CN103484552A - Simplified Sanger gene sequencing method - Google Patents
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- CN103484552A CN103484552A CN201310469440.7A CN201310469440A CN103484552A CN 103484552 A CN103484552 A CN 103484552A CN 201310469440 A CN201310469440 A CN 201310469440A CN 103484552 A CN103484552 A CN 103484552A
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- C12Q2535/101—Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
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Abstract
Belonging to the field of biotechnology, the invention discloses a simplified Sanger gene sequencing method. Based on fluorescent quantitative PCR and nucleic acid purification technology, the invention carries out great improvement on the Sanger gene sequencing method, reduces flows and operation steps in the Sanger gene sequencing method and shortens gene sequencing time. The sequencing method provided by the invention is simple and safe for operation, economical and efficient, and does not require expensive reagent; the method can effectively shorten operation time and complete the sequencing process within 4-6 h; and the gene sequencing process can be completely carried out in a 96 orifice plate or8 connected tubes, and does not require test tube transfer, thereby effective preventing pollution.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of Sanger method gene order surveying method of simplification.
Background technology
In molecular biology research, the sequential analysis of DNA is the basis of further research and transformation goal gene.Mainly contain at present the Sanger dideoxy chain termination (Chain Termination Method) of Frederick Sanger invention for the technology of DNA sequencing.The Sanger method is to start at a certain fixing point according to Nucleotide, at some specific bases place, stop at random, and carry out fluorescent mark in each base back, a series of Nucleotide of four groups of different lengthss that generation finishes with A, T, C, G, so just formed a large amount of ends by the extension products of fluorescently-labeled, different in size (termination site difference).Then, then separate these extension products with high-resolution capillary gel electrophoresis, by the differentiation to four kinds of different fluorescence colors of extension products end, computer software is " reading " DNA sequence dna automatically, thereby obtains visible DNA base sequence.Sanger gene sequencing technology has been passed through the development of 30 years and perfect, can the DNA fragmentation that reach 1,000bp be checked order now, and each base read to accuracy rate up to 99.999%.Owing to having the very high accuracy rate that reads, the order-checking of Sanger method becomes the gold standard of the genetic analysiss such as transgenation, single nucleotide polymorphism.But owing to needing before the order-checking of Sanger method that sample is carried out to early stage, process, the treatment time is long, and operation steps is many, easily pollutes in treating processes, thereby has limited the use of Sanger method gene sequencing in clinical.
Therefore, this area active demand exploitation Sanger method order-checking process simplification scheme, for reducing Sanger method order-checking flow process, shorten the Sanger method order-checking time.
Summary of the invention
The object of the present invention is to provide a kind of Sanger method gene order surveying method of simplification, it is basis that quantitative fluorescent PCR and nucleic acid purification technology are take in the present invention, Sanger method gene sequencing technology is significantly improved, reduce sanger method gene sequencing flow process and operation steps, shortened the gene sequencing time.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
A kind of Sanger method gene order surveying method of simplification, it comprises the steps:
(1) goal gene fluorescent quantitative PCR: adopt fluorescent quantitative poly to connect reaction, obtain quantitative result and the PCR product of goal gene;
(2) according to the quantitative result in step (1), the PCR product that step (1) is obtained carries out purifying and to checked order PCR reaction of the product after purifying: the mixed solution that adds exonuclease I and alkaline phosphatase in the PCR product obtained in step (1), after mixing, add again sealing liquid, then add sequencing primer, Bigdye and Bigdye buffer on sealing liquid, operation PCR program, the PCR product obtains checking order;
(3) utilize the order-checking PCR product that paramagnetic particle method obtains step (2) to carry out purifying, obtain the order-checking PCR product after purifying;
(4) order-checking PCR product application of sample to the sequenator after step (3) purifying is carried out to sequential analysis.
In such scheme, in the described fluorescent quantitative PCR of step (1), upstream primer 5 ' end inserts general M13 sequence: TGTAAAACGACGGCCAGT, and downstream primer 5 ' end inserts general M13 sequence C AGGAAACAGCTATGACC; The described sequencing primer of step (2) is universal sequence: TGTAAAACGACGGCCAGT.
In such scheme, the addition of the described exonuclease I of step (2) and alkaline phosphatase is: in every microlitre PCR product, add the exonuclease I of 1U and the alkaline phosphatase of 0.4U.
In such scheme, the described sealing liquid of step (2) is that whiteruss or other have the chemical substance of same alike result.
In such scheme, the described PCR order-checking of step (2) program is: 37 ℃ 15 minutes, 80 ℃ 2 minutes; 96 ℃ 1 minute; 96 ℃ 10 seconds, 50 ℃ 5 seconds, 60 ℃ 75 seconds, 40 circulations; 4 ℃ 5 minutes.
In such scheme, the detailed process of the described paramagnetic particle method purifying order-checking of step (3) PCR product is: add sodium chloride solution in order-checking PCR product, then add Virahol and magnetic bead, after mixing, under room temperature standing 3～5 minutes, then be placed in the standing 1～3min of magnetic frame; Abandon waste liquid, and then add the ethanol that volumetric concentration is 70%, mix, standing 1～3min under room temperature, then be placed in magnetic frame standing 1～3 minute; Abandon waste liquid, liquid is blotted under rear room temperature and dries; Then add distilled water, mix, place 3～5min, then be placed in the standing 1～3min of magnetic frame for 60 ℃; Get supernatant, then supernatant is placed in to 95 ℃, 5min, then be placed on-20 ℃, 5min, obtain the order-checking PCR product after purifying.
The invention provides a kind of Sanger gene order surveying method of simplification, the method can complete whole gene sequencing process in 96 orifice plates or 8 connecting legs, and the method step that can simplify the operation shortens the operating time, and can greatly prevent from polluting.
Beneficial effect of the present invention:
(1) simple to operate, the safety of sequence measurement of the present invention, do not need the use of more expensive reagent simultaneously, economical and efficient;
(2) sequence measurement of the present invention can effectively shorten the operating time, and the order-checking process can complete in 4～6 hours;
(3) gene sequencing process of the present invention can all complete in 96 orifice plates or 8 connecting legs, does not need to shift test tube, thereby can effectively prevent from polluting.
The accompanying drawing explanation
Fig. 1 is traditional sanger gene sequencing schema.
Fig. 2 is the sanger gene sequencing schema that the present invention simplifies.
Fig. 3 is embodiment 1 fluorescent quantitative PCR curve.
Fig. 4 is embodiment 1 sequencing result peak figure.
Embodiment
In order to understand better the present invention, further illustrate content of the present invention below in conjunction with accompanying drawing, embodiment, but content of the present invention not only is confined to following example.
Embodiment 1
1, the primer probe is synthetic
To EGFR gene 18,19,20 and 21 exons design primer and probe, by the method for synthetic, synthesize following primer and probe:
EGFR18F:TTGTCTCTGTGTTCTTGTCCC
EGFR18R:TTCCCAAACACTCAGTGAAAC
EGFR18P:FAM-CCCAGCTTGTGGAGCCTCTTACACCC-BHQ1
EGFR19F:ACATTATCAGGCTTAGGTGCG
EGFR19R:CAGACAGTAGAAAAGGTGGGC
EGFR19P:FAM-CTCCACAGCCCCAGTGTCCCTCA-BHQ1
EGFR20F:TCCCTGTGCTAGGTCTTTTG
EGFR20R:TCCCTTCCCTGATTACCTTT
EGFR20P:FAM-CGATCTGCACACACCAGTTGAGC-BHQ1
EGFR21F:CTTTCATGCGCCTTTCCAT
EGFR21R:GCCACCTCCTTACTTTGCCT
EGFR21P:FAM-ACGTTCGCCAGCCATAAGTCCTCG-BHQ1
Above-mentioned primer and probe sequence are 5 ' end to 3 ' end, and F means upstream primer, and R means downstream primer, and P means probe, and FAM means fluorophor in probe, and BHQ1 means quenching group.All upstream primer 5 ' ends all add universal sequence: TGTAAAACGACGGCCAGT, and downstream primer 5 ' end all adds universal sequence: CAGGAAACAGCTATGACC.
2, detect the fluorescent quantitative PCR of sample DNA
(1) extract sample DNA: adopt Qiagen company commercialization paraffin DNA extraction test kit, the concrete steps of extracting sample DNA are as follows:
1.1 cut the thick paraffin section of 8 left and right, 8～10 μ m from the paraffin sample, put into the 1.5ml centrifuge tube;
1.2 add 1ml dimethylbenzene, cover centrifuge tube lid, vortex mixes to wax and significantly dissolves, room temperature, 4000rpm, centrifugal 2min, carefully abandon supernatant with pipettor, notes tissue not being removed;
1.3 add the 1ml dehydrated alcohol, vortex mixes (ethanol removes residual dimethylbenzene), 14000rpm, and centrifugal 2min, carefully abandon supernatant with pipettor, notes tissue not being removed;
1.4 room temperature (15～25 ℃) is dried or 37 ℃ of metal baths are dried residual ethanol;
Whether 1.5 add 180 μ l ATL(to detect, have precipitation to occur, precipitation is used 70 ℃ of water-baths to dissolve) damping fluid mixes, then add 20 μ l Proteinase Ks, vortex mixes;
1.656 ℃ water-bath 1h above (preferably spending the night), until sample is by cracking (can shake several times) fully midway;
1.790 a ℃ water-bath 1h(adds 90 ℃ of insulations of ATL damping fluid, can repair to a certain extent the change of formaldehyde to nucleic acid construct, but soaking time is long or excess Temperature, can cause DNA break.If only have a well heater, after 56 ℃ of end, sample is placed on to room temperature, equitemperature is put into well heater by sample after rising to 90 ℃ again);
1.8 of short duration centrifugal, add the AL damping fluid (detect and whether to have precipitation to occur, precipitation is used 70 ℃ of water-baths to dissolve) of 200 μ l, vortex mixes; Then add 200 μ l dehydrated alcohols, vortex mixes (mixing of sample, AL damping fluid and ethanol wanted rapidly, if extract great amount of samples simultaneously, can first AL damping fluid and ethanol be mixed, and then adds together sample to mix);
1.9 of short duration centrifugal, whole lysates are transferred in the purification column that test kit provides to (purification column is packed in the collection tube of 2ml), cover the collection tube lid, the centrifugal 1min of 8000rpm, put into new collection tube by purification column;
1.10 add 500 μ l AW1 damping fluids (check in AW1 and whether added ethanol) to purification column, cover the collection tube lid, the centrifugal 1min of 8000rpm, outwell liquid in collection tube, and purification column is put into to collection tube;
1.11 add 500 μ l AW2 damping fluids (check in AW2 and whether added ethanol) to purification column, cover the collection tube lid, the centrifugal 1min of 8000rpm, outwell liquid in collection tube, and purification column is put into to collection tube;
1.1214000rpm centrifugal 3min, make purification column film complete drying (can consider room temperature is uncapped place 2～5 minutes so that fully dry);
1.13 purification column is put into the 1.5ml centrifuge tube (providing for oneself) of new sterilizing, to purification column film center, adds 20～100 μ l ATE or ddH2O, room temperature is placed 2～5min, the centrifugal 1min of 14000rpm, collect liquid and obtain the paraffin sample DNA, 4 ℃ of preservations, long-term storage is placed on-20 ℃.
(2) utilize above-mentioned synthetic upstream primer, downstream primer and probe, adopt the fluorescent quantitative PCR sample DNA, obtain the fluorescent quantitative PCR product, PCR reaction system (5 μ l) is:
The fluorescent quantitative PCR program:
(3) obtain fluorescent PCR amplification curve (see figure 3) and pcr amplification product, judge having or not and specificity of pcr amplification product according to fluorescent PCR amplification curve (Fig. 3): the pcr amplification curve becomes typically " S " type curve, line smoothing, without obvious background signal, specific pcr amplification product has been described.
3, the order-checking PCR of the purifying of PCR product and the product after purifying reaction
Add 1U/ μ l alkaline phosphatase 2 μ l and 5U/ μ l exonuclease I 1 μ l in above-mentioned PCR product (every pipe 5 μ l), add a small amount of Witco 70 after mixing, then add 1.6 μ l bidgye and 1.9 μ l bigdyebuffer, form following order-checking PCR system:
Operation PCR response procedures is as follows:
Because, during to the primer of EGFR gene 18,19,20 and 21 exons design, its upstream primer 5 ' end all adds universal sequence: TGTAAAACGACGGCCAGT, downstream primer 5 ' end all adds universal sequence: CAGGAAACAGCTATGACC; So the sequencing primer of above-mentioned 4 exons is universal sequence: TGTAAAACGACGGCCAG, the benefit of doing like this is: avoided each exon to adopt different sequencing primers, handled easily.
4, adopt paramagnetic particle method purifying order-checking PCR product
(1) add 54 μ l0.75M NaCl in above-mentioned order-checking PCR product, add 30 μ l Virahols and 10 μ l magnetic beads, after mixing, under room temperature standing 3 minutes;
(2) be placed in the standing 1min of magnetic frame, abandon waste liquid;
(3) add again 200 μ l70% ethanol, mix, standing 1min under room temperature, then be placed in magnetic frame standing 1 minute;
(4) abandon waste liquid, liquid feed is blotted, room temperature is dried;
(5) then add 20 μ l distilled waters, mix, place 3min for 60 ℃, then be placed in the standing 1min of magnetic frame, get supernatant 10 μ l, be placed in 95 ℃, 5min, then be placed on-20 ℃, 5min, obtain the order-checking PCR product after purifying.
5, order-checking PCR product application of sample to the sequenator after purifying checks order
Order-checking PCR product application of sample to sequenator after above-mentioned 10 μ l purifying is checked order, sequenator is ABI company 3500 sequenators, the order-checking pattern is mode standard or quick mode, obtain sequencing result and see Fig. 4, in Fig. 4, the sequencer map peak value is between 1000～1500, without obviously assorted peak, dyestuff peak and alcohol peak, show that sequencing result is reliable.
Obviously, above-described embodiment is only to be the example that clearly explanation is done, and is not the restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And the apparent variation of therefore amplifying or change are still within the protection domain in the invention.
Claims (6)
1. the Sanger method gene order surveying method of a simplification, is characterized in that, comprises the steps:
(1) goal gene fluorescent quantitative PCR: adopt fluorescent quantitative poly to connect reaction, obtain fluorescent quantitation result and the PCR product of goal gene;
(2) according to the quantitative result in step (1), the PCR product that step (1) is obtained carries out purifying and to checked order PCR reaction of the product after purifying: in the PCR product obtained in step (1), add exonuclease I and alkaline phosphatase, after mixing, add again sealing liquid, then add sequencing primer, Bigdye and Bigdye damping fluid on sealing liquid, operation PCR program, the PCR product obtains checking order;
(3) utilize the order-checking PCR product that paramagnetic particle method obtains step (2) to carry out purifying, obtain the order-checking PCR product after purifying;
(4) order-checking PCR product application of sample to the sequenator after step (3) purifying is carried out to sequencing analysis.
2. gene order surveying method according to claim 1, is characterized in that the addition of the described exonuclease I of step (2) and alkaline phosphatase is: in every microlitre PCR product, add the exonuclease I of 1U and the alkaline phosphatase of 0.4U.
3. gene order surveying method according to claim 1, is characterized in that step (2) is described
Sealing liquid is whiteruss.
4. gene order surveying method according to claim 1 is characterized in that the described PCR program of step (2) is: 37 ℃ 15 minutes, 80 ℃ 2 minutes; 96 ℃ 1 minute; 96 ℃ 10 seconds, 50 ℃ 5 seconds, 60 ℃ 75 seconds, 40 circulations; 4 ℃ 5 minutes.
5. gene order surveying method according to claim 1, it is characterized in that in the described fluorescent quantitative PCR of step (1), upstream primer 5 ' end inserts general M13 sequence: TGTAAAACGACGGCCAGT, and downstream primer 5 ' end inserts general M13 sequence C AGGAAACAGCTATGACC; The described sequencing primer of step (2) is universal sequence: TGTAAAACGACGGCCAGT.
6. gene order surveying method according to claim 1, the detailed process that it is characterized in that the described paramagnetic particle method purifying order-checking of step (3) PCR product is: in order-checking PCR product, add sodium chloride solution, add again Virahol and magnetic bead, after mixing, under room temperature standing 3～5 minutes, then be placed in the standing 1～3min of magnetic frame; Abandon waste liquid, and then add the ethanol that volumetric concentration is 70%, mix, standing 1～3min under room temperature, then be placed in magnetic frame standing 1～3 minute; Abandon waste liquid, liquid is blotted under rear room temperature and dries; Then add distilled water, mix, place 3～5min, then be placed in the standing 1～3min of magnetic frame for 60 ℃; Get supernatant, then supernatant is placed in to 95 ℃, 5min, then be placed on-20 ℃, 5min, obtain the order-checking PCR product after purifying.
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Cited By (3)
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| CN104293771A (en) * | 2014-10-29 | 2015-01-21 | 北京大学 | High-flux simple and convenient purification method by magnetic beads for sequencing product |
| CN107365841A (en) * | 2017-07-13 | 2017-11-21 | 广州金域医学检验中心有限公司 | For detecting pcr amplification primer thing, kit and the detection method of MPL genes W515 mutation |
| CN107841538A (en) * | 2017-11-23 | 2018-03-27 | 合肥金域医学检验所有限公司 | For detecting the primer and detection method of CEBPA gene mutations |
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Cited By (3)
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| CN104293771A (en) * | 2014-10-29 | 2015-01-21 | 北京大学 | High-flux simple and convenient purification method by magnetic beads for sequencing product |
| CN107365841A (en) * | 2017-07-13 | 2017-11-21 | 广州金域医学检验中心有限公司 | For detecting pcr amplification primer thing, kit and the detection method of MPL genes W515 mutation |
| CN107841538A (en) * | 2017-11-23 | 2018-03-27 | 合肥金域医学检验所有限公司 | For detecting the primer and detection method of CEBPA gene mutations |
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