Summary of the invention
The present invention relates to the fluorescently-labeled Nucleotide of class 5 '-terminal phosphate, provide molecular structure and synthetic method.This quasi-molecule can better identify by archaeal dna polymerase, and realizing fluorescent signal by the fast transition of closing to opened condition by polysaccharase identification and after correct combination under alkaline phosphatase is assisted, thus can be applied in the fields such as determining nucleic acid sequence as polymerase substrate.More detailed description is had in the patent CN 201510155218.9 that the synthetic method of fluorescence molecule concrete in this case is applied for applicant.
The invention provides the fluorescently-labeled nucleotide structure of a kind of terminal phosphate, it is characterized in that: there is structure shown in following general formula (1),
Wherein R
a, R
bindependently can be selected from-H ,-OH; N for being more than or equal to 1, and is less than or equal to the integer of 5;
Wherein, the fluorophor in general formula (1) possesses general formula (2) described structure below,
Wherein, R in general formula (2)
1, R
5independently can be selected from the C1-C6 alkoxyl group of-H, fluorine, chlorine, bromine, aryl, substituted aryl, C1-C6 alkyl, the C1-C6 alkyl of replacement, C1-C6 alkoxyl group, replacement;
R
2-4, R
6-8, R
11-13can independently be selected from-H, fluorine, chlorine, bromine, aryl, substituted aryl, heteroaryl ,-CO
2h ,-CO
2r ,-SO
3h ,-SO
3r ,-CH
2cO
2h ,-CH
2cO
2r ,-CH
2sO
3h ,-CH
2sO
3r ,-CH
2nH
2,-CH
2nHR ,-NO
2, C1-C6 alkyl, the C1-C6 alkyl replaced, C1-C6 alkoxyl group, the C1-C6 alkoxyl group replaced, C1-C6 alkoxy aryl, the C1-C6 alkoxy aryl replaced, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzoyl, wherein R is selected from C1-C6 alkyl, the C1-C6 alkyl replaced, C1-C6 alkoxyl group, the C1-C6 alkoxyl group replaced, C1-C6 alkoxy aryl, the C1-C6 alkoxy aryl replaced, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzoyl,
R
9, R
10independently can be selected from C1-C6 alkyl, the C1-C6 alkyl of replacement, C1-C6 alkoxyl group, the C1-C6 alkoxyl group of replacement, C1-C6 alkoxy aryl, the C1-C6 alkoxy aryl of replacement, benzyl, replacement benzyl.
The invention provides a kind of method of synthesizing the fluorescently-labeled nucleotide structure of terminal phosphate, it is characterized in that comprising the following steps:
A the hydroxyl reaction of () trimetaphosphate and fluorescent dye groups, generates the three metaphosphoric acid intermediates that fluorophor is modified;
B three metaphosphoric acid intermediate reactions that () selects nucleosides and fluorophor with 5 '-phosphoric acid chain to modify, change into product 5 '-polyphosphoric acid Terminal fluorescent labels Nucleotide;
Wherein, the fluorescently-labeled nucleotide structure of described terminal phosphate has structure shown in following general formula (1):
Wherein R
a, R
bindependently can be selected from-H ,-OH; N for being more than or equal to 1, and is less than or equal to the integer of 5.The method of the fluorescently-labeled nucleotide structure of synthesis terminal phosphate provided by the invention, it special requirement for fluorescence molecule, oxa anthracenes, tonka bean camphor, resorufin class fluorescence molecule such as containing hydroxyl.
According to a preferred embodiment of the invention, the described structure described in fluorophor preferred formula (2).
According to another preferred embodiment of the present invention, n value is 3,4 or 5.
Wherein, in general formula (1), base can be selected from VITAMIN B4 (A), guanine (G), cytosine(Cyt) (C), thymus pyrimidine (T), uridylic (U), the nucleoside base modified or non-natural nucleoside base.
According to a preferred embodiment of the invention, in the step (a) of the fluorescently-labeled nucleotide structure method of synthesis terminal phosphate, trimetaphosphate is activated under activating reagent exists, then with the hydroxyl reaction of fluorescent dye groups, generate three metaphosphoric acids that intermediate fluorophor is modified.Activating reagent can be acyl chlorides, SULPHURYL CHLORIDE, chlorination sulfone, sulfur oxychloride, thionyl chloride etc.
According to a preferred embodiment of the invention, in the fluorescently-labeled nucleotide structure method of synthesis terminal phosphate, described nucleotide structure is 2 '-deoxynucleoside, 5 '-monophosphate (dNMP), 2 '-deoxynucleoside, 5 '-bisphosphate (dNDP) or 2 '-deoxynucleoside, 5 '-triphosphoric acid (dNTP).
According to another preferred embodiment of the present invention, the fluorescence molecule that described fluorophor is corresponding is following structure,
According to a preferred embodiment of the invention, in the fluorescently-labeled nucleotide structure method of synthesis terminal phosphate, first fluorophor is first reacted with the trimetaphosphate of activation, generate fluorophor three metaphosphoric acid mixture intermediate, then react with nucleosides 5 '-monophosphate or nucleosides 5 '-bisphosphate or nucleosides 5 '-triphosphoric acid or 5 '-Tripyrophosphoric acid.
According to a preferred embodiment of the invention, described method is a cooking-pot type reaction, and centre does not need intermediate described in separation and purification.Described method does not comprise the amino of described nucleosides and/or the protection of hydroxyl and/or goes protection.
The fluorescently-labeled nucleotide structure of terminal phosphate provided by the present invention, can as DNA or RNA polymerase substrate, is incorporated in DNA or RNA chain and goes, and discharge the polyphosphoric acid molecule of fluorophor mark by DNA or RNA polymerase identification.The fluorescently-labeled nucleotide structure of described terminal phosphate can be continued to decompose as the substrate of alkaline phosphatase, until all phosphate group departs from from fluorescence molecule.
The fluorescently-labeled nucleotide structure of described terminal phosphate is being decomposed and be fluorescence closing condition before discharging fluorophor, does not significantly send fluorescent signal during the rayed that is excited.The fluorescently-labeled nucleotide structure of described terminal phosphate discharges fluorophor after being decomposed by polysaccharase and alkaline phosphatase, significantly sends fluorescent signal when excitation light irradiation.The excitation wavelength scope of described fluorophor is 530-590nm, and wavelength of transmitted light scope is 550-650nm.
According to the preferred embodiment of the present invention, the fluorescently-labeled nucleotide structure of formula (1) described terminal phosphate is 5 '-end-ζ-phosphoric acid fluorescently-labeled Nucleotide six phosphoric acid ester, 5 '-end-η-phosphoric acid fluorescently-labeled Nucleotide seven phosphoric acid ester or 5 '-end-θ-phosphoric acid fluorescently-labeled Nucleotide eight phosphoric acid ester.
The present invention also provides the fluorescently-labeled nucleic acid molecule of a kind of terminal phosphate and synthetic method thereof, and its structure is that fluorophor connects 5-8 phosphoric acid molecules, is then connected to ribose and base successively; Wherein said fluorescently-labeled nucleic acid molecule is the hydroxyl reaction by trimetaphosphate and fluorescent dye groups, and then reacts with the nucleosides with 5 '-phosphoric acid chain and obtain; The fluorescently-labeled nucleic acid molecule of described terminal phosphate can identify by archaeal dna polymerase, and fluorescent signal can be realized change to opened condition by closing under alkaline phosphatase is assisted after reaction bonded.
One of advantage of the present invention is that in invented nucleic acid molecule structure, fluorescence marker groups is only at 5 '-polyphosphoric acid terminal position, molecule scar is not produced, so do not affect the new DNA molecular structure produced when being thus used in the related application such as DNA synthesis as archaeal dna polymerase substrate.Two of advantage is that fluorescence-labeled nucleotides molecule provided by the invention was fluorescence closing condition before archaeal dna polymerase association reaction, and the hydrolysis after reacting with Phosphoric acid esterase changes fluorescence opened condition into.Advantage three for the invention provides a kind of reaction method preparing long-chain fluorescent mark polyphosphoric acid Nucleotide easily, it is one of initial feed that present method make use of the Trisodium trimetaphosphate that commercialization is easy to get, first it is combined with fluorescence molecule, define the fluorescent mark cyclisation phosphoric acid ester of active state easily, target molecule is prepared, the advantage that Tripyrophosphoric acid chain length is longer again in the Tripyrophosphoric acid nucleosides reaction be easy to get.The reaction method preparing fluorescent mark polyphosphoric acid Nucleotide provided by the invention, than the method being prepared fluorescent mark polyphosphoric acid Nucleotide by Nucleotide annulation, not only method is simple, it also avoid into the generation of by product after ring, improve preparation efficiency, product yield is significantly improved, has the advantage that Tripyrophosphoric acid chain length is longer.
Embodiment
Before open description compound of the present invention, mixture, reaction system, device, system/or method, should be appreciated that these specific synthetic method mentioned in embodiment, article used, devices, special reagent (unless providing in addition) etc. is only further illustrate for of the present invention, the term used only for describing particular form, does not form limiting factor of the present invention yet.
Embodiment 1:
Provide a kind of fluorescein analogue---the fluorescein that carbon bridge replaces or anthracene class fluorescein, such material has long wavelength, fluorescence can generation, the characteristic such as derivatize is simple, optical extinction coefficient and quantum yield are high, has having structure general formula (1):
With the fluorescence dye that anthracene structure is parent, it is characterized in that: there is structure, wherein R as Suo Shi general formula (1)
0-H can be selected from, phosphate, the phosphate of replacement, R
1, R
5independently can be selected from the C1-C6 alkoxyl group of-H, fluorine, chlorine, aryl, substituted aryl, C1-C6 alkyl, the C1-C6 alkyl of replacement, C1-C6 alkoxyl group, replacement, R
2-4, R
6-8, R
11-13can independently be selected from-H, fluorine, chlorine, bromine, aryl, substituted aryl, heteroaryl ,-CO
2h ,-CO
2r ,-SO
3h ,-SO
3r ,-CH
2cO
2h ,-CH
2cO
2r ,-CH
2sO
3h ,-CH
2sO
3r ,-CH
2nH
2,-CH
2nHR ,-NO
2, C1-C6 alkyl, the C1-C6 alkyl replaced, C1-C6 alkoxyl group, the C1-C6 alkoxyl group replaced, C1-C6 alkoxy aryl, the C1-C6 alkoxy aryl replaced, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzoyl, wherein R is selected from C1-C6 alkyl, the C1-C6 alkyl replaced, C1-C6 alkoxyl group, the C1-C6 alkoxyl group replaced, C1-C6 alkoxy aryl, the C1-C6 alkoxy aryl replaced, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzoyl, R
9, R
10independently can be selected from C1-C6 alkyl, the C1-C6 alkyl of replacement, C1-C6 alkoxyl group, the C1-C6 alkoxyl group of replacement, C1-C6 alkoxy aryl, the C1-C6 alkoxy aryl of replacement, benzyl, substituted benzyl.
The synthetic method of compound is as follows:
(1) by the adjacent halo m-hydroxybenzaldehyde with hydroxy-protective group or react with other substituent congeners and with hydroxy phenyl metal reagent between hydroxy-protective group or with hydroxy phenyl metal reagent between substituent, adduct and benzhydrol derivative is obtained.
(2) above-mentioned benzhydrol derivative is converted into benzophenone derivates through peroxidation.
(3) above-mentioned benzophenone derivates and metal reagent are reacted, obtain the diphenylmethane derivatives that dialkyl group replaces.
(4) above-mentioned dialkyl group diphenylmethane derivatives process is reacted at low temperatures with metal reagent, generate arylide.
(5) above-mentioned arylide and benzaldehyde derivative are reacted, generate adduct and benzyl alcohol derivative.
(6) above-mentioned benzyl alcohol derivative is generated methanone derivatives through peroxidation.
(7) above-mentioned methanone derivatives process is removed phenolic hydroxyl protecting group group, obtain bisphenol compound, be obtained by reacting target fluorescent thing by dehydration ring closure.
Embodiment 2.
Compound according to embodiment 1, it is specifically as follows:
Embodiment 3.
Compound according to embodiment 2, its synthetic method specifically can be as follows:
Reagent and reaction conditions: i) THF ,-40 DEG C; Ii) DCM, PCC, diatomite; Iii) ZnMe
2, TiCl
4, DCM-40 DEG C; Iv) t-Buli, THF; O-tolualdehyde; V) DCM, PCC; Vi) BBr
3; MeSO
3h
1). synthesis (the bromo-5-p-methoxy-phenyl of 2-)-3-methoxy benzyl alcohol 1a
In the dry round-bottomed bottle of the 250ml being equipped with constant pressure funnel; the 80ml dry tetrahydrofuran solution of m-methoxyphenyl magnesium chloride is chilled to-40 DEG C; under argon shield, the 20ml dry tetrahydrofuran solution of 3-methoxyl group o-bromobenzaldehye (10.8g) is dropwise added in reaction flask by constant pressure funnel;-40 DEG C are kept to stir 2-6 hour; by TLC monitoring reaction; stopped reaction after raw material 3-methoxyl group o-bromobenzaldehye disappears, adds 20ml saturated aqueous ammonium chloride cancellation reaction.Reaction mixture removes after most tetrahydrofuran (THF) through Rotary Evaporators and adds 100ml methylene dichloride at twice, and after separatory, merge organic phase, organic phase is through saturated common salt water washing, Na
2sO
4the concentrated rear silica gel column chromatography of dry, Rotary Evaporators, developping agent petrol ether/ethyl acetate ~ 10/1, collects Rf ~ 0.3 product point, obtains 15g colourless liquid, yield 93%.
2). synthesis (the bromo-5-p-methoxy-phenyl of 2-)-3-methoxy benzophenone 1b
Getting (the bromo-5-p-methoxy-phenyl of 2-)-3-methoxy benzyl alcohol 1a (6.5g) is dissolved in 80ml methylene dichloride, 10g pyridinium chloro-chromate (PCC) is added again under rapid stirring, 2-5h is reacted under room temperature, TLC monitors this reaction until raw material stops after substantially disappearing, reaction solution filters through diatomite drainage short column, filtrate is collected after eluent methylene chloride, through silicagel column column chromatography after Rotary Evaporators is concentrated, developping agent petrol ether/ethyl acetate ~ 10/1, collect Rf ~ 0.6 product point, micro-yellow solid 6.1g is obtained, yield 95% after concentrated.
3). the bromo-4-methoxyl group of synthesis 1--2-(2-(3-p-methoxy-phenyl sec.-propyl)) benzene 1c
In the 250ml round-bottomed bottle of drying, add 70ml methylene dichloride, be chilled to-40 DEG C, add TiCl
4(9ml), under stirring, be slowly added dropwise to zinc methide solution (the toluene solution 80ml of 1M), stir 15 minutes under-40 DEG C of conditions under argon shield.Getting (the bromo-5-p-methoxy-phenyl of 2-)-3-methoxy benzophenone 1b (6.1g) is dissolved in 30ml methylene dichloride, this solution is added dropwise in above-mentioned solution, keep-40 DEG C of stirring reactions 3 hours, slowly be warming up to 0 DEG C afterwards and continue reaction again 5 ~ 10 hours, TLC monitors this reaction until raw material stops after substantially disappearing.Pour cancellation reaction in trash ice under being stirred by this brown reaction solution into, mixture through dichloromethane extraction, Na
2sO
4drying, Rotary Evaporators concentrates rear silica gel column chromatography, developping agent petrol ether/ethyl acetate ~ 15/1, collects Rf ~ 0.6 product point, concentrates to obtain 5.5g colourless liquid, yield 87%.
4). synthesis (4-methoxyl group-2-(2-(3-p-methoxy-phenyl) sec.-propyl) phenyl) (2-tolyl) methyl alcohol 1d
Compound 1c (3g) is added in the 100ml round-bottomed bottle of drying; add 30ml anhydrous tetrahydro furan again to be dissolved;-78 DEG C are chilled under argon shield; the hexane solution (9 ~ 10mmol) of tert-butyl lithium is added dropwise under stirring; maintain the temperature at after dropwising within the scope of-78 DEG C ~-60 DEG C and react 1 hour; again o-tolualdehyde (11.5mmol) is dissolved in 10ml dry tetrahydrofuran; slowly be added in the reaction solution of compound 1c of-78 DEG C with syringe, after dropwising, make its slow free temperature reaction 2 ~ 6 hours.Add 10ml saturated ammonium chloride solution cancellation reaction.Remove after most tetrahydrofuran (THF) through Rotary Evaporators and add 100ml dichloromethane extraction again three times, organic phase drying, after concentrated, obtain crude Compound 1d, be directly used in next step reaction.
5). synthesis (4-hydroxyl-2-(2-(3-hydroxy phenyl) sec.-propyl) phenyl) (2-tolyl) ketone 1e
Previous step compound 1d is dissolved in 40ml methylene dichloride, 4g pyridinium chloro-chromate (PCC) is added again under rapid stirring, 2-4h is reacted under room temperature, TLC monitors this reaction until raw material stops after substantially disappearing, and reaction solution filters through diatomite drainage short column, collects filtrate after eluent methylene chloride, through silicagel column column chromatography purification after Rotary Evaporators is concentrated, developping agent petrol ether/ethyl acetate ~ 7/1, collects Rf ~ 0.5 product point, obtains micro-yellow solid 2.9g after concentrated.
6). synthesising target compound 1 " Beijing orange " (Peking Orange), is labeled as PO.
Get compound 1e (2.9g) and be dissolved in 40ml dry methylene chloride, drip boron tribromide (2-5eq) under ice-water bath cooling, dropwise and continue reaction 2 ~ 5 hours.Carefully add the cancellation of 20ml frozen water after completion of the reaction, mixture continues stirring 30 minutes, uses saturated NaHCO
3the aqueous solution regulates PH to ~ 7, adds 150ml dichloromethane extraction, organic phase through washing, saturated common salt water washing, anhydrous Na
2sO
4after drying, concentrate through Rotary Evaporators, then through vacuum oil pump decompressing and extracting, obtain micro-yellow oil.In this oily matter, add 5ml methylsulphonic acid, under stirring, be heated to 80 ~ 100 DEG C, keep the lower reaction of heating to stop for 1 hour.Pour in trash ice under being stirred by reactant, the solid of precipitation is collected after filtration, then through washing, vacuum-drying, obtains target product 1 crude product and be about 2.2g.Crude product, through silica gel column chromatography separating-purifying, obtains 1.8g Orange red solid " Beijing orange " (Peking Orange), yield 72%.
On this basis, the compound obtaining the general formula (1) of other substituted radicals belongs to the routine techniques means of chemical field.
Embodiment 4.
Synthesis 5 '-ζ-terminal phosphate fluorescently-labeled 2 '-deoxynucleoside six phosphoric acid ester
1) synthetic dyestuff three metaphosphoric ester active intermediate
Get Trisodium trimetaphosphate (355mg, 1.16mmol) be dissolved in appropriate amount of deionized water, this solution is replaced into three metaphosphoric acid tributyl amine salt through ion exchange column (BioRad AG-50W-XB Zeo-karb) process, by the aqueous solution of this amine salt through lyophilize, drain 12h through oil pump vacuum again, obtain three dry metaphosphoric acid tributyl amine salt solids.Under argon atmosphere, this solid is put into dry 25mL round bottom reaction flask, the 10mL acetonitrile solution of anhydrous drying is added in this reaction flask, this solution is cooled under agitation and (cools 10 ~-10 DEG C, here 0 DEG C is selected), in solution, be added dropwise to thionyl chloride (or other activating reagents) (109uL) and DMAP (to Dimethylamino pyridine) (8mg), add 430mg tributylamine subsequently.This solution stirs 1 ~ 30 minute at 0 DEG C subsequently, then by fluorescent marker dyes ' Beijing orange ' 1 (164mg, 0.5mmol) join in above-mentioned reaction solution, stirring reaction 1 ~ 5 hour under this mixed reaction solution arrives the condition of room temperature at 0 DEG C, until HPLC detects the disappearance of most fluorescence dye raw material get final product stopped reaction, this solution comprises midbody compound 2.This solution can be kept in-20 DEG C of refrigerators stand-by.
2) 5 '-ζ-terminal phosphate fluorescently-labeled 2 '-deoxynucleoside six phosphoric acid ester is synthesized
Get 2 '-Desoxyadenosine-5 '-triphosphoric acid sodium salt (50umol) and be dissolved in 1ml deionized water solution, this solution is processed through ion exchange column (BioRad AG-50W-XB Zeo-karb), be converted to the aqueous solution of 2 '-Desoxyadenosine-5 '-triphosphoric acid Tributylamine salt, this aqueous solution low temperature rotary evaporation under high vacuum removes most aqueous solution, remaining solution adds the DMF of the anhydrous drying of 1mL, low-temperature rotary evaporation of solvent under a high vacuum again, this process repeats twice again, then by this except anhydrate 2 '-Desoxyadenosine-5 '-triphosphoric acid Tributylamine salt is dissolved in the anhydrous dry DMF of 2mL, in stirring at room temperature situation, by step 1) in the acetonitrile solution 2mL of fluorescently-labeled three metaphosphoric acid intermediates 2 that obtains extracted by syringe and add in this reaction solution, add anhydrous MgBr subsequently
2.2Et
2the DMF solution of O (0.4umol), this mixture is stirring reaction 8-24 hour at room temperature, and reaction process uses HPLC tracing detection, treats that product peak no longer increases, gets final product stopped reaction.By this reaction solution DMF solvent that low-temperature rotary evaporation removing is most under a high vacuum, again residue is dissolved in the acetic acid triethylamine buffer solution (PH 7.6) of the 100mM of 3mL, this solution is through reverse phase separation chromatographic purification, adopt in Agela and press preparative chromatograph, AQ C-18-60g preparative column is separated.Gradient elution: mobile phase A is 50mM acetic acid triethylamine buffer solution, and PH is 7.5; Mobile phase B is trifluoroacetic acid aqueous solution, elution program is: 0-15min is increased to 20%B by A, and 15-35min B component is increased to 40%, flow velocity 15mL/min, collect containing target component cut, concentrating under reduced pressure, through efficient liquid phase chromatographic analysis purity about 90%, meticulous separation and purification adopts highly effective liquid phase chromatographic system, the separation and purification of Inertsil C18 semipreparative column, flow velocity is 3mL/min, and mobile phase A is 50mM acetic acid triethylamine buffer solution, and PH is 7.5; Mobile phase B is trifluoroacetic acid aqueous solution, and gradient elution program is: 0-15min is increased to 20%B by A, and 15-25min is increased to 30%, 25-35min by B component and is increased to 50%.Collect pure product fractions, cryoconcentration, be positioned in-20 DEG C of refrigerators after measuring concentration and save backup.Sterling through MALDI-TOF mass spectroscopy, target product ' Beijing orange-dA6P ' (or be called for short PO-dA6P) molecular composition and calculated value C
33h
37n
5o
22p
6, m/z 1041.0375. measured value 1040.1097 (M-H).Its yield is about 70%.
Other three kinds of 5 '-ζ-terminal phosphate fluorescently-labeled 2 '-deoxynucleosides six phosphoric acid ester (PO-dG6P, PO-dC6P, PO-dT6P) are synthesized respectively according to method described in the present embodiment 4.Its structure is as follows:
Embodiment 5
5 '-δ-terminal phosphate fluorescently-labeled 2 '-deoxynucleoside four phosphate synthesis carries out according to method described in the present embodiment 4, only 2 '-deoxynucleoside-5 '-triphosphoric acid sodium salt need be replaced with 2 '-Desoxyadenosine-5 '-monophosphate sodium salt.
Embodiment 6
5 '-ε-terminal phosphate fluorescently-labeled 2 '-deoxynucleoside five phosphate synthesis carries out according to method described in the present embodiment 4, only 2 '-deoxynucleoside-5 '-triphosphoric acid sodium salt need be replaced with 2 '-Desoxyadenosine-5 '-bisphosphate sodium salt.
Embodiment 7
Four kinds of 5 '-ζ-terminal phosphate fluorescently-labeled nucleosides six phosphoric acid ester (PO-dN6P) application in determined dna sequence according to embodiment 4.
First by immobilized to glass-chip on the surface by the mode of asymmetric Solid phase PCR for the ssDNA template being used for sequential test: chip surface has been planted in the passage being implanted with primer and has injected PCR mixed solution, comprising the primer 1 of 1uM; The primer 2 of 125nM; The DNA profiling of 100nM; The archaeal dna polymerase (Platinum Taq (Life technology)) of 0.3U/uL, the magnesium ion of 3mM, 1x standard Taq damping fluid.First be pre-amplification (95 DEG C of 30s of 15 ~ 20 circulations, 65 DEG C of 15s, 72 DEG C of 45s), to produce abundant single-stranded DNA templates, then amplification (95 DEG C of 30s of 20 ~ 30 circulations are made, 65 DEG C, 150s) so that the ssDNA of pre-amplification and chip surface primer annealing also effectively extend.After PCR, pure water cleaning chip surface 10min, the final chip surface obtained about containing 5fmol/mm2 sequence concentration to be measured.
Before order-checking starts, four kinds of 5 '-ζ-terminal phosphate fluorescently-labeled 2 '-deoxynucleoside six phosphoric acid ester PO-dN6P to be individually positioned in the sample bottles of 4 DEG C of coolings and to be diluted to 1uM (use damping fluid is 50mM Tris-HCl pH 7.9,50mM NaCl, 0.1%tween-20,1mM MnCl
2, 1mM DTT), also comprise alkaline phosphatase (CIP), archaeal dna polymerase (Bst) in sample bottle simultaneously.During order-checking, a kind of PO-dN6P is added each time in chip channel, then the work of 65 DEG C of initiated polymerization enzymes is warming up to, discharge with fluorescently-labeled polyphosphoric acid fragment chain, this polyphosphoric acid chain of alkaline phosphatase fast decoupled also discharges fluorescence dye core element, after polyreaction completes, reduction temperature is to room temperature, produces fluorescent signal under excitation light, and the base quantity that this signal and polysaccharase correctly identify and are attached in template is linear.Settling signal is taken pictures and is gathered post-flush chip channel (50mM Tris-HCl pH 7.9,50mM NaCl, 0.1%tween-20,1mMEDTA, 1mM DTT), carry out adding of next labeled substrate PO-dN6P, so circulation is carried out, until checked order successively.This sequence measurement does not limit to specific sequence, only needs to build storehouse process accordingly according to actual sample source, and on specific reaction chip, carries out surface sample increase.Present method has the sequencing reaction cycle short (often wheel reaction and signals collecting are no more than 3 minutes), signal to noise ratio high (the long excitation/emission wavelength with more than 540nm of fluorescence molecule avoids the background fluorescences such as chip material and pollutes), accuracy is high, reads the features such as long.
Embodiment 8
2 '-deoxynucleoside four phosphoric acid ester according to embodiment 5, gene sequencing is used as under the identical order-checking condition described in embodiment 7, its sequencing reaction speed (each is taken turns sequencing reaction and completes the required time) will be slower than PO-dN6P, whole result shows as the acquisition identical order-checking time of reading needed for length longer, its reason is that the identification of polysaccharase to the Nucleotide with different lengths phosphoric acid chain is different with in conjunction with speed, and have influence on thus order-checking accuracy and the reaction times extends cause mispairing equal error increase.
Embodiment 9
On the basis of embodiment 4, if the activity link group of fluorescence molecule changes amino or other groups into by hydroxyl, the fluorescently-labeled nucleotide structure of then formed terminal phosphate can not excise by Phosphoric acid esterase, thus cannot discharge fluorescence molecule, cause producing fluorescent signal.
The specific embodiment of the present invention is further describing for technical solution of the present invention, in order to the effect better describing application claims protection content is selected, is not the restriction for summary of the invention.Its step, parameter etc., in such as embodiment 4, design parameter is that those skilled in the art can select according to design of the present invention.