, can also remove primer dimer and the primer not consumed, and the purity for reclaiming DNA be improved, so as to improve sequencing
Success rate and accuracy rate.
Description of the drawings
Fig. 1 is electrophoretogram of the low concentration PCR primer Jing after two kinds of distinct methods concentrations in specific embodiment.
Fig. 2 is sequencing of the low concentration PCR primer of 1,2, No. 3 samples in specific embodiment Jing after the inventive method concentration
Collection of illustrative plates.
Fig. 3 is the common DNA product purification examination of low concentration PCR primer Jing Tiangeng of 1,2, No. 3 samples in specific embodiment
Sequencing chromatogram after the concentration of agent box.
Specific embodiment
The technical solution adopted in the present invention is comprised the following steps：
1. 100ulPCR products are taken, and (settling agent is to add the settling agent of 3M sodium acetates that 1/10 volume pH is 5.2 and 2ul
The artificial sequence synthetic nucleic acid of 20ng/ul, sequence is cctaagaggttttaactcccccgcgtgctggca
atcgcagatacatacacacgctaacgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa, SEQ ID NO:1), turn upside down mixing；
2. the dehydrated alcohol of 2 volumes, mixing of turning upside down are added；
3. the 40%PEG8000 of 300ul, mixing of turning upside down, -20 DEG C of placement 15min are added；
4.4 DEG C of 12000rpm are centrifuged 10min, are carefully removed from supernatant, suck all of drop on tube wall；
5. 1ml70%4 degree Celsius of pre-cooled ethanol is added, is gently mixed, 4 DEG C of 12000rpm are centrifuged 5min, are carefully removed from
Clear liquid, sucks all of drop on tube wall；
6. the EP pipes uncapped are placed at room temperature, make evaporating to dry, macroscopic white precipitate change for residual
For transparence；
7. appropriate bulk hydrops or TE buffer solutions precipitation DNA are added.
Embodiment 1
1.PCR expands urethral secretionies DNA
3 urethral secretionies sample DNAs are taken, according to following condition performing PCR reaction is entered.
1.1 prepare reactant liquor according to PCR reaction systems in table 1.
The PCR reaction systems of table 1
1.2PCR amplification reaction condition
Table 2 is sequenced PCR reaction conditions
2. using the inventive method and column purification concentrated reagent box (the common DNA product purification kit (DP204) of Tiangeng)
Two methods concentrate PCR primer simultaneously, and the PCR for concentrating above-mentioned 3 different sample DNAs simultaneously with both method for concentration respectively is produced
Thing, parallel comparison.
2.1 method of the present invention concentrate PCR primer
2.1.1 the μ lPCR products of 2 pipe 50 are taken, a pipe is combined into.
2.1.2 (settling agent is artificial for 20ng/ul's to add the settling agent of 3M sodium acetates that 1/10 volume pH is 5.2 and 2ul
Synthetic nucleic acid sequence, sequence is cctaagaggttttaactcccccgcgtgctggcaatcgcagatacatacacacgcta ac
gcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
Aaaaaaaaaaaaaaaaaaaaaaaaa, SEQ ID NO:1), turn upside down mixing.
2.1.3 the dehydrated alcohol of 2 volumes is added, mixing of turning upside down.
2.1.4 the 40%PEG8000 of 300ul, mixing of turning upside down, -20 DEG C of placement 15min are added.
2.1.54 DEG C 12000rpm centrifugation 10min, are carefully removed from supernatant, suck all of drop on tube wall.
2.1.6 1ml70%4 degree Celsius of pre-cooled ethanol is added, is gently mixed, 4 DEG C of 12000rpm are centrifuged 5min, are carefully removed from
Supernatant, sucks all of drop on tube wall.
2.1.7 the centrifuge tube uncapped is placed at room temperature, makes evaporating to dry for residual, it is macroscopic white heavy
Shallow lake is changed into transparence.
2.1.8 30 μ lTE buffer solutions are added to precipitate DNA.
2.2 concentrate PCR primer with the common DNA product purification kit (DP204) of Tiangeng
2.2.1 the balance liquid BL of 500 μ l, 12000rpm centrifugation 1min, in outwelling collecting pipe are added in adsorption column CB2
Waste liquid, adsorption column CB2 is placed back in collecting pipe.
2.2.2 the μ lPCR products of 2 pipe 50 are taken, a pipe is combined into.The combination liquid PB of 500 μ l is added thereto to, is fully mixed.
2.2.3 previous step resulting solution is added in an adsorption column CB2 (adsorption column is put in collecting pipe), room temperature is placed
2min, 12000rpm are centrifuged 60sec, outwell the waste liquid in collecting pipe, and adsorption column CB2 is put in collecting pipe.
2.2.4 600 μ l rinsing liquid PW, 12000rpm centrifugation 60sec, in outwelling collecting pipe are added in adsorption column CB2
Waste liquid, adsorption column CB2 is put in collecting pipe.
2.2.5 repetitive operation step 4.
2.2.6 adsorption column CB2 is put back in collecting pipe, 12000rpm centrifugation 2min remove rinsing liquid as far as possible.By adsorption column
CB2 is placed in room temperature and places several minutes.
2.2.7 adsorption column CB2 is put in a clean centrifuge tube, is washed to the hanging μ l of Deca 30 in adsorbed film centre position
De- buffer EB, room temperature places 2min.12000rpm centrifugation 2min collect DNA solution.
3. electrophoresis (1.0% agarose gel) contrasts two kinds of method for concentration and processes the slice result after PCR primer, as a result
As shown in Figure 1.
Well 1：No. 1 sample PCR primer product Jing after the inventive method concentration；
Well 2：No. 2 sample PCR primers product Jing after the inventive method concentration；
Well 3：No. 3 sample PCR primers product Jing after the inventive method concentration；
Well 4：DL2000DNA Maker；
Well 5：No. 1 sample PCR primer product Jing after the common DNA product purification kit concentration of Tiangeng；
Well 6：No. 2 sample PCR primers product Jing after the common DNA product purification kit concentration of Tiangeng；
Well 7：No. 3 sample PCR primers product Jing after the common DNA product purification kit concentration of Tiangeng.4. it is sequenced：
PCR primer send Suzhou Jin Weizhi bio tech ltd to be sequenced after concentration, and NCBI BLAST compare online sequencing
As a result with the homology of genes of interest, and the quality of Sequencing chromatogram is observed.The Sequencing chromatogram of product is shown in after the inventive method concentration
Fig. 2, the Sequencing chromatogram of product is shown in Fig. 3 after the common DNA product purification kit concentration of Tiangeng.
5. interpretation of result：
As shown in Figure 1, after the inventive method concentration after the DNA product purification kit concentration more common than Tiangeng of the band of product
The band of product is bright, thus proves production concentration DNA product purification kit more common than Tiangeng after the inventive method concentration
Production concentration is high after concentration.
From Fig. 2 and Fig. 3, product Sequencing chromatogram is identical in quality after two methods concentration.Meanwhile, sequence Jing compare after,
98% is all higher than with the homology of genes of interest.Thus prove that low concentration PCR primer could be used for Jing after two methods concentration straight
Connect sequencing.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that still can make
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, specification and drawings are considered as illustrative
And it is nonrestrictive.
SEQUENCE LISTING
<110>Jiangsu Rui Bo bio tech ltd
<120>A kind of low concentration PCR primer method for concentration for direct Sequencing
<130> 20161110
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 160
<212> DNA
<213>Artificial sequence
<400> 1
cctaagaggt tttaactccc ccgcgtgctg gcaatcgcag atacatacac acgctaacgc 60
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 160
Claims (12)
1. a kind of low concentration PCR primer method for concentration for direct Sequencing, it is characterised in that：
(1) 100ulPCR products are taken, adds 1/10 volume pH to be 5.2 3M sodium acetates and the nucleic acid settling agent of 2ul, turned upside down
Mix；
(2) dehydrated alcohol of 2-2.5 volumes, mixing of turning upside down are added；
(3) 40%PEG8000 of 300ul, mixing of turning upside down, -20 DEG C of placement 10-30min are added；
(4) 4 DEG C of 12000rpm are centrifuged 5-20min, are carefully removed from supernatant, suck all of drop on tube wall；
(5) 1ml70%4 degree Celsius of pre-cooled ethanol is added, overturns and mix, 4 DEG C of 12000rpm are centrifuged 2-10min, are carefully removed from
Clear liquid, sucks all of drop on tube wall；
(6) centrifuge tube uncapped is placed at room temperature, makes evaporating to doing for residual, macroscopic white precipitate is changed into
Transparence；
(7) appropriate bulk hydrops or TE buffer solutions precipitation DNA are added.
2. method according to claim 1, it is characterised in that the concentration of described addition sodium acetate is 3M.
3. method according to claim 1, it is characterised in that the described nucleic acid settling agent that adds is one section of synthetic
DNA sequence, its sequence is SEQ ID NO:Nucleotide sequence shown in 1.
4. method according to claim 1, it is characterised in that the volume of described addition nucleic acid settling agent is 2ul.
5. method according to claim 1, it is characterised in that the volume of described addition dehydrated alcohol be PCR primer and
2-2.5 times of sodium acetate cumulative volume, preferably 2 times.
6. method according to claim 1, it is characterised in that the concentration of described addition PEG8000 is 40%.
7. method according to claim 1, it is characterised in that the volume of described addition PEG8000 is 300ul.
8. method according to claim 1, it is characterised in that after described addition dehydrated alcohol, the times of -20 DEG C of placements
For 10-30min, preferred 15min.
9. method according to claim 1, it is characterised in that after described addition dehydrated alcohol is placed, 4 DEG C
12000rpm centrifugation times are 5-20min, preferred 10min.
10. method according to claim 1, it is characterised in that 4 degrees Celsius of pre-cooled ethanol concentration of described addition 1ml
For 70%.
11. methods according to claim 1, it is characterised in that 4 DEG C after described 4 degrees Celsius of pre-cooled ethanols of addition
12000rpm centrifugation times are 2-10min, preferred 5min.
12. methods according to claim 1, it is characterised in that described pcr amplification product is 100-500bp.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109280662A (en) * | 2018-09-03 | 2019-01-29 | 海宁市公安局 | One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit |
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| CN103484451A (en) * | 2013-09-29 | 2014-01-01 | 中国热带农业科学院热带生物技术研究所 | Method for rapidly extracting plant miRNA |
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2016
- 2016-11-15 CN CN201611013263.1A patent/CN106591434A/en active Pending
Patent Citations (3)
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| CN102352351A (en) * | 2011-10-17 | 2012-02-15 | 西安金磁纳米生物技术有限公司 | Method utilizing magnetic particles to purify deoxyribonucleic acid (DNA) from samples containing trace nucleic acid |
| CN103484451A (en) * | 2013-09-29 | 2014-01-01 | 中国热带农业科学院热带生物技术研究所 | Method for rapidly extracting plant miRNA |
| CN105176969A (en) * | 2015-07-13 | 2015-12-23 | 杭州优思达生物技术有限公司 | Nucleic acid extraction method and nucleic acid extraction reagent for biological samples |
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Cited By (1)
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| CN109280662A (en) * | 2018-09-03 | 2019-01-29 | 海宁市公安局 | One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit |
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