CN101519687A

CN101519687A - Method for analyzing sequence by utilizing high-fidelity DNA polymerase-mediated alternative primer extension reaction

Info

Publication number: CN101519687A
Application number: CN200810052351A
Authority: CN
Inventors: 李凯; 张佳
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-02-29
Filing date: 2008-02-29
Publication date: 2009-09-02

Abstract

The invention discloses a method for analyzing sequence by utilizing high-fidelity DNA polymerase-mediated alternative primer extension reaction, and the steps are as follows: (1) primers which are resistant to 3'-5' exonuclease digestion are prepared; (2) fluorescently-labeled ddNTP with the single type and non-fluorescently-labeled dNTP with the same type are taken as substrates, the partial chain termination primer extension reaction is carried out under the catalysis of DNA polymerase without 3'-5' exonuclease activity; (3) the alternative primer extension reaction is carried out on the dNTP which is resistant to 3'-5' exonuclease digestion under the catalysis of the high-fidelity DNA polymerase with the 3'-5' exonuclease activity; and (4) the partial chain termination primer extension reaction and the alternative primer extension reaction are coupled and repeated. The primers which need to be extended are not significantly reduced along with the sequencing in the method, so each sequencing primer can determine longer sequence and be compatible with a plurality of technologies, thereby being applicable to the chip-based sequence analysis.

Description

Utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique

Technical field

The present invention relates to a kind of dna sequence analysis method, particularly relate to and a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique.

Background technology

Gene order-checking is a very important job in the current life science.From the nineteen ninety Human Genome Project

(HGP) start to calendar year 2001 and finish substantially, genome research has had development by leaps and bounds.Be to disclose gene function, develop the evolutionary relationship between personalized medicine and clear and definite living species and the similarities and differences of different organism vital movements, need check order more genome.In this sense, gene order-checking only is the beginning of genome times afterwards comprehensively.The appearance of the development need new-generation sequencing technology of genome research.

Recently, NIH (NIH) provides " revolutionary gene order-checking technology " (Revolutionary GenomeSequencing Technologies) fund of project, initiated new challenge to biotechnology: planned 2009, the whole genome sequence that reaches a people of every mensuration only spends 100,000 dollars target, and by 2014, expense will be reduced to 1000 dollars." 1000 dollars of genomes " contained meaning is, promises to undertake that dna sequencing will be inexpensive as to the individual and be enough to afford, and makes and it is believed that this can being worth the expenditure once in all one's life of oneself genome sequence deposit confession doctor reference.In addition, cheap sequencing technologies can enlarge Study on Genome person troop, supplies with the more genome of researchist so that the individual difference between patient and the healthy person is understood in contrast, thereby makes gene order information more valuable.At present, the obstacle of all these application is cost of manufacture and the effective accuracy that gene map is high.

The relevant technologies progress:

Deriving of Sanger sequence measurement: these class methods are prolonged with the chain end of two deoxidations and are ended the method principle by the fluorescent mark dideoxy nucleotide, and associating capillary electrophoresis separation technology obtains genomic information.This method has had advantage accurately and reliably always since invention.Although this method has been made contribution to improving the human genome order-checking, during owing to its material therefor costliness, check fee, limited (1,2) in the works that it is used in 1000 dollars of order-checkings.

Sequencing by hybridization (Sequencing by hybridization): the representative technology based on the hybridization principle is biochip technology, and gene chip has potential sudden change detectivity equally.This technology depends on a large amount of particular probe and the mark sample is hybridized, by detecting the quantity of target molecule in the hybridization signal power whether match and then the judgement sample.Just because of the identification of this single-point battle array, hindered its utilization in the high-throughput gene sequencing.Sum up reason, one reaches 3,000,000,000 concerning the gene order-checking of base for finishing the mankind, and gene chip needs the probe of a large amount of astronomical quantity; Its two, gene chip is (3,4) that are difficult to measure for the sample with highly repetitive sequence.

DNA synthesis method order-checking (Sequencing by synthesis): by dna mimic molecular replication process, join in the new complementary strand, finish the detection of signal with the dNTP replacement ddNTP of tape label.Report is arranged recently, and a kind of based on synthesis method, the utilization microscope separation detection not technology platform of the dNTPs of isolabeling is applied to (5) in the chip.And polysaccharase is cloned in the order-checking again that successfully is used in bacterial genomes (6).Have new trial that polysaccharase is cloned on the crosslinked strain shape particulate in being immersed in emulsion form in addition, after amplified reaction finished, each particulate all can carry many copies of target DNA molecule and then check order simultaneously (7,8).If can be organically in conjunction with these technology, the gene sequencing expense will be reduced significantly becomes possibility.

The physical chemistry method: at present, a kind of new method that is known as the nanometer order-checking is risen, and the physical properties difference based between 4 kinds of bases is transformed into the signal that can detect with this difference, thereby checks order.Be in theory can according to the chemical quality of each base, size, chemical bond and electrically charged type of coming the identification form base.But the drawback that this method may exist is that with respect to full genome, the preparation meeting of its purpose single stranded DNA is difficulty (9,10) relatively.

From medical angle, perhaps we do not need to determine the sequence of 3,000,000,000 pairs of bases in the full gene group, accounts in the human genome 10% coding region and only need concentrate on, and can obtain enough biomedical relevant informations.In view of this consideration, in the near future, people can promptly obtain individual's genome encoding information at birth and be benefited all one's life.With regard to technical feasibility, we think that the popularization of biochip will make 1000 U.S. dollar order-checking plans come true.According to the black luxuriant and rich with fragrance law of making in the integrated chip, the density of chip will be increased to certain level.Comprised 100,000 to 300,000 arrays when a substrate can be prepared as, and 1000 Nucleotide can be discerned in each site, when the time comes, 1000 dollars of plans of measuring complete genome sequence will can not be dreams.Certainly, the research of at present relevant 1000 U.S. dollar order-checking plans does not touch key problem and does not also find suitable methods involving in other words.Even if in this platform of chip, the single-basic extension method can be finished the mensuration of most gene group sequence, but also far from the target of 1000 U.S. dollars order-checking, and can't measure simple repeated sequence.

It is believed that the bottleneck on the sequencing technologies has been to use the mode of " gene amplification " to carry out " reading DNA " sequence, if can get around the method that the mode of " gene amplification " reads " dna sequence dna ", can make the shortening time and reduce cost becomes possible (11).In order to quicken the progress of 1000 U.S. dollar order-checking plans, October in 2006 U.S. on the 4th X otsa gene can to have set up total value be the awards of 1,000 ten thousand U.S. dollars, to reward the research team that finally finishes this target (12).The research of 1000 U.S. dollar order-checking plans will promote the fast development of related-art technology and carrying out in an all-round way of individuality medicine.

The gordian technique of 1000 U.S. dollars order-checking plan comprises that the length of high-throughput and each tested sequence reaches up to a hundred even thousands of bases, has reached the density that every chip contains 1,000,000 short sequences based on the current chip manufacturing technology.Therefore, how making single determined sequence obtain more information, is that the order-checking of 1000 U.S. dollars has the difficult problem of reality the most to be solved in the works.At present, we are attempting the on/off molecular switch and are combining with chip inactivation/activation technique, in the hope of reaching the target of 100 bases of every site identification.If target can realize, 1000 U.S. dollars are surveyed the whole genome sequence plan and will be finished in following 10 years so, and undoubtedly, this will be not only for individualized treatment provides strong assurance, and provides foundation for improving the human survival quality.

And the order-checking scheme based on the nanoscale molecular switch that we propose, except the mutational site being carried out the gene type, it is still can disposable identification several to ten Nucleotide (13,14) that each extends primer.This base walking method is used for order-checking, and is similar on effect with the sequence measurement of ligase enzyme mediation to the probe hybridization method, all can only obtain short arrangement set, will have a plurality of unknown nucleotide sequences when being applied to gene order-checking unavoidablely.Therefore, if based on the on/off molecular switch, two kinds of different primer extension reactions of coupling are the part chain termination and replace extension in a reaction system, will make one on the chip to extend the hundreds of even longer base sequence of primer decoding.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, provide a kind of and utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique.

Technical scheme of the present invention is summarized as follows:

A kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, comprise the steps:

(1) but prepare the primer of anti-3 '-5 ' exonuclease digestion and three terminal free hydroxyl of these primer actual participations reactions carried out quantitative assay;

(2) with the same kind dNTP of the fluorescently-labeled ddNTP of single kind and non-fluorescent label for mixing substrate, not having under 3 '-5 ' the exonuclease activity archaeal dna polymerase catalysis, carry out part chain termination primer extension reaction;

(3) the replaceability primer extension reaction that under high-fidelity DNA polymerase catalysis, carries out of the dNTP of anti-3 '-5 ' exonuclease digestion of synthetic with 3 '-5 ' exonuclease activity;

(4) coupling part chain termination primer extension reaction and replaceability primer extension reaction are also reused this coupling process.Described primer is the three end modified primers or the primer of three terminal unmodifieds.Described three end modified primers are the modification of anti-3 '-5 ' exonuclease digestion.

Described high-fidelity DNA polymerase with 3 '-5 ' exonuclease activity is Vent high-fidelity DNA polymerase, DeepVent high-fidelity DNA polymerase, Pfu high-fidelity DNA polymerase, Sso7d-Pfu high-fidelity DNA polymerase or Kapa high-fidelity DNA polymerase.

It is described that not have 3 '-5 ' exonuclease activity archaeal dna polymerase be TaqDNA polysaccharase, T7 Sequenase, Vent ^-Archaeal dna polymerase, DeepVent ^-Archaeal dna polymerase.

Acy-NTP that the PS-dNTP that the dNTP of anti-3 '-5 ' exonuclease digestion of described synthetic modifies for sulfuration, cyclisation are modified or lock nucleic acid.

The maximum of the concentration ratio of the dNTP of described fluorescently-labeled ddNTP and non-fluorescent label single base in sequence to be measured is 1/3 to 1/30 when the repetition degree is less than 10 continuously.

The maximum of the dNTP concentration ratio of described fluorescently-labeled ddNTP and non-fluorescent label single base in sequence to be measured is 1/31 to 1/300 when the repetition degree is less than 100 continuously.

The maximum of the dNTP concentration ratio of described fluorescently-labeled ddNTP and non-fluorescent label single base in sequence to be measured is 1/301 to 1/3000 when the repetition degree is less than 1000 continuously.

Advantage of the present invention is:

1. the present invention is used to check order for first coupling part chain termination extension in the world and replaceability primer extension reaction, and this design is a feasibility of the present invention and have a high-throughout basis.

2. fluorescently-labeled ddNTP only is used to the intermediate reaction process, after signal (base identification) obtains, utilize 3 ' of high-fidelity DNA polymerase-5 ' correct functioning to remove inductile base in the new synthetic chain, its 3 ' end is activated, can continue another reaction process.

3. in addition, the dNTP of anti-3 '-5 ' exonuclease digestion combines the replaceability primer extension reaction that is carried out with the high-fidelity DNA polymerase with 3 '-5 ' exonuclease activity, can remove fluorescence ddNTP, reduces background noise, and makes the reaction synchronization.

4. this method adopts fluorescence ddNTP mature technology to replace the non-mature technology of fluorescence dNTP that uses in the present synthesis method order-checking, makes classical chain termination sequencing method and synthetic sequencing seem inconsistent technology for two kinds in conjunction with the replaceability primer extension reaction and is coupled in the reaction system.

5. this method is compatible with the multiple technologies platform, is suitable for the sequential analysis based on chip especially.

Description of drawings

Fig. 1 is a method principle of work synoptic diagram of the present invention.

Fig. 2 is the compatible synoptic diagram with chip technology of the present invention.

Embodiment

The key of the inventive method is carried out part inactivation and activated circulating reaction again for the primer of anti-3 '-5 ' exonuclease digestion that needs are extended.As shown in Figure 1.

Prepare the primer of anti-3 '-5 ' exonuclease digestion and but three terminal free hydroxyl of these primer actual participation reactions are carried out quantitative assay; (three terminal free hydroxyl are carried out quantitative assay be meant that it is that substrate carries out single base extension and utilizes 3 '-5 ' exonuclease to excise the base of the band fluorescence that primer three ends newly add subsequently with fluorescently-labeled ddNTP that utilization does not have 3 '-5 ' exonuclease activity archaeal dna polymerase mediation.)

By treat extend primer (primer of anti-3 '-5 ' exonuclease digestion) with the same kind dNTP of the fluorescently-labeled ddNTP of single kind and non-fluorescent label for mixing substrate, do not have under 3 '-5 ' the exonuclease activity archaeal dna polymerase catalysis, carry out part chain termination primer extension reaction, the 3 ' end that is connected to the nascent DNA chain by ddNTP and ddNTP in the dNTP mixing substrate stops extension.This reaction of (ddNTP is a bi-deoxyribose Nucleotide) (the dNTP mixture of fluorescently-labeled ddNTP and non-fluorescent label is ddATP/dATP or ddTTP/dTTP or ddCTP/dCTP or ddGTP/dGTP) is mediated by Sequenase such as T7 Sequenase (also can adopt the TaqDNA polysaccharase or the Vent of no corrective action ^-Archaeal dna polymerase or DeepVent ^-Archaeal dna polymerase mediates).Do not cause the termination of extension owing to ddNTP and the dNTP in the dNTP mixing substrate, therefore, can adjust the ratio of ddNTP/dNTP according to the continuous repetition degree of single base in the sequence to be measured, be generally between 1/10 to 1/1000: when single base in the sequence to be measured maximum continuously when the repetition degree is less than 10, this ratio is 1/3 to 1/30; When single base in the sequence to be measured maximum continuously when the repetition degree is less than 100, this ratio is 1/31 to 1/300; When single base in the sequence to be measured maximum continuously when the repetition degree is less than 1000, this ratio is 1/301 to 1/3000.But when the continuous repetition degree of the single base of specific region in the sequence to be measured during greater than 1000, error can appear in sequencing result of the present invention.After part chain termination primer extension reaction, remove the ddNTP fluorescent signal that remains in the reaction system by repeatedly washing, when using the chip order-checking, can add the one times of concentration PCR reaction buffer that does not contain ddNTP and dNTP to chip, hatched 30 seconds to one minute.Repeated washing 2-5 time.Detect the fluorescence signal intensity of waiting to extend on the primer by the fluorescent scanning device then.When a kind of reaction of base is finished, clean and finish and after detection finished, 3 ' end of nascent DNA molecule was in two states, wherein a part of three ends contain the ddNTP residue and do not have extendible 3 ' terminal free hydroxyl; Another part then contains the dNTP residue and has extendible 3 ' terminal hydroxyl.Therefore, for making nascent strand when using another kind of ddNTP/dNTP mixing substrate, have enough quantity and quantity hydroxyl substantially invariable to be extended, and remove and the background fluorescence signal when reducing another new base and detecting, present method achieves the above object by introducing the replaceability primer extension reaction.

The replaceability primer extension reaction is by carrying out under the high-fidelity DNA polymerase catalysis with 3 '-5 ' exonuclease activity.High-fidelity DNA polymerase with 3 '-5 ' exonuclease activity can be selected Vent high-fidelity DNA polymerase (also can select DeepVent high-fidelity DNA polymerase, Pfu high-fidelity DNA polymerase, Sso7d-Pfu high-fidelity DNA polymerase or Kapa high-fidelity DNA polymerase for use) for use.In the replaceability primer extension reaction, the ddNTP of not anti-3 '-5 ' exonuclease digestion and dNTP residue are eliminated, the dNTP of anti-3 '-5 ' exonuclease digestion then is integrated in the new synthetic dna molecular, the result of this reaction is ddNTP and the dNTP residue that the dNTP of anti-3 '-5 ' exonuclease digestion has replaced not anti-3 '-5 ' exonuclease digestions of nascent DNA molecule three ends, thereby make the reaction system synchronization, activated those and mixed three ends of inactivation because of ddNTP.Along with fluorescently-labeled ddNTP is digested, the original fluorescent signal in the reaction system just rinse method of available physical is removed, and the one times of concentration PCR reaction buffer that does not contain fluorescence ddNTP is still used in rinsing, general rinsing 2-5 time, each 30 seconds to one minute.Then, carry out the reaction of another kind of base successively.The reaction sequence of four kinds of bases is fixed, can select for use specific a kind of arrangement in its 16 kinds of arrangements as program.

The continuous circulation of above-mentioned part chain termination primer extension reaction and replaceability primer extension reaction and respectively four kinds of component A, T, C, G that constitute DNA being reacted successively obtains the character and the quantity of base to be determined by the inactivation of primer; By the replaceability primer extension reaction part primer of inactivation is activated again, carry out thereby this reaction can be circulated.From the angle that signal obtains, the part chain termination is the reaction process of direct decoding or order-checking.This step and Sanger ' s order-checking principle is consistent, all may occur extension products differ with the difference of fluorescence color with extension products length in a kind of primer.In the order-checking of Sanger ' s enzyme process, the different lengths product is recognized by electrophoresis, but when the chip sequencing reaction, because extension carries out at solidus surface, reaction product can not electrophoresis.Here, the length of extension products is reflected by detected fluorescence signal intensity.For example, when the ratio of ddNTP/dNTP was 1/1000, the fluorescence signal intensity of the extension products of the different lengths tumor-necrosis factor glycoproteins of single kind of base was a quantization unit change (table two) with 1/1000 in repeat number less than 32 o'clock basically.For the chip sequencing reaction, the stdn of signal can reach by two aspects.At first, as shown in Figure 2, be the single base extension of substrate, to obtain saturated or maximum fluorescence intensity, as 100% reference value by carrying out with 100% fluorescently-labeled ddNTP to chip.In addition, can the mean value of the highest low signal as the basic quantized signal of a base extension appear by choosing the close and frequency of strength of signal, when using ddNTP/dNTP to be 1/1000, it is 1/1000 of 100% reference value that the mean value of this basic quantized signal approaches the saturation type maximum fluorescence intensity.

The order-checking of Sanger ' s enzyme process can't directly apply to the chip order-checking owing to require electrophoresis.In addition, Sanger ' s enzyme process checks order readable length generally only for the 400-800 base, after a bit be owing to can be used for the reason that 3 ' terminal hydroxyl constantly consumes that contains of extension in the terminal cessation method sequencing reaction process.Compare with the order-checking of Sanger ' s enzyme process, though the order-checking of present circulation synthesis method has certain advantage in theory, because of introducing multiple new compound, the extending length of nascent strand is subjected to very big restriction, only be that its practical application is restricted between the 10-100 base.In view of this, the present invention has used ddNTP/dNTP mixing substrate in conjunction with the polymerase-mediated terminal cessation method of Sanger ' s, is applied in the part chain termination primer extension reaction.Simultaneously, the present invention combines the principle that the synthetic sequencing of chain is treated each the base proceed step by step reaction of order-checking row, by the dNTP and replaceability primer extension reaction that high-fidelity DNA polymerase mediated with 3 '-5 ' exonuclease activity of invention by anti-3 '-5 ' exonuclease digestion, making the terminal termination order-checking of Sanger ' s enzyme process and these two kinds of couplings of chain synthesis cycle reaction order-checking is a reaction system.The continuous circulation of part chain termination primer extension reaction and replaceability primer extension reaction, respectively four kinds of component A, T, C, G that constitute DNA are reacted successively and circulate, can make the length of nascent strand reach and surpass terminal termination of Sanger ' s enzyme process and check order.

As shown in Fig. 2, one of stdn of fluorescence signal intensity is for resultant by single base extension: the order-checking microchip is hatched in containing four kinds of blended fluorescent mark ddNTP and template reaction liquid to be measured, make on the chip each and corresponding template bonded primer all extend a base, detect its strength of signal as reference equivalent 100%.Then, chip moves in the reaction solution that contains 3 '-5 ' exonuclease and hatches, and allow chip return original state, and whether detection signal strength approaches zero.This step is used for the calibration of laboratory apparatus, and has detected the working efficiency of high-fidelity DNA polymerase.The single-basic extension chip technology is used widely.Different with common order-checking the primer is that when the present invention was applied to the chip technology platform, primer 3 ' the terminal palpus on its chip have the characteristic of anti-3 '-5 ' exonuclease digestion through modifying.

Give an example as Fig. 2, at selected specific primer point, add the mixed reaction solution contain ddATP/dATP when hatching to chip, wherein ddATP is a green fluorescence mark and dATP is a non-fluorescent label, if template to be measured sequence herein contains two T bases, new synthetic dna sequence dna then can two A bases of corresponding extension.After part chain termination primer extension reaction is finished, add the one times of concentration PCR reaction buffer that does not contain ddNTP and dNTP on chip, repeated washing 2-5 time is removed background fluorescence signal and the unnecessary template that remains in the reaction system by repeatedly washing.Detect its strength of signal then, to judge the number of sequence current position VITAMIN B4.Suppose that this point of chip contains 10000 primers of having an appointment, need to extend 2 VITAMIN B4, and the concentration ratio of ddATP/dATP is 1/100, when first A base of primer extension, nearly 9900 dATP enter new synthetic dna molecular (10000 x 99/100), have 100 primer 3 ' ends to be introduced into the ddATP of green fluorescence mark (10000 x 1/100) simultaneously approximately.Because the 3 ' end of ddATP does not contain free hydroxyl and reaction terminating, therefore when extending second A base, have only remaining 9900 primers can continue to extend, wherein there are 99 primers to be introduced into the ddATP of green fluorescence mark (9900 x 1/100) again, the T base of template sequence has been finished reproduction process but has been lacked other three kinds of bases in the reaction solution and caused the reaction of this point to proceed at this moment, the fluorescence ddATP that is integrated in the nascent DNA chain in its green fluorescence intensity and this step reaction process is directly related, according to the intensity that detects resulting green fluorescence, can judge that it is 2 that the A base is extended number.Then, the damping fluid from the special VITAMIN B4 that contains 3 '-5 ' exonuclease activity high-fidelity DNA polymerase and the modification of anti-3 '-5 ' exonuclease digestion to chip and the template to be measured that add are hatched, the ddATP of green fluorescence mark and the dATP of non-fluorescent label are digested, primer 3 ' end terminal position is introduced into the modification VITAMIN B4 of anti-3 '-5 ' exonuclease digestion, finishes the special VITAMIN B4 of modification and replaces extension.So far, green fluorescence mark ddATP and common dATP are digested, and are together removed by the physics rinsing with unnecessary template, and only there is the modification VITAMIN B4 of anti-3 '-5 ' exonuclease digestion that is introduced in primer 3 ' terminal position.The fluoroscopic examination strength of signal is reduced to levels of background noise after the rinsing, and so far A base extension finishes, 2 A bases that this has put successful primer extension, and these all primers of point are returned to again and have 3 ' terminal free hydroxyl and enter extensible state simultaneously.

After the extension of A base finishes, adding the mixed reaction solution and the template to be measured that contain ddTTP/dTTP to chip hatches, wherein ddTTP is a red fluorescence mark and dTTP is a non-fluorescent label, in Fig. 2 example template to be measured herein sequence only contain an A base, new synthetic dna sequence dna then can T base of corresponding extension, and continues to extend other required three kinds of bases and cause reaction terminating because of lacking primer in the reaction solution.On chip, add rinsing liquid, behind the removal background fluorescence signal, detect its strength of signal, judge the number of sequence current position dTTP.Same this point of chip of working as contains 10000 primers, need to extend a T base, and the concentration ratio of ddTTP/dTTP is 1/100, when primer extension the time, then nearly 9900 dTTP enter new synthetic dna molecular (10000 x 99/100) and have 100 primer 3 ' ends to be introduced into the ddTTP of red fluorescence mark (10000 x 1/100) simultaneously approximately.Extend reaction end that required other three kind bases cause this point because primer 3 ' end can't obtain this moment from reaction solution.Its red fluorescence intensity is about 1% of resulting saturated fluorescence intensity in the single base extension process, by detecting the intensity of red fluorescence, can judge that it is 1 that the T base is extended number.Then, adding the special dTTP damping fluid and the template to be measured that contain 3 '-5 ' exonuclease activity high-fidelity DNA polymerase and the modification of anti-3 '-5 ' exonuclease digestion to chip hatches, the ddTTP of red fluorescence mark and the dTTP of non-fluorescent label are digested, the special dTTP that anti-3 '-5 ' exonuclease digestion is modified replaces and extends, primer 3 ' end terminal position is introduced into the modification dTTP of anti-3 '-5 ' excision enzyme digestion, at this moment only there is the modification dTTP of anti-3 '-5 ' exonuclease digestion that is introduced in primer 3 ' terminal position, and makes the red fluorescence detection signal strength reduce to levels of background noise after the rinsing.So far T base reaction finishes.

After T base extension finishes, adding the mixed reaction solution and the template to be measured that contain ddCTP/dCTP to chip hatches, wherein ddCTP is a non-fluorescent label for blue fluorescent mark dCTP, and template to be measured sequence alkali-free base G herein in Fig. 2 example, new synthetic dna sequence dna then do not have the C base and extend.Detect fluorescence signal intensity by after the corresponding rinsing, its fluorescence intensity shows as background noise and does not have specific peaks, can judge the no C base extension in this secondary response of this primer point.Though this primer point contains in previous step and does not have primer extension in the ddCTP/dCTP mixed reaction solution in the chip, contain 3 '-5 ' exonuclease activity high-fidelity DNA polymerase and hatch so that other has the primer point of extension to replace extension on the chip but still need to add, and finish corresponding rinse cycle with the damping fluid of the special dCTP of anti-3 '-5 ' exonuclease digestion modification to chip.

After C base extension finishes, adding the mixed reaction solution and the template to be measured that contain ddGTP/dGTP to chip hatches, wherein ddGTP is a fluorescent mark and dGTP is a non-fluorescent label, when template to be measured herein sequence contain 1 C base, new synthetic dna sequence dna then can 1 G base of corresponding extension, and continues to extend other required three kinds of bases and cause reaction terminating because of lacking primer in the reaction solution.After the part chain termination reaction is finished, remove the background fluorescence signal, detect its strength of signal, judge the number of sequence current position dGTP by rinsing.Then, the damping fluid that adds the special dGTP that contains 3 '-5 ' exonuclease activity high-fidelity DNA polymerase and the modification of anti-3 '-5 ' exonuclease digestion to chip is hatched, the dGTP of fluorescently-labeled ddGTP and non-fluorescent label is digested, the special dGTP that anti-3 '-5 ' exonuclease digestion is modified replaces and extends, primer 3 ' end terminal position is introduced into the modification dGTP of anti-3 '-5 ' exonuclease digestion,, and finish corresponding rinse cycle.So far G base extension finishes.

In sum, kind and intensity by the analysis of fluorescence signal, can obtain primer 3 ' terminal kind and the number of introducing base: in reaction, participate in the part ddNTP of extension, the new base kind that adds of its fluorescence kind decoding, the fluorescence intensity decoding newly adds the quantity of base.With regard to the representative primer point in the illustration 2, through the i.e. order-checking circulation of continuous four-step reaction, primer 3 ' end is introduced into AATG, finishes the order-checking task of one section sequence cleverly.This method constantly extends primer by constantly reacting respectively at each base and circulation continuously, and the sequence on the template to be measured is constantly decoded thereupon, and therefore, the present invention can be long sequence template order-checking.

The present invention is further illustrated below by specific embodiment.

Embodiment 1

Design of hereditary breast cancer genes involved (Brca-1) part sequencing primer and signal analysis

Brca-1 is that relevant gene takes place for a kind of and hereditary breast cancer, and its being numbered in gene pool (NM_065379.3) is contained 8 exons.Should use-case with the exons 1 of Brca-1,2 and 3 is example, and the applying step of the present invention in actual sequence is measured is described.

(1) but prepare the primer of corresponding anti-3 '-5 ' exonuclease digestion and three terminal free hydroxyl of these primer actual participations reactions carried out quantitative assay.The order-checking of target gene can be divided into genome sequence mensuration and encoding sequence is measured.This example with chip platform to the design of checking order of first three exons coding sequence of target sequence Brca-1.Form output intent option from Genbank can directly obtain the intron sequences at corresponding exon and exon two ends, and the sequencing primer of modifying with five terminal amino groups (seeing Table 1) is modified in synthetic three terminal sulfurations earlier.The chip glass coating formation covalent attachment that the amino group of sequencing primer (concentration at 80nM between the 50 μ M) by its five end and epoxidation are handled is with damping fluid (pH=8.0) point sample that contains the 150mM sodium phosphate.Air-dry overnight under the room temperature was dried 30 minutes for 65 ℃, was containing 0.1%SDS, and flushing can be used in about 1 minute in the hot mixed solution of 5% ethanol and distilled water.The PCR instrument that utilization can be carried out original position PCR carries out single-basic extension and the excision reaction of extending base to chip: use 100 μ l and contain four kinds of fluorescently-labeled ddNTp and template to be checked order and treat and extend primer and carry out single-basic extension, but quantitatively wait to extend three terminal free hydroxyl numbers of primer three end actual participations reaction with resulting fluorescence intensity, afterwards by chip and the nuclease that contains 3 '-5 ' exonucleolytic activity being hatched altogether the single base that reaches excision band fluorescence.

(2), be to mix in the reaction mixture of substrate and template to be measured to carry out part chain termination primer extension reaction with the fluorescently-labeled ddNTP and the same kind dNTP of non-fluorescent label of single kind not having 3 '-5 ' exonuclease activity archaeal dna polymerase mediation.In the reaction of this example, used ddNTP/dNTP mixing substrate is a ddATP/dATP mixing substrate, its ratio adopts 1/100, and primer extension is by the mediation of T7 Sequenase, and temperature of reaction and time are controlled between 52 degrees centigrade to 64 degrees centigrade respectively and between 30 seconds in second to 90.This step is similar to the terminal cessation method order-checking of Sanger ' s on principle, and difference is that this experiment do not adopt the public mixing substrate that contains four kinds of ddNTP and four kinds of dNTP, but adopts ddATP/dATP respectively; DdTTP/dTTP; The mixing substrate that ddCTP/dCTP is different with ddGTP/dGTP four classes.The terminal cessation method order-checking of Sanger ' s has been widely used in various fields such as molecular biology, genetics and gene order-checking at present.After part chain termination primer extension reaction is finished, add the one times of concentration PCR reaction buffer that does not contain ddNTP and dNTP on chip, repeated washing 2-5 time is removed background fluorescence signal and the unnecessary template that remains in the reaction system by repeatedly washing.Detect its strength of signal then, to judge the number of sequence current position VITAMIN B4.

(3) the replaceability primer extension reaction that under high-fidelity DNA polymerase catalysis, carries out of the dNTP of anti-3 '-5 ' exonuclease digestion of synthetic with 3 '-5 ' exonuclease activity.In the replaceability primer extension reaction in this example, the PS-dATP substrate that adopts sulfuration to modify, its working concentration is 200 μ M dATP, reaction is mediated by having 3 '-5 ' exonuclease activity Pfu high-fidelity DNA polymerase, and the primer extension reaction temperature and time is controlled between 52 degrees centigrade to 64 degrees centigrade respectively and between 30 seconds in second to 90.

(4) coupling part chain termination primer extension reaction and replaceability primer extension reaction are also reused this coupling process.As shown in table 1, this example has shown each circulation all respectively at the base A of each particular types, T, and C, G carry out part chain termination primer extension reaction and replaceability primer extension reaction.By constantly repeating this working cycle, with the longer sequence of decoding.This working cycle is finished the order-checking task for the basis that the present invention is used for sequential analysis by part chain termination primer extension reaction and the continuous circulation of two linked reactions of replaceability primer extension reaction.After part chain termination primer extension reaction was finished, the then corresponding working concentration respectively of replaceability primer extension was 200 μ M dATP, 200 μ M dTTP, and 200 μ M dCTP, 200 μ MdGTP are to finish the replaceability primer extension reaction.

By above-mentioned four steps, this example has provided wild-type sequence in last column of table 1.

Table 1, BRCA1

Illustrate: the base that the black matrix in the sequencing primer underlines mark is sulfuration modification person.Listed fluorescence intensity derives from the part chain termination reaction and uses the ddNTP/dNTP ratio to be 1:100 in this table.Its meaning difference of proportional concentration of different ddNTP/dNTP, for example 1:10 gained strength of signal is stronger, very fast to the long sequence decay of the bunchiness of single base, and 1:100 or corresponding the dying down of 1:1000 gained strength of signal, but slower to the long sequence decay of the bunchiness of single base.In fact, when this ratio is 1:0 (promptly only using four kinds of blended ddNTP), can only extend a base; And when this ratio was 0:1 (promptly only using four kinds of blended dNTP), then extension can carry out continuously.

Mix the total fluorescence intensity (is that the single-basic extension fluorescence intensity of substrate is 1 with pure ddNTP) of the fluorescence ddNTP in the nascent DNA chain in each step at single base when table 2, ddNTP/dNTP different ratios are measured the bunchiness base

Become string length

(base pure ddNTP 1,/10 1/,100 1/1000 a pure dNTP

Number)

1 1.000 0.100 0.010 0.001 0.000

2 0.190 0.020 0.002 0.000

3 0.271 0.030 0.003 0.000

4 0.344 0.039 0.004 0.000

5 0.410 0.049 0.005 0.000

6 0.469 0.059 0.006 0.000

7 0.522 0.068 0.007 0.000

8 0.570 0.077 0.008 0.000

9 0.613 0.086 0.009 0.000

10 0.651 0.096 0.010 0.000

11 0.686 0.105 0.011 0.000

12 0.718 0.114 0.012 0.000

13 0.746 0.122 0.013 0.000

14 0.771 0.131 0.014 0.000

15 0.794 0.140 0.015 0.000

16 0.815 0.149 0.016 0.000

17 0.833 0.157 0.017 0.000

18 0.850 0.165 0.018 0.000

19 0.865 0.174 0.019 0.000

20 0.878 0.182 0.020 0.000

21 0.891 0.190 0.021 0.000

22 0.902 0.198 0.022 0.000

23 0.911 0.206 0.023 0.000

24 0.920 0.214 0.024 0.000

25 0.928 0.222 0.025 0.000

26 0.935 0.230 0.026 0.000

27 0.942 0.238 0.027 0.000

28 0.948 0.245 0.028 0.000

29 0.953 0.253 0.029 0.000

30 0.958 0.260 0.030 0.000

31 0.962 0.268 0.031 0.000

32 0.966 0.275 0.032 0.000

33 0.969 0.282 0.032 0.000

34 0.972 0.289 0.033 0.000

35 0.975 0.297 0.034 0.000

The base that black matrix in the table 1 in the sequencing primer underlines mark is the sulfuration modified base.Listed fluorescence intensity derives from part chain termination primer extension reaction and uses the ddNTP/dNTP ratio to be 1:100 in this table.Its meaning difference of proportional concentration of different ddNTP/dNTP, for example 1:10 gained strength of signal is stronger, very fast to the long sequence decay of the bunchiness of single base, and 1:100 or corresponding the dying down of 1:1000 gained strength of signal, but slower to the long sequence decay of the bunchiness of single base.In fact, when this ratio is 1:0 (promptly only using four kinds of blended ddNTP), can only extend a base; And when this ratio was 0:1 (promptly only using four kinds of blended dNTP), then extension can carry out continuously.This proportional concentration according to ddNTP/dNTP obtains the varying strength fluorescent signal and fluorescence signal intensity has detailed reflection with the situation of the long sequence decay of the bunchiness of single base in table 2 in the order-checking process.

Reference

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Claims

1. one kind is utilized high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, it is characterized in that comprising the steps:

(4) coupling part chain termination primer extension reaction and replaceability primer extension reaction are also reused this coupling process.

2. according to claim 1ly a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, it is characterized in that described primer is the three end modified primers or the primer of three terminal unmodifieds.

3. according to claim 2ly a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, it is characterized in that described three end modified primers are the modification of anti-3 '-5 ' exonuclease digestion.

4. according to claim 1ly a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, it is characterized in that described high-fidelity DNA polymerase with 3 '-5 ' exonuclease activity is Vent high-fidelity DNA polymerase, DeepVent high-fidelity DNA polymerase, Pfu high-fidelity DNA polymerase, Sso7d-Pfu high-fidelity DNA polymerase or Kapa high-fidelity DNA polymerase.

5. according to claim 1ly a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, it is characterized in that described not have 3 '-5 ' exonuclease activity archaeal dna polymerase be TaqDNA polysaccharase, T7 Sequenase, Vent ^-Archaeal dna polymerase, DeepVent ^-Archaeal dna polymerase.

6. according to claim 1ly a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, it is characterized in that the PS-dNTP that the dNTP of anti-3 '-5 ' exonuclease digestion of described synthetic modifies for sulfuration, acy-NTP or the lock nucleic acid that cyclisation is modified.

7. according to claim 1ly a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, the concentration ratio of dNTP that it is characterized in that described fluorescently-labeled ddNTP and non-fluorescent label single base maximum continuously in sequence to be measured is 1/3 to 1/30 when the repetition degree is less than 10.

8. according to claim 1ly a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, the dNTP concentration ratio that it is characterized in that described fluorescently-labeled ddNTP and non-fluorescent label single base maximum continuously in sequence to be measured is 1/31 to 1/300 when the repetition degree is less than 100.

9. according to claim 1ly a kind ofly utilize high-fidelity DNA polymerase-mediated replaceability primer extension reaction to carry out sequencing technique, the dNTP concentration ratio that it is characterized in that described fluorescently-labeled ddNTP and non-fluorescent label single base maximum continuously in sequence to be measured is 1/301 to 1/3000 when the repetition degree is less than 1000.