CN105256003A - DNA sequencing method based on acid-sensitive modified nucleotide - Google Patents
DNA sequencing method based on acid-sensitive modified nucleotide Download PDFInfo
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Abstract
The invention discloses a DNA sequencing method based on an acid-sensitive modified nucleotide. The method comprises steps: the acid-sensitive modified nucleotide shown in the formula (1) is employed as a reversible termination agent, and participates in a DNA chain extension reaction, and the formula (1) is shown in the specification. The acid-sensitive connection unit in the acid-sensitive modified nucleotide is fractured. The fluorescein is selected from BODIPY, rhodamine, coumarin, xanthenes, cyanin, pyrene, phthalocyanine, Alexa, a squaring dye, energy transduction generation dye or combination thereof. The base is selected from the following bases: U, T, C, A and G. Compared with the prior art, the provided extension method has a high accuracy, and has a high extension efficiency and a shearing efficiency especially when a template is a plurality of continuous same bases.
Description
Technical field
The invention belongs to chemosynthesis and biochemical field, relate to the compound that can be used for DNA sequencing, be specifically related to a kind of DNA sequencing method based on acid-sensitive modified nucleotide.
Background technology
DNA sequencing technology is one of means important in modern biology research.After the Human Genome Project completes, DNA sequencing technology obtains and develops rapidly.DNA sequencing (DNAsequencing) refers to the base sequence analyzing specific DNA fragments, namely the arrangement mode of VITAMIN B4 (A), thymus pyrimidine (T), cytosine(Cyt) (C) and guanine (G).Development is accurate, the DNA sequencing method of high-throughput, low cost has very important significance for biology, medical science etc.
Synthesis method order-checking (SequencingBySynthesis, SBS) is one of DNA sequencing technology of new generation.Synthesis method sequence measurement is by being fixed template DNA fragment tested in a large number, and hybridization, in conjunction with general DNA primer, controls four kinds of Nucleotide extensions on DNA primer respectively in immobilized DNA sequencing template.By detecting extension process or extending Nucleotide, realize the detection of the DNA sequence dna information of high-flux parallel.
In synthesis method order-checking, first want four kinds of nucleotide material of synthetic DNA chain extension, i.e. reversible terminator (reversibleterminator).This kind of Nucleotide, except requiring 3 '-hydroxyl and blocking, in order to not affect extension and the identification of next labeled nucleotide, also requires that the connector element can sheared by is coupled together Nucleotide and fluorescein.Then, before next labeled nucleotide extends, under mild conditions the fluorescein of mark is sheared completely, so that next labeled nucleotide continues to participate in DNA chain extension, thus read base sequence.The read long and efficiency of this connector element on order-checking has very important impact, and therefore, people are also devoted to develop new cleavable connector element, to improve the efficiency of DNA sequencing always.
The read long and efficiency of connector element on DNA sequencing can be sheared and have important impact, and existing connector element exists, and shearing condition is gentle not, efficiency is not high, read during for checking order to grow the shortcomings such as too short, therefore, design, synthesize new connector element, and explore suitable shearing condition for improve order-checking efficiency, develop new sequence measurement and have very important meaning.
The disulfide linkage Reversible terminal dUTP-SS-TAMRA usually adopted in prior art, when the template of continuous multiple base A, once extensible two reversible terminators, and cannot ensure that an extension only extends a reversible terminator.At present in the art, in order to ensure once only extending a reversible terminator, the modified nucleotide that use 3 '-OH protects often all is needed.But the modified nucleotide synthesis of 3 '-OH protection is complicated, productive rate is low, product purification difficulty; And in actual sequencing procedure, when using the reversible terminator of 3 '-OH protection to extend, need two reaction site to rupture simultaneously, that is, also need while fluorescein 100% being sheared, with 100% efficiency, 3 '-OH is gone protection.But in sequencing procedure, these two sites often can not be sheared simultaneously, or cleavage reaction also not exclusively, and this certainly will cause the mistake in sequencing procedure to be accumulated, and what final impact was checked order reads length and efficiency.
In sum, this area is high in the urgent need to a kind of accuracy, particularly can have very high extension efficiency and the DNA sequencing method of shear efficiency when template is continuous multiple identical base.
Summary of the invention
The object of the invention is to the deficiency overcoming the existence of above-mentioned prior art, provide a kind of accuracy high, particularly can have higher extension efficiency and the extension method of shear efficiency when template is continuous multiple identical base.
The present invention relates to a kind of DNA sequencing method, described method comprises:
A () is reversible terminator with the acid-sensitive modified nucleotide shown in formula (I), participate in DNA chain extension reaction and
B () is ruptured to the acid-sensitive connector element in described acid-sensitive modified nucleotide;
Wherein,
Described fluorescein is selected from: the combination of one or more in BODIPY, rhodamine, tonka bean camphor, cluck ton, cyanine, pyrene, phthalocyanine, Alexa, Squaring dyestuff, generate energy transferred dyes;
Described Base (base) expression is selected from following base: U, T, C, A or G.
Preferably, described fluorescein is selected from: the combination of one or more in Cy2, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, FITC, TexasRed-X, AF594, Bodipy-FL, R6G, ROX, Bodipy-650, AlexaFluor594, DyLight594, AlexaFluor647, DyLight649, AlexaFluor546, DyLight549.
Preferably, described acid-sensitive modified nucleotide is selected from:
Preferably, in step (a), in described DNA chain extension reaction, enzyme used comprises Klenow, 9 ° of N or Theminator.
Preferably, in step (b), described fracture acid used comprises dilute hydrochloric acid, dilute sulphuric acid or various lewis acid.
Preferably, in step (b), the breaking temperature of described fracture is 25 DEG C ~ 60 DEG C.Be more preferably 37 ± 2 DEG C.
Preferably, in step (b), described in break at pH and carry out for 2 ~ 5 times.
Preferably, in step (b), the rupture time of described fracture is 0.5 ~ 10min.Be more preferably 1 ~ 3min.
Preferably, 3 '-OH of described acid-sensitive connector element does not have protecting group, or 3 '-OH of described acid-sensitive connector element is unprotected.
The present inventor provides a kind of preparation method of acid-sensitive connector element in Chinese patent application 201410186697.6, and by such connector element for the synthesis of corresponding fluorescence-labeled nucleotides and reversible terminator.Prove that fluorescein can cut away by such reversible terminator under mild conditions with non denatured glue further, there are the potentiality being applied to DNA sequencing, and prove when extending efficiency and not yet reaching 100% with sex change glue and sequencing gel, when template is continuous multiple identical base, a reversible terminator once can only be extended.Sequencing gel Figure 37 in Chinese patent application 201410186697.6 is the extension products of fluorescent nucleotide dUTP, this figure shows that the primary product extended is for only to extend a reversible terminator, but can be clear that to there is a large amount of by products in the process from this Figure 37.And the Figure 38 in Chinese patent application 201410186697.6 is fluorescently-labeled dCTP reversible terminator DNA chain extension reaction in embodiment 20, from this Figure 38, really a reversible terminator is once only extended, but therefrom also can find easily, extension products is also incomplete, a lot of primer is still had not react, in biochemical reaction, those skilled in the art will know that when reactant not yet completely dissolve time, second time extension generally can not occur, that is cannot illustrate when extending efficiency and reaching 100% in Chinese patent application 201410186697.6, once can only extend a reversible terminator, Figure 39 and Figure 40 in Chinese patent application 201410186697.6 is respectively dGTP and dATP reversible terminator DNA chain extension reaction sex change glue figure, equally all when extension is incomplete, once can only extend a reversible terminator.
The present invention is under the condition repeatedly optimizing biological assessment, pay a large amount of creative works, the efficiency of extension being reached 100%, when there is multiple repetition base in template, a fluorescent nucleotide and reversible terminator can be extended at an extension.
Compared with prior art, the present invention has following beneficial effect:
(1) method of the present invention under mildly acidic conditions, and the fluorescein of mark is by while quick and complete cutting away, and the protecting group of 3 '-hydroxyl also can go protection with the efficiency of 100%, have the potential value being applied to DNA sequencing.
(2) the present invention compares detailed evaluation by the performance and advantage such reversible terminator being applied to DNA sequencing, find that the fluorescein of mark not only can cut away by such fluorescence-labeled nucleotides under mildly acidic conditions, and this connector element all quick and completely can rupture in the basic conditions and under neutrallty condition.
(3) shear efficiency of the inventive method is very high, all can reach 100% at shear efficiency that is acid, alkaline and neutrallty condition downward-extension product; Cleavage reaction is very thoroughly clean, generates without any by product.
(4) at the reversible terminator of the order-checking widely used disulfide linkage in field, after reductive agent DTT fracture disulfide linkage, must be sulfhydryl protected by what generate with iodo-acid amide, otherwise can permutoid reaction be there is with the reversible terminator newly added in sulfydryl.But the inventive method, in the fluorescein process of excision mark, can not cause damage to DNA chain, thus without the need to protecting.
(5) the present invention's sequencing gel proves that the first time of such reversible terminator extends efficiency, second time extends efficiency and the 4th extension efficiency all can reach 100%, and corresponding fracture efficiency is also close to 100%, and prove by phenol chloroform MALDI-MS further, the structure of its extension products and cleavage product is correct.
(6) and under identical condition, disulfide linkage Reversible terminal dUTP-SS-TAMRA, when template be continuous multiple base is A, once can extend two Reversible terminal.And adopt method of the present invention, when template is continuous multiple A, C, G, T, its extension products is and once only extends a reversible terminator containing U, G, C, A base.
(7) under similarity condition, azo reversible terminator dUTP-azo-TAMRA is when template is continuous multiple identical base A, and extension products there will be the situation once extending one and two reversible terminator.And adopt method of the present invention, when template is continuous multiple A, C, G, T, its extension products is and once only extends a reversible terminator containing U, G, C, A base.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 is the exemplary plot of sequence measurement of the present invention;
Fig. 2 is the sequencing gel figure of the extension products of embodiment 2;
Fig. 3 is the sequencing gel figure of the extension products of embodiment 3;
Fig. 4 is the sequencing gel figure of embodiment 4;
Fig. 5 A is the MALDI-MS spectrogram of the extension products of embodiment 5, and Fig. 5 B is the MALDI-MS spectrogram of the cleavage product of embodiment 5;
Fig. 6 A extends the schematic diagram with cleavage reaction in embodiment 6.1, Fig. 6 B is the electrophorogram of product;
Fig. 7 is the electrophorogram of the cleavage product of embodiment 6.2;
Fig. 8 is the sequencing gel figure of reversible terminator dCTP-DMKL-BODIPY-FL, dATP-DMKL-Cy5;
Fig. 9 is the sequencing gel figure of reversible terminator dGTP-DMKL-Cy3.5;
Figure 10 is the sequencing gel figure that four look fluorescent marks, four different nucleotide bases extend four times continuously in reaction system;
Figure 11 is that on magnetic bead, first time extends (a), first time fracture (b), second time extends (c), second time fracture (d), third time extends (e), third time fracture (f) and extend the fluorogram of (g) for the 4th time.
Figure 12 is the sequencing gel figure of the reversible terminator dUTP-SS-TAMRA of disulfide linkage of comparative example 1;
Figure 13 is the sequencing gel figure of the reversible terminator dUTP-azo-TAMRA of azo of comparative example 2.
Embodiment
The present inventor is based on long-term and deep research, provide a class DNA sequencing method, described method adopts specific Reversible terminal to participate in extending, and under appropriate conditions specificity extension is carried out to DNA chain, extend efficiency and can reach 100%, when particularly there is continuous multiple base in a template, once only extend a base.And without the need to protecting template in extension process.Based on above-mentioned discovery, contriver completes the present invention.
Based on the reversible terminator of acid-sensitive connector element
The object of the present invention is to provide a kind of DNA sequencing method (as Fig. 1) based on acid-sensitive modified nucleotide, also can shear modified nucleotide for one (also to claim, the reversible terminator of acid-sensitive) purposes in DNA sequencing, be included in the efficiency in DNA chain extension reaction, shear efficiency and again extend efficiency; And such reversible terminator is when template is continuous multiple identical base, still only a reversible terminator can be extended, and it extends efficiency can reach 100%, under mildly acidic conditions, its shear efficiency can reach 100%, second time extends efficiency can reach 100% equally, until extending efficiency the 4th time is still 100%.By contrast, traditional disulfide linkage Reversible terminal when DNA profiling is continuous multiple identical base first time extension once can connect two reversible terminators, these data suffice to show that acid-sensitive Reversible terminal has the extraordinary prospect and value being applied to DNA sequencing.The present invention demonstrates with MALDI-MS and sequencing gel dual mode the good result that it is applied to DNA sequencing first.
The structural formula of the reversible terminator of acid-sensitive of the present invention is such as formula shown in (I):
The reversible terminator based on acid-sensitive connector element that the present invention proposes, its biological assessment shows, under mildly acidic conditions, the fluorescein of mark is cut away by quick and complete, has the value being applied to DNA sequencing.The present invention compares detailed evaluation by the performance and advantage such reversible terminator being applied to DNA sequencing, find that the fluorescein of mark not only can cut away by such fluorescence-labeled nucleotides under mildly acidic conditions, and this connector element all quick and completely can rupture in the basic conditions and under neutrallty condition.And shear efficiency is very high, cleavage reaction is very thoroughly clean, generates without any by product.By contrast at the reversible terminator of the order-checking widely used disulfide linkage in field, after reductive agent DTT fracture disulfide linkage, must be sulfhydryl protected by what generate with iodo-acid amide, otherwise can permutoid reaction be there is with the reversible terminator newly added in sulfydryl.Based on experimental fact of the present invention, certainly, such reversible terminator based on acetal and ketal structure, in the process of being cut away by the fluorescein of mark, can not cause damage to DNA chain.The present invention's sequencing gel method proves that the first time of such reversible terminator extends, second time extends and extend efficiency for the 4th time all can reach 100%, and fracture efficiency also can reach 100%; And prove that its structure is correct by phenol chloroform MALDI-MS further.More pleasurable, when template is continuous multiple identical base A, C, G, T, its extension products is and once only extends a reversible terminator containing U, G, C, A base.
Below in conjunction with specific embodiment, set forth the present invention further.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, conveniently condition in the following example, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
embodiment 1, fluorescein-labelled connector element velocity of shear of shearing compare
1.1 acid-sensitive connector elements connect the investigation of compound Y016 and Y039 crack velocity under pH=4.0 condition of fluorescein:
1) pH=4.0 buffer preparation
Pipette 5.7mL0.2mol/LNa respectively
2hPO
4solution, 14.3mL0.1mol/L citric acid solution, 20mL Chromatographic Pure Methanol, pH meter actual measurement pH=4.07;
2) fracture experiment
Weigh Y016 (0.88mg, 0.00152mmol) and Y039 (1mg, 0.00153mmol) respectively, add in 5mL round-bottomed flask, under lucifuge condition, add 1mLpH=4.07 buffered soln respectively.Respectively at 2min, 4min, 6min, 8min, 10min, after pipetting 45 μ L reaction solutions, add the saturated NaHCO of 20 μ L
3with cancellation reaction solution in solution, analysis mode HPLC analyzes breaking effect;
3) disrupt data
Record Y016 and the Y039 that do not rupture of different time respectively, shared its adds the per-cent of total amount separately.Conclusion: under pH=4.07 condition, Y016 crack velocity is faster than Y039.
| Sample | 2min | 4min | 6min | 8min | 10min |
| Y016 | 26.894％ | 5.638％ | 1.816％ | 0.516％ | 0.001％ |
| Y039 | 36.276％ | 6.239％ | 2.877％ | 1.109％ | 0.387％ |
1.2 acid-sensitive connector elements connect the investigation of compound Y016 and Y039 crack velocity under pH=3.5 condition of fluorescein
1) pH=3.5 buffer preparation
Pipette 3.44mL0.2mol/LNa respectively
2hPO
4solution, 16.56mL0.1mol/L citric acid solution, 30mL Chromatographic Pure Methanol, pH meter actual measurement pH=3.5;
2) fracture experiment
Weigh Y016 (0.86mg, 0.0015mmol) and Y039 (1mg, 0.00153mmol) respectively, add in 5mL round-bottomed flask, under lucifuge condition, add 1mLpH=3.5 buffered soln respectively.Respectively at 2min, 4min, 6min, 8min, 10min, after pipetting 45 μ L reaction solutions, add the saturated NaHCO of 20 μ L
3with cancellation reaction solution in solution, analysis mode HPLC detects its crack velocity;
3) disrupt data
Record Y016 and the Y039 that do not rupture of different time respectively, shared its adds the per-cent of total amount separately.Conclusion: under pH=3.5 condition, Y016 crack velocity is obviously fast than Y039.
| Sample | 2min | 4min | 6min | 8min | 10min |
| Y016 | 2.014％ | 0.464％ | 0％ | ||
| Y039 | 12.501％ | 1.755％ | 0.391％ | 0.071％ | 0.006％ |
, can not mispairing be there is in embodiment 2, reversible terminator Y017 energy specific recognition base A
The primer of base pair complementarity and template are dissolved in Tris-EDTA damping fluid (TE, pH7.5), anneal according to following condition: 95 DEG C are incubated 3 minutes, are then cooled to 4 DEG C with the speed of 0.1 DEG C/sec and are incubated.Annealed primer and template form double-strand, can be used for DNA extension.Add successively in PCR pipe: 5 μ L1xThermoPol reaction buffers, 5 μ L sodium chloride solutions (1M), the primer that 2 μ L are annealed and template (1 μ g/ μ L), 10 μ LdUTP-DMKL-TAMRA (1mM), 1 μ L (2U) TherminatorDNA polysaccharase, 27 μ L aqua sterilisas, form the reaction system that cumulative volume is 50 μ L.After mixing, this reaction system be heated to 72 DEG C and be incubated 8.5 minutes, then naturally cooling to room temperature.Through phenol chloroform and alcohol settling, obtain extension products.Extension products is processed 90 seconds with a small amount of hydrochloric acid (10mM) at 37 DEG C, fluorophore (TAMRA) on dUTP-DMKL-TAMRA is ruptured, then adds and with the sodium hydroxide solution of hydrochloric acid equivalent (10mM), cleavage reaction is stopped.The cleavage product obtained can be used for extension next time.
The sequencing gel figure of extension products as shown in Figure 2, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4, Lane5, Lane6.
Lane1：24nt
Lane2：25nt
Lane3:Y017 first time extension products (template is the oligo3 of an A)
Lane4:Y017 does not extend (template is the oligo11 of a C)
Lane5:Y017 does not extend (template is the oligo7 of a G)
Lane6:Y017 does not extend (template is the oligo15 of a T)
·Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCGTCGAoligo7(SEQIDNO.6)
·Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCTACGAoligo15(SEQIDNO.12)
·Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCCTColigo11(SEQIDNO.8)
·Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCATCGATCGCCATGTCGOilgo3(SEQIDNO.3)
Conclusion: the reversible terminator dUTP-DMKL-TAMRA of acid-sensitive can specific recognition base A, mispairing can not occur.
embodiment 3, reversible terminator participate in DNA chain extension, and when template is continuous multiple A, one time extension can only extend a reversible terminator, and extension products and shearing product have identical Rf value on sequencing gel.That is they cannot distinguish on sequencing gel, this is because acid-sensitive connector element ruptures in the process of running glue, and with MALDI-MS, follow-up embodiment 5 proves that the structure of extension products and cleavage product is correct.
The experimental implementation of this embodiment, each step agents useful for same and condition are with embodiment 2.
The sequencing gel figure of extension products as shown in Figure 3, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4, Lane5, Lane6.
Lane1：24nt：
Lane2：25nt：
Lane3:Y017oligo2-3 extension products; (template Oilgo3 is containing an A)
Lane4:Y017oligo2-4 extension products; (continuous two A of template Oilgo4)
Lane5:Y017oligo2-6 extension products; (continuous four A of template Oilgo6)
Lane5:Y017oligo2-1 extension products; (continuous 12 A of template Oilgo1)
Lane6:Y017oligo2-4 extension/cleavedproduct;
Conclusion: 1., when template is continuous multiple A, fluorescent nucleotide dUTP has successfully carried out extension, and extension can only extend a fluorescence-labeled nucleotides and reversible terminator each time.
2. extension products acid-sensitive connector element in dehybridization procedure ruptures, sequencing gel is observed whole be post-rift product, cannot extension products be observed.
3.Y017 the efficiency participating in DNA chain extension reaction is 100%.
·5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·3’-CTCCTTTCCCTTCCCTTTCCTTCCATCGATCGCCATGTCGOilgo3(SEQIDNO.3)
·Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAACGATCGCCATGTGCOligo4(SEQIDNO.4)
·Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAAAAATCGCCATGTGCOligo6(SEQIDNO.5)
·Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAAAAAAAAAAAAGTGCOligo1(SEQIDNO.1)
embodiment 4, reversible terminator participate in DNA chain extension, its first time extend to extend with second time efficiency all with the efficiency that ruptures all close to 100%
The reversible terminator dUTP of the acid-sensitive that the present embodiment relates to, participates in DNA chain extension reaction and cleavage reaction evaluation, and for dUTP-DMKL-TAMRA, its schematic diagram participating in DNA chain extension reaction and cleavage reaction is as follows:
The exemplary plot of the present embodiment and sequencing gel figure as shown in Figure 4, from left to right, are followed successively by Lane1, Lane2, Lane3, Lane4.
Lane1：24nt
Lane2：25nt
Lane3:acidY017 first time extension/cleavedproduct
Lane4:acidY017 second time extension/cleavedproduct
That observes on sequencing gel for acid-sensitive Y017 only has cleavage product, because all extension products are when unwinding double-strand as strand, acetonylidene connector element ruptures.
·Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
·Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAACGATCGCCATGTGCOligo4(SEQIDNO.4)
Above-mentioned sequencing gel the results show, the efficiency of extension is 100% for the first time, and fracture efficiency is also 100%; And the efficiency of second time extension is similarly 100%.
embodiment 5, MALDI-MA confirm that the structure of DNA chain first time extension product, cleavage product, second time extension products and the 4th extension products is correct further.
The primer used in extension and template as follows:
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCATCGATCGCCATGTCGOilgo3(SEQIDNO.3)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAACGATCGCCATGTGCOligo4(SEQIDNO.4)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAAAAGTCGCCATGTGCOligo6(SEQIDNO.5)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAAAAAAAAAAAAGTGCOligo1(SEQIDNO.1)
The mass spectrum of extension products as shown in Figure 5.
In first time extension products, the peak m/z7663 completely dissolve of former primer oligo2, extension products m/z is 8518 appearance (Fig. 5 A); In extension products, adding acid, the completely dissolve of former extension products 8518, there is (Fig. 5 B) in the peak m/z8090 of cleavage product; First time extends efficiency close to 100% as can be seen from Figure 5A, from Fig. 5 B, and fracture efficiency 100%.This MALDI-MS spectrogram is not through purifying, and in figure, m/z11963 is the peak (oligo4) of template.From this experimental result MALDI-MS spectrogram, extend efficiency with fracture efficiency all close to 100%.
embodiment 6: when template is continuous four A, extension is same with the efficiency of cleavage reaction reaches 100%, and the reversible terminator synthesized by this result proves has the value and potentiality that are applied to DNA sequencing.
The reversible terminator used in 6.1 embodiments 6.1 is: dUTP-DMKL-TAMRA
The sequence of the primer that extension uses and template is as follows:
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAAAAATCGCCATGTGCOligo6(SEQIDNO.5)
As shown in Figure 6A, this schematic diagram comprises 4 times and extends and the process of 4 cleavage reactions and the product of generation the schematic diagram of its extension and cleavage reaction.Fig. 6 B shows the electrophorogram of product, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4, Lane5, Lane6, Lane7, Lane8, Lane9, Lane10.The corresponding relation of each swimming lane is as follows:
Lane1：24nt
Lane2：25nt
Lane3: extension products for the first time
Lane4: cleavage product for the first time
Lane5: second time extension products
Lane6: second time cleavage product
Lane7: extension products for the third time
Lane8: cleavage product for the third time
Lane9: the four extension products
Lane10: the four cleavage product
As can be seen from Fig. 6 B, when template is continuous four A, first time is 100% to the efficiency of the 4th extension, and first time is also 100% to the efficiency of the 4th cleavage reaction.No matter be extension or cleavage reaction simultaneously, all very clean, complete, do not observe any by product and generate.Illustrate that such reversible terminator has the great potential and value being applied to DNA sequencing.
The tolerance test of 6.2DNA chain to this shearing condition under the acid shearing condition of reversible terminator
The electrophorogram of cleavage product as shown in Figure 7, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4, Lane5, Lane6, Lane7.Wherein, the corresponding relation of each swimming lane is as follows:
Lane1：0
Lane2：0.5h
Lane3：1h
Lane4：3h
Lane5：6h
Lane6：12h
Lane7：24h
As can be seen from Figure 7, within 6h, DNA is not subject to obvious loss.Prove that this reversible terminator is feasible for checking order in acid condition.
the reversible terminator of fluorescence-labeled nucleotides of embodiment 7, other base (C, A, G) participates in the evaluation of DNA chain extension reaction
DNTP (AP
3) the DNA chain extension reaction (N=C, A, G) of-DMKL-Fluorophore
Oligo2 is combined respectively with corresponding template and forms DNA double chain, i.e. 2-7,2-8 to 2-18: get oligo2 and other oligo template 5uL in PCR pipe, then 95 DEG C of 3min and to be down to 15 DEG C of preservations with 0.1 DEG C/S stand-by.Prepare capillary electrophoresis glue again.
1) for reversible terminator dCTP-DMKL-BODIPY-FL, template used as follows:
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCGTCGAoligo7(SEQIDNO.6)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCGGCGAoligo8(SEQIDNO.7)
For reversible terminator dATP-DMKL-Cy5, template used as follows:
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCTAoligo15(SEQIDNO.13)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCTTCGAoligo16(SEQIDNO.14)
Reversible terminator dCTP-DMKL-BODIPY-FL and dATP-DMKL-Cy5 of fluorescein-labelled Nucleotide is used for DNA chain extension reaction.
In eppendorf pipe, the DNA chain extension reaction of Reversible terminal is set up according to following system:
Cumulative volume 100 μ L, is placed in 30 DEG C, 15min, 75 DEG C, 10min by reaction system, 16 DEG C of preservations.Phenol chloroform, after alcohol settling simmer down to solid, be dissolved in the water of respective volume and make its concentration reach 40ng/ μ L, add 0.1MNaOH, after 95 DEG C of 5min process, sex change glue (i.e. sequencing gel) as shown in Figure 8, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4, Lane5, Lane6.
Lane1：24nt：
Lane2：25nt：
Lane3:oligo2-7, when template only has a G, can only connect a dCTP-DMKL-BODIPY-FL; Extend efficiency 100%.
Lane4:oligo2-8, when template has two G continuously, is also connect a dCTP-DMKL-BODIPY-FL; Extend efficiency 100%.
Lane5:oligo2-15, when template only has a T, can only connect a dATP-DMKL-Cy5; Extend efficiency 100%.
Lane6:oligo2-16, when template has two T continuously, is also connect a dATP-DMKL-Cy5; Extend efficiency 100%.
As can be seen from Figure 8, when oligo2 is primer, when template is continuous two G or T, reversible terminator dCTP-DMKL-BODIPY-FL and dATP-DMKL-Cy5 all can only extend one, and extension efficiency is 100%.
1) for reversible terminator dGTP-DMKL-Cy3.5, template used as follows:
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCCTCGAoligo11(SEQIDNO.9)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCCCCGAoligo13(SEQIDNO.10)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCCCCCGoligo14(SEQIDNO.11)
Fluorescein-labelled reversible terminator dGTP-DMKL-Cy3.5 is used for DNA chain extension reaction.
In eppendorf pipe, the DNA chain extension reaction of reversible terminator is set up according to following system:
Cumulative volume 100 μ L, is placed in 30 DEG C, 15min, 75 DEG C, 10min by reaction system, 16 DEG C of preservations.Phenol chloroform, after alcohol settling simmer down to solid, is dissolved in the water of respective volume and makes its concentration reach 40ng/ μ L, add 0.1MNaOH, after 95 DEG C of 5min process, carry out capillary electrophoresis analysis.Result as shown in Figure 9, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4.
Lane1：24nt：
Lane2：25nt：
Lane3:oligo2-11, dGTP-DMKL-Cy3.5, extension products; (template C)
Lane4:oligo2-13, dGTP-DMKL-Cy3.5, extension products; (template 3 C)
As can be seen from Figure 9 oligo2 is primer, and when template is continuous 3 C, reversible terminator dGTP-DMKL-Cy3.5 can only extend one, and extension efficiency is 100%.
Conclusion: from above experiment, when template is many T, when many G, many C, each extension all can only extend a corresponding fluorescence-labeled nucleotides and reversible terminator.
the reversible terminator of fluorescence-labeled nucleotides of embodiment 8, four different bases (U, C, A, G) participates in the evaluation of DNA chain extension reaction
Oligo2 is combined respectively with corresponding template and forms DNA double chain, i.e. 2-3: get oligo2 and oligo3 template 5uL in PCR pipe, then 95 DEG C of 3min and to be down to 15 DEG C of preservations with 0.1 DEG C/S stand-by.Prepare capillary electrophoresis glue again.
Template used as follows:
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCATCGATCGCCATGTCGOligo3
Four fluorescence-labeled nucleotides mixing are added reaction system and carries out DNA chain extension reaction.
In eppendorf pipe, the DNA chain extension reaction of Reversible terminal is set up according to following system:
Cumulative volume 100 μ L, is placed in 30 DEG C, 15min, 75 DEG C, 10min by reaction system, 16 DEG C of preservations.Phenol chloroform, after alcohol settling simmer down to solid, be dissolved in the water of respective volume and make its concentration reach 40ng/ μ L, add 0.1MNaOH, after 95 DEG C of 5min process, sex change glue (i.e. sequencing gel) as shown in Figure 10, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4, Lane5, Lane6.
Lane1：24nt：
Lane2：25nt：
Lane3: extension products for the first time
Lane4: second time extension products
Lane5: extension products for the third time
Lane6: the four extension products
As can be seen from Figure 10, the fluorescein-labelled Nucleotide mixing of four different bases adds reaction system, can extend four times continuously, and extends efficiency and fracture efficiency is all very high at every turn.
with the experiment of magnetic bead, embodiment 9: when template is continuous four A, proves that extension is same with the efficiency of cleavage reaction and reaches 100%, this result proves that synthesized reversible terminator has the value and potentiality being applied to DNA sequencing further again.
Supernatant liquor is taken out after 5 microliters of magnetic beads (10mg/mL) are washed with 1X damping fluid in 150 microlitre reaction tubess, add 10 microlitre 2X damping fluids, then 1 microlitre DNA profiling (100 μMs) is added, add 9 microlitre Tris-HCl (10mM again, pH7.5), oscillatory reaction 15 minutes at 25 DEG C.Then with magnet, magnetic bead is inhaled bottom reaction tubes, take out supernatant liquor, use 1X buffer solution, take out supernatant liquor.In above-mentioned magnetic bead, add 1.5 l primer solution (100 μMs), then add 10 microlitre Tris-HCl (10mM, pH7.5), react 3 minutes at 95 DEG C, then naturally cool to room temperature.Take out supernatant liquor, use 1X buffer solution, take out supernatant liquor.In above-mentioned magnetic bead, add 3 microlitre DNA polymerase buffer liquid successively, 1.5 microlitre acid-sensitive dNTPs (1mM), 1 microlitre TherminatorDNA polysaccharase, 14 microliters of sterile water, form the reaction system of 20 microlitres.At 72 DEG C, oscillatory reaction 8 minutes, takes out supernatant liquor, uses 1X buffer solution, takes out supernatant liquor.Finally add the damping fluid that pH value is 2.3, oscillatory reaction 2 minutes.Take out supernatant liquor, take out supernatant liquor with after 1X buffer solution.Figure 11 is first time extension, for the first time fracture, second time extends, rupture for the second time, third time extends, third time ruptures and the fluorogram of the 4th extension.
Reversible terminator of the present invention comprises all structures described in patent 201410186697.6, these Reversible terminal are applied to being distinguished as of DNA sequencing, the actual conditions of extension and cleavage reaction that each compound participates in DNA chain has a difference a little, but their extension efficiency and shear efficiency all can reach 100%, and the fluorescein that all will be able to mark fast, completely under acidity, alkalescence and neutrallty condition is sheared.These experimental results (MALDI-MS and sequencing gel dual mode) show, such Reversible terminal all can perform well in order-checking, show as each time extend efficiency all up to 100% with each time the fluorescein of mark is cut away the shear efficiency that all can realize 100%.Its concrete structure is as follows:
comparative example 1: the reversible terminator dUTP-SS-TAMRA of disulfide linkage extends the evaluation (when template is continuous multiple A, once extensible two reversible terminators, and only can not extend) of efficiency.
Patent 201310401580.0 and 201310015235.3 is seen about the structure of the reversible terminator dUTP-SS-TAMRA of disulfide linkage, synthesis and sign thereof.Sequencing gel result as shown in Figure 12, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4, Lane5.
Lane1：24nt：
Lane2：25nt：
Lane3:oligo2-3, when template only has an A, extends a Reversible terminal dUTP-SS-TAMRA; Extending efficiency is 100%.
Lane4:oligo2-4, when template has two A continuously, extends two dUTP-SS-TAMRA; Extending efficiency is 100%.
Lane5:oligo2-3, when template is AT, can only extend a reversible terminator dUTP-SS-TAMRA, extends efficiency 100%.Second reversible terminator dATP-SS-Cy5, when the previous reversible terminator dUTP-SS-TAMRA extended is not sheared, can not extend.Only have reversible terminator when previous extension by cleaved after, next reversible terminator could continue to extend.
5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
3’-CTCCTTTCCCTTCCCTTTCCTTCCATCGATCGCCATGTCGOilgo3(SEQIDNO.3)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAACGATCGCCATGTGCOligo4(SEQIDNO.4)
Remarks:
The advantage that the reversible terminator of disulfide linkage compared by the reversible terminator of acid-sensitive of the results show the present invention synthesis of this comparative example is, when template is continuous multiple A, the reversible terminator of acid-sensitive can only extend one at every turn, and disulfide linkage can extend two reversible terminators.And as can be seen from Figure 12 also, the extension of the reversible terminator of disulfide linkage and cleavage reaction all have by product to generate, reaction is clean not, complete.For when surveying multiple base continuously for the needs that check order veritably in the future, immeasurable noise can be brought, have a strong impact on its order-checking efficiency and read length.
But also illustrate simultaneously, the reversible terminator of disulfide linkage is not before the previous reversible terminator extended is sheared, next reversible terminator can not extend, the reversible terminator of previous extension is only had to be sheared, next reversible terminator could extend smoothly, so the reversible terminator of disulfide linkage also may be used for order-checking, but its reaction is complete not, has by product to generate.Because when template is base AT, if the fluorescein in the dUTP-SS-TAMRA of first time extension is not sheared, then second reversible terminator dATP-SS-Cy5 can not extend; After the fluorescein of the reversible terminator dUTP-SS-TAMRA of first time extension is sheared, then second reversible terminator dATP-SS-Cy5 can extend smoothly with the efficiency of 100%.
comparative example 2: the reversible terminator dUTP-azo-TAMRA of azo-based connector element is when first time, extension not yet reached 100%, and second time extension products occurs in a large number.(when template is continuous multiple A, cannot ensure once only to extend a reversible terminator).
Patent 201310462509.3 is seen about the structure of the reversible terminator dUTP-azo-TAMRA of azo, synthesis and sign thereof.Sequencing gel result as shown in Figure 13, from left to right, is followed successively by Lane1, Lane2, Lane3, Lane4.
Lane1：24nt：
Lane2：25nt：
Lane3:oligo2-3, when template only has an A, extends a reversible terminator dUTP-AZO-TAMRA; Part primer is still had not extend.
Lane4:oligo2-4, when template has two A continuously, when primer does not have completely dissolve, comprises the product of one and two dUTP-AZO-TAMRA in extension products; Extend efficiency not high.
5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
3’-CTCCTTTCCCTTCCCTTTCCTTCCATCGATCGCCATGTCGOilgo3(SEQIDNO.3)
Primer：5’-GAGGAAAGGGAAGGGAAAGGAAGGOligo2(5’-Dylight800)(SEQIDNO.2)
Template：3’-CTCCTTTCCCTTCCCTTTCCTTCCAACGATCGCCATGTGCOligo4(SEQIDNO.4)
Can find out that the reversible terminator that the reversible terminator based on acid-sensitive connector element of the present invention compares the reversible terminator of disulfide linkage and azo-based connector element all has the great potential and good prospect that are better applied to DNA sequencing from comparative example 2.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.In addition, all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (9)
1. a DNA sequencing method, is characterized in that, described method comprises:
A () is reversible terminator with the acid-sensitive modified nucleotide shown in formula (I), participate in DNA chain extension reaction and
B () is ruptured to the acid-sensitive connector element in described acid-sensitive modified nucleotide;
Wherein,
Described fluorescein is selected from: BODIPY, rhodamine, tonka bean camphor, xanthene, cyanine, pyrene, phthalocyanine, Alexa, Squaring dyestuff, generate energy transferred dyes, or its combination;
Described Base represents and is selected from following base: U, T, C, A, G.
2. DNA sequencing method as claimed in claim 1, it is characterized in that, described fluorescein is selected from Cy2, Cy3, Cy3.5, Cy5, Cy5.5, TAMRA, FITC, TexasRed-X, AF594, Bodipy-FL, R6G, ROX, Bodipy-650, AlexaFluor594, DyLight594, AlexaFluor647, DyLight649, AlexaFluor546, DyLight549, or its combination.
3. DNA sequencing method as claimed in claim 1, it is characterized in that, described acid-sensitive modified nucleotide is selected from:
4. DNA sequencing method as claimed in claim 1, it is characterized in that, in step (a), in described DNA chain extension reaction, enzyme used comprises Klenow, 9 ° of N, Bst or Theminator.
5. DNA sequencing method as claimed in claim 1, is characterized in that, in step (b), described fracture acid used comprises dilute hydrochloric acid, dilute sulphuric acid or lewis acid.
6. DNA sequencing method as claimed in claim 1, it is characterized in that, in step (b), the breaking temperature of described fracture is 25 DEG C ~ 60 DEG C.
7. DNA sequencing method as claimed in claim 1, is characterized in that, in step (b), described in break at pH and carry out for 2 ~ 5 times.
8. DNA sequencing method as claimed in claim 1, it is characterized in that, in step (b), the rupture time of described fracture is 0.5 ~ 10min.
9. DNA sequencing method as claimed in claim 1, it is characterized in that, 3 '-OH of described acid-sensitive connector element does not have protecting group, or 3 '-OH of described acid-sensitive connector element is unprotected.
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