CN100463973C - Molecular seal sequencing device and sequencing method - Google Patents

Abstract

Translated fromChinese

分子印章测序装置和测序方法涉及一种基于焦磷酸测序原理的方法，该装置是包括DNA延伸部分、化学发光部分和机械连接三部分。该分子印章测序方法是将滚环扩增单链DNA模板或PCR扩增的单链模板和DNA聚合酶固定在的DNA延伸基底的一面；而化学发光相关酶、化学发光辅助物则固定于发光透明基片的一面；光信号检测装置则置于发光透明基片的另一面。当延伸基底面与加有发光酶的透明发光基片接触压印后，焦磷酸基团转移进入加有发光酶的发光透明基片，就会在该基片的相应位置上释放一定强度的化学反应光信号，通过发光检测装置就可以实时检测到该信号的有无及强弱。当四种dNTP单核苷酸依次循环加入该系统中，就可以达到对DNA延伸基底面上每一DNA序列进行分析测序。

The molecular seal sequencing device and the sequencing method relate to a method based on the principle of pyrosequencing, and the device includes three parts: a DNA extension part, a chemiluminescent part and a mechanical link. The molecular stamp sequencing method is to immobilize rolling circle amplified single-stranded DNA templates or PCR-amplified single-stranded templates and DNA polymerase on one side of the DNA extension substrate; One side of the transparent substrate; the optical signal detection device is placed on the other side of the luminescent transparent substrate. When the extended basal surface is in contact with the transparent luminescent substrate added with luminescent enzyme for imprinting, the pyrophosphate group is transferred into the luminescent transparent substrate added with luminescent enzyme, and a certain intensity of chemical will be released on the corresponding position of the substrate. In response to the light signal, the existence and strength of the signal can be detected in real time through the luminescence detection device. When four dNTP single nucleotides are sequentially added to the system in a circular manner, the analysis and sequencing of each DNA sequence on the base surface of the DNA extension can be achieved.

Description

Molecular seal sequencing device and sequence measurement

Technical field

The present invention relates to a kind of molecular seal sequencing device and sequence measurement, relate in particular to a kind of low cost, high-throughout microarray molecular seal sequencing device and sequence measurement based on tetra-sodium order-checking principle.

Background technology

Finishing of the Human Genome Project is to a great extent owing to the development of sequencing technologies and the reduction of order-checking cost.The dna sequencing cost is reduced to about ten cents of present each bases by about ten dollars of each bases before more than ten years.But, along with going deep into to human genome research, people more and more clearly realize that: people will really will study the function of gene, crack this branch sub-routine of evolving naturally and producing of genome, and then on gene level, carry out clinical diagnosis, disease prevention and susceptibility and assess, only detect,, understand this a part programming language by the more different phenotype mankind's genome difference for the full genomic information of a large amount of individuals.That is to say, be familiar with the difference of different ethnic groups, crowd and dna sequence between individual, and then accurately infer of the influence of these differences disease generation, the rule of development by the detection of the full genomic information of a large amount of individuals.This just needs further to reduce significantly the cost of human genome sequencing.Have the expert to propose the target of 1000 dollars of genome sequencings, that is to say, the cost of dna sequencing will further descend 1,000,000 times.Therefore, this is great challenge for the mankind, has met the individual medical science and the challenge in pharmacy epoch since the human high-throughput dna sequencing technology of being badly in need of a kind of brand-new Ultra Low Cost of development.

At present, the sequence measurement of commonplace use is the dideoxy sequencing method of Sanger invention in 1977, though this method is through development and the improvement in 29 years, existingly aspect the dna sequence dna length improve greatly reading, flux is low, speed slow and need electrophoresis or carry out weakness such as marker detection but it still has.And early stage Maxam and Gilbert chemical cracking sequencing, though the ability that checks order is more by force also arranged, owing to use deleterious chemical substance in this order-checking process, and do not obtain practical application.Sequencing by hybridization also seldom is employed owing to its serious non-specific hybridization signal influences the accuracy of order-checking.In addition, also have multiple other sequence measurement, just in the laboratory study stage, need time mostly from application.

The tetra-sodium sequencing technologies is a dna sequence analysis technology of new generation, this method is under the cascade katalysis of four kinds of enzymes (being archaeal dna polymerase, sulfurylase, luciferase and apyrase) in same reaction system, ssDNA to be measured goes up base of the every generation of bonded primer and extends, and the fluorescent signal that then has certain intensity is released.Take turns in the sequencing reaction at each like this, add one of four kinds of dNTP (being dATP, dTTP, dCTP and dGTP) successively,, just can reach the purpose of The real time measure dna sequence dna by detecting having or not and intensity of each fluorescence release.This technology need not electrophoresis to the sequential analysis of DNA, and dna fragmentation also need not mark, therefore, has the automatization height, advantages such as good reproducibility.The tetra-sodium sequencing technologies is widely used at aspects such as the Rapid identification of the research of single nucleotide polymorphism, nosetiology analysis, pathogenic micro-organism, legal medical expert's evaluations.At present, use this technology to check order both at home and abroad and mainly contain two kinds of methods.A kind of is the tetra-sodium sequence measurement that generally adopts the single tube reaction, promptly carries out the sequencing of a sample at every turn, and high throughput testing is restricted.Another kind is external parallel tetra-sodium sequence measurement based on 96 orifice plates, it is that 96 different samples are placed 96 holes respectively, then these samples are checked order, though the analysis throughput of this method increases, the preparation of sample is cumbersome and the consumption medicine is bigger.Though more than two kinds of tetra-sodium sequencing devices can realize mensuration to the target DNA sequence because their reaction system is all huge and specimen preparation trouble, the very big raising of the cost that causes checking order.What is more important, because tetra-sodium sequencing reaction system is a liquid-phase system, each sequencing reaction all needs an independently reaction chamber, this has just limited the flux of dna sequencing widely.For this reason, we have proposed application molecular seal method based on the tetra-sodium principle that checks order, and have developed that a kind of cost is low, reagent consumes the little and simple high-throughput dna sequencing of specimen preparation method, this method will greatly reduce the cost of dna sequencing, can be used in the genomic detection of human individual.

Summary of the invention

Technical problem: the invention provides a kind of novel molecular seal sequencing device and sequence measurement, it can carry out low cost, high-throughput, high-sensitivity analysis to DNA purpose fragment on biochip.

Technical scheme: biochip sequencing device of the present invention extends substrate, chemoluminescence transparent substrate and mechanical connection arm three parts by DNA and constitutes; Wherein, DNA extends substrate, the chemoluminescence transparent substrate is separately fixed on the mechanical connection arm; It is a solid material substrate that DNA extends substrate, extend one side relative in the substrate at DNA and extend basal surface for the DNA that places DNA and polysaccharase with the chemoluminescence transparent substrate, on the chemoluminescence transparent substrate, extend the relative one side of substrate for placing the luminescence transparent substrate face of luminous relevant enzyme and luminous subsidiary with DNA, outside at the another side of chemoluminescence transparent substrate is provided with luminescence detector, the output termination signal analysis device of luminescence detector.

The material that DNA extends substrate is one of glass, quartz, metal, plastics, pottery, rubber, gel, paper wood, nylon, or by two kinds in them or the multiple mixture of forming, and the DNA extension substrate one side relative with the chemoluminescence transparent substrate is a horizontal plane; Extend not relative with chemoluminescence transparent substrate one side in the substrate at DNA, the laying temperature regulation device is as heating electrode, semiconductor temperature-control device, optics heating, air-flow temperature control.The material of placing the substrate face of luminous relevant enzyme and luminous subsidiary is one of materials such as slide, rubber, quartz, gel, or by two kinds in them or the multiple mixture of forming.Luminescence detector is a kind of among photoelectric coupled device CCD, photomultiplier PMT and the complementary matal-oxide semiconductor CMOS.Two cards are arranged on the mechanical connection arm, and they are used for fixing DNA respectively and extend substrate, chemoluminescence transparent substrate; These two cards can be that one of them fastens on the mechanical connection arm, and another card slides up and down along the mechanical connection arm.

The sequence measurement of biochip sequencing device of the present invention is, dna profiling to be measured and archaeal dna polymerase are placed on DNA and extend on the basal surface, and luminous relevant biological enzyme and luminous subsidiary then are placed on the luminescence transparent substrate face; After adding dNTP on the DNA extension basal surface, above-mentioned two portions are carried out contact printing face-to-face, the tetra-sodium that extension produced extends basal surface from DNA and transfers on the luminescence transparent substrate face, and on this luminescence transparent substrate face with luminous associated biomolecule enzyme and luminous subsidiary effect, emit the optical signal of certain intensity; The above-mentioned luminescence detector of this optical signals detects.

The dna profiling of placing on the DNA extension basal surface in this method to be measured is a pcr amplification product, or the rolling circle amplification product, or other any type of sequence DNA to be measured; This dna profiling to be measured can directly be fixed on double-stranded DNA on the face of substrate, treated again acquisition single stranded DNA, and each single stranded DNA is hybridized with corresponding sequencing primer more then; Also can be after double-stranded DNA is processed into single stranded DNA earlier, to be fixed on the basal surface of extension, each single stranded DNA be hybridized with corresponding sequencing primer more again.The adding method of the required dNTP of DNA extension is for to carry out application of sample by the method for ulv spraying, or transfer printing application of sample from four different seals respectively.Luminous organism enzyme on the luminescence transparent substrate face is a luciferase, can comprise a kind of or two kinds of compositions in ATP sulfurylase and the adenosine triphosphate bis phosphoric acid degrading enzyme simultaneously; Luminous subsidiary is adenosine phosphinylidyne sulfuric acid and fluorescein.Contact printing is DNA to be extended substrate fix, and with mechanical manipulator chemoluminescence transparent substrate face is impressed in DNA and extends on the basal surface; Otherwise or the chemoluminescence transparent substrate fixed, with mechanical manipulator DNA is extended basal surface and impresses on chemoluminescence transparent substrate face.

Beneficial effect: the molecular seal sequence measurement that the present invention proposes a kind of novelty, this method is based on tetra-sodium sequencing technologies principle, single stranded DNA sequence to be measured is combined with sequencing primer, each base is extended all with the coupling of a chemiluminescence signal phase on the primer, the institute that obtains according to luminescence detection apparatus discharges the power that has that it's too late of optical signal, can determine dna sequence dna to be measured.The present invention compares with existing sequencing technologies and has the following advantages: a. high-throughput.Thousands of dna sequencing fragments to be measured are fixed on and form microarray extended DNA substrate on the substrate, and therefore, this method can realize the high-throughput of a large amount of dna sequence dnas to be measured and parallelism are measured.B. low-cost.Present method need not to consume the more reaction solution of volume, but archaeal dna polymerase and three kinds of luminous relevant enzyme and subsidiary directly are added to substrate, and can access repetition, thus reduced reaction enzymes consumption, reduce the order-checking cost; Nucleic acid monomer has also reduced consumption of raw materials with the method for little spray or impression.In addition, DNA to be measured can directly be fixed on by cheap acrylamide gel and extend on the substrate.C. easy and simple to handle.This device is realized easily automatization, DNA to be measured need not mark also need not electrophoresis, thereby avoided loaded down with trivial details glue, run processes such as glue and scanning.

Description of drawings

Fig. 1 is the tetra-sodium sequencing device synoptic diagram based on molecular seal that the present invention proposes.

Fig. 2 is that DNA extends basal surface (11) front schematic view among the present invention.

Fig. 3 adds each lattice point DNA of dNTP base extension synoptic diagram takes place.

Have among the above figure: DNA extends substrate 1, DNA extendsbasal surface 11, chemoluminescence transparent substrate 2, luminescencetransparent substrate face 21, mechanical connection arm 3, luminescence detector 4, signal analysis device 5, dna profiling to be measured 6, archaeal dna polymerase 7, the little lattice point numbering 8 of DNA, DNA base extension number 9.

Embodiment

The present invention is based on tetra-sodium order-checking principle, proposed a kind of molecular seal sequencing device and method based on biochip.At first, with the amplified production of chemically modified be prepared into single stranded DNA and with corresponding sequencing primer hybridization after, be fixed in solid substrate material (as: slide by certain media, quartzy, metal, plastics, pottery, rubber, gel, paper wood, one of nylon etc., or by two kinds in them or the multiple mixture of forming) the surface on, (or refabrication became single stranded DNA after the amplified production of chemically modified was fixed in substrate earlier, and with the hybridization of corresponding sequencing primer), again archaeal dna polymerase is positioned in this substrate, constitutes to contain and wait to check order dna profiling microarray DNA extension substrate; By ulv spraying or method for stamping one of four kinds of dNTP Nucleotide are evenly sprayed again or impress and extend substrate surface in DNA, under the effect of archaeal dna polymerase, extend suprabasil sequencing primer if the extension of base takes place, at the extended position generation tetra-sodium group of substrate.Then that chemoluminescence is required enzyme and subsidiary are added on another reaction substrate (or in); After extension substrate and the substrate that is added with luminous enzyme pass through contact printing, the tetra-sodium radical transfer enters the substrate that is added with luminous enzyme, will on corresponding position, there be the chemiluminescence signal of certain intensity to discharge, just can detecting the power that has that it's too late of this signal by luminescence detection apparatus in real time.Add in this system when four kinds of dNTP circulate successively, the dna sequence dna that just can reach extending each lattice point in the substrate carries out accurate multianalysis purpose.

This sequence measurement that hits the biochip sequencing device is characterized in that dna profiling to be measured and archaeal dna polymerase are placed on DNA and extend on thebasal surface 11, and luminous relevant biological enzyme and luminous subsidiary then are placed on the luminescencetransparent substrate face 21; After adding dNTP on the DNA extensionbasal surface 11, above-mentioned two portions are carried out contact printing face-to-face, the tetra-sodium that extension produced extendsbasal surface 11 from DNA and transfers on the luminescencetransparent substrate face 21 that contains luminous relevant enzyme and luminous subsidiary, and, emit the optical signal of certain intensity in thesubstrate face 21 and luminous relevant enzyme and luminous subsidiary effect of luminous relevant enzyme and luminous subsidiary; The above-mentioned luminescence detector 4 of this optical signals detects.

Dna profiling 6 to be measured can be a pcr amplification product on the DNA extensionbasal surface 11, also can be the rolling circle amplification product, or other any type of sequence DNA to be measured.The fixed form of DNA to be measured can be directly the DNA amplification product to be fixed on basal surface, obtains single stranded DNA through certain processing again, and each single stranded DNA is hybridized with corresponding sequencing primer more then; Also can be the DNA amplification product is processed into single stranded DNA earlier outside substrate after, to be fixed on the substrate, single stranded DNA be hybridized with sequencing primer more then again.The preparation method of single stranded DNA can be by behind the acrylamide gel amplified production that fixedly acrylamide is modified, and is removing revocable single stranded DNA acquisition through alkaline denaturation; Also can be after magnetic bead that Streptavidin is modified is fixed with the amplified production of biotin modification; Also can be after being fixed with the amplified production of biotin modification by the dextran microballon that Streptavidin is modified, to remove revocable strand through alkaline denaturation.The described archaeal dna polymerase of this part can be Klenow Fragmentpolymerase (exo-), also can be the Sequenase Sequenase through point mutation.

The adding method of the required dNTP of DNA extension (dATP, dCTP, dGTP and dTTP) is for to carry out application of sample by the method for ulv spraying, or transfer printing application of sample from four different seals respectively.

Be placed with luminous relevant biological enzyme and luminous relevant subsidiary on the chemoluminescence substrate face 21.For example, on chemoluminescence transparent substrate face, be placed with the combination of three kinds of luminous involved enzyme such as ATP sulfurylase and adenosine phosphinylidyne sulfuric acid, luciferase and fluorescein and adenosine triphosphate bis phosphoric acid degrading enzyme etc. and luminous relevant subsidiary, or on chemoluminescence transparent substrate face, be placed with the combination of luminous relevant biological enzyme such as two kinds of ATP sulfurylase and adenosine phosphinylidyne sulfuric acid, luciferase and fluoresceins etc. and luminous relevant subsidiary, or by luciferase with and fluorescein form.Wherein ATP sulfurylase, luciferase and adenosine triphosphate bis phosphoric acid degrading enzyme etc. are luminous relevant biological enzyme.Adenosine phosphinylidyne sulfuric acid is luminous relevant subsidiary with fluorescein.

It is fixing that the contact printing of above-mentioned DNA extensionbasal surface 11 andchemoluminescence substrate face 21 extends DNA substrate 1 exactly, with mechanical manipulator chemoluminescencetransparent substrate face 21 impressed in DNA and extend on thebasal surface 11; Otherwise or chemoluminescence transparent substrate 2 is fixing, with mechanical manipulator DNA is extendedbasal surface 11 and impresses on chemoluminescencetransparent substrate face 21.

Example 1. usefulness rolling circle amplification products carry out sequencing to 50 dna fragmentations.

1. carry out enzyme with corresponding restriction enzyme respectively at 50 dna fragmentations and cut, and a chain in the endonuclease bamhi is connected into cyclic DNA with a universal adapter (dna sequence dna that contains 36 bases) under the effect of target DNA.

2. respectively above-mentioned cyclic DNA is carried out two primer rolling circle amplifications with the universal amplification reverse primer (5 ' end has acrylamide to modify and holds 18 base complementrities with adapter 3 ') of one 18 base and the universal amplification forward primer of another 18 base (with adapter 5 ' end 18bps complementation); Then the rolling circle amplification product is mixed with acrylamide gel solution, and with the chip point sample instrument with 50 sample spot of handling on sheet glass, and aggregate into the gel dot matrix, be prepared into the micro-array chip of order-checking usefulness, see shown in Figure 2; Pass through alkaline denaturation, electrophoresis and flushing again, with revocable DNA, primer, single-stranded loop and other Impurity removal just obtain DNA and extend microarray;

3. use the single stranded DNA sequence hybridization to be measured on universal sequencing primer thing (identical) and the above-mentioned extension basal surface with amplification forward primer sequence, and unnecessary sequencing primer on the electrophoresis eccysis dna profiling.

4. archaeal dna polymerase evenly is positioned on the dna microarray that extends substrate, simultaneously luminous relevant enzyme (ATP sulfurylase, luciferase and apyrase) and luminous subsidiary (adenosine phosphinylidyne sulfuric acid) is fixed on the luminous substrate of another transparent glass.

5. the deoxyribonucleotide reaction solution with one of an amount of dNTP is dispersed on the dna microarray that extends basal surface by ulv spraying.If the base complementrity of the most close sequencing primer of corresponding position dna sequence dna on the dNTP of this time adding and the template, this moment, the polymerization extension took place in this base and DNA bonded sequencing primer to be measured under the effect of archaeal dna polymerase, produced equimolar pyrophosphate salt simultaneously.

6. rapidly DNA is extended basal surface and impress on another luminescence transparent substrate, pyrophosphate salt is transferred on the luminescence transparent substrate face.Under the synergy of ATP sulfurylase and luciferase etc., the fluorescent signal that then can produce respective strengths discharges.By CCD imaging,, just can determine whether this dNTP the number of polymerization and primer extension base takes place at certain lattice point to the detection with intensity of having or not of each lattice point fluorescence at luminescence transparent substrate another side.Fig. 3 is behind dNTP of adding, the synoptic diagram of the primer generation base extension on each lattice point of molecular seal chip.X-coordinate is represented this lattice point sequence number among the figure, and ordinate zou is represented base extension number.The figure shows, after this time adds the dNTP reaction solution,lattice point 1,4,6,7,9,12 ... Deng the extension that a base has taken place; Latticepoint 3,11 ... Deng the extension that two bases have taken place; Lattice point 8 ... Deng the extension that three bases have taken place; Lattice point 2,5,10,13 ... Deng the extension that base does not then take place.

7. DNA being extended dNTP unnecessary on the substrate removes.

8. repeating step 5 to 7, four kinds of different dNTP are circulated to add successively extend substrate, and write down each dNTP generation optical signal that polymerization discharges.After four kinds of monomer dATP, dGTP, dCTP and dTTP reaction liquid circulate the adding micro-array chip successively, then can carry out sequencing to each dna fragmentation on the microarray simultaneously.For example, after first lattice point passes through successively, the polymerization of the polymerization of two bases, the polymerization of zero base, a base and the polymerization of four bases have taken place respectively in dATP, dGTP, dCTP and dTTP circulate for the first time.Thus, seven bases that obtain on the sequencing primer taking place extending are 5 '-AACTTTT-3 ', thereby determine this lattice point DNA based composition be 5 '-AAAAGTT-3 ', finally just can obtain DNA full sequence to be measured on first lattice point through several times circulation back.In like manner can acquire other each lattice point dna sequence dna.Several times reaction result on last each lattice point of combined reaction chip just can obtain extending each lattice point dna sequence dna composition on the substrate, thereby realize 50 DNA; Segmental parallel high-flux sequence.

Example 2. utilizes the PCR product that 100 SNP genotype are detected.

1. at first, extract people's genomic dna, and, obtain 100 pcr amplification product fragments that contain different SNP site respectively by round pcr.5 ' end at the reverse primer of above-mentioned PCR product use biotin modification, and makes preceding 18 base sequences of this reverse primer identical, and the base of remainder is each segmental distinguished sequence part to be amplified.

2. these 100 PCR products are fixed with the magnetic bead that Streptavidin is modified respectively, carry out denaturing treatment with NaOH again, and the damping fluid flushing obtains single stranded DNA.

3. extend basal surface with the DNA that has magnetic above-mentioned single stranded DNA is fixed into 10 * 10 arrays.

4. extend suprabasil microarray DNA with universal sequencing primer thing and this and hybridize, and unnecessary primer is rinsed well.

5. archaeal dna polymerase evenly is applied in DNA and extends in the microarray extension substrate of substrate, simultaneously luminous relevant enzyme and luminous subsidiary are placed on another luminescence transparent substrate.

6. an amount of dNTP is transferred on the extended DNA microarray substrate with method for stamping, and DNA extended basal surface and another luminescence transparent substrate carries out contact printing.

7. above-mentioned microarray DNA is extended each lattice point DNA in the substrate produce optical signal to have that it's too late strong and weak detects and record with luminescence detection apparatus, provide the number of base that each lattice point extends simultaneously by software analysis.

8. more respectively with dTTP, the operation of dATP anddGTP repeating step 6 and 7.

9. like this four kinds of dNTP being circulated successively time impresses on the DNA that extends basal surface, and the power of the each optical signal that each lattice point discharges of record, provides the sequence of each lattice point DNA again by the analysis-by-synthesis of software.

10. at last 100 SNP site mutation types are judged.

Example 3. usefulness rolling circle amplification products carry out the mensuration again of intestinal bacteria complete genome DNA sequence.

1. extract colibacillary genomic dna, and by the ultrasonic method fragmentation.By the end in the dna fragmentation is combined with the adapter (dna sequence dna that contains 36 bases) of general a 5 ' end phosphoric acid under the effect of target DNA, make a chain in the dna fragmentation connect into cyclic DNA, and carry out suitable dilution, form the colibacillary genome sequencing template solution of single cyclic DNA.

2. the universal amplification reverse primer (5 ' end has acrylamide to modify and holds 18 base complementrities with adapter 3 ') with one 18 base carries out two primer rolling circle amplifications to above-mentioned cyclic DNA respectively; Then the rolling circle amplification product is mixed with acrylamide gel solution, spreads on the sheet glass equably, and aggregate into ultrafine gel coat, be prepared into contain a large amount of unit molecule multiple copieies roll ring product micro-array chip.

3. the unique DNA template on universal sequencing primer thing and the micro-array chip is hybridized, and unnecessary sequencing primer on the electrophoresis eccysis dna profiling, just obtain DNA and extend microarray.

4. archaeal dna polymerase evenly is positioned on the dna microarray that extends substrate, simultaneously luminous relevant enzyme (ATP sulfurylase, luciferase and apyrase) and luminous subsidiary (adenosine phosphinylidyne sulfuric acid) is fixed on the luminous substrate of another transparent glass.

5. the deoxyribonucleotide reaction solution with one of an amount of dNTP is dispersed on the dna microarray extension basal surface by ulv spraying.If the base complementrity of the most close sequencing primer of corresponding position dna sequence dna on the dNTP of this time adding and the template, this moment, the polymerization extension took place in this base and DNA bonded sequencing primer to be measured under the effect of archaeal dna polymerase, produced equimolar pyrophosphate salt simultaneously.

6. will extend basal surface rapidly and impress on another luminescence transparent substrate, pyrophosphate salt is transferred on the luminescence transparent substrate face, under the synergy of ATP sulfurylase and luciferase etc., the fluorescent signal that then can produce respective strengths discharges.By CCD imaging,, just can determine whether this dNTP the number of polymerization and primer extension base takes place at certain lattice point to the detection with intensity of having or not of each lattice point fluorescence at luminescence transparent substrate another side.

7. DNA being extended dNTP unnecessary on the basal surface removes.

8. repeating step 5 to 7, four kinds of different dNTP are circulated to add successively extend basal plane, and write down each dNTP generation optical signal that polymerization discharges.After four kinds of monomer dATP, dGTP, dCTP and dTTP reaction liquid circulate the adding micro-array chip successively, then can carry out sequencing to each dna fragmentation on the microarray simultaneously.Several times reaction result on last each lattice point of combined reaction chip just can obtain extending each unique DNA sequence composition in the substrate, thereby has realized the segmental high-flux parallel order-checking of complete genome DNA.By with reference to known colibacillary genome sequence, the assembling of the dna fragmentation that carries out being checked order realizes that colibacillary full genome checks order again.

Claims (8)

Translated fromChinese

1.一种生物芯片测序装置，其特征在于该装置由DNA延伸基底(1)、化学发光透明基片(2)、机械连接臂(3)、发光检测器(4)、信号分析装置(5)构成；其中，DNA延伸基底(1)、化学发光透明基片(2)分别固定在机械连接臂(3)上；DNA延伸基底(1)为一固体材料基底，在DNA延伸基底(1)上与化学发光透明基片(2)相对的一面为放置DNA及聚合酶的DNA延伸基底面(11)，在化学发光透明基片(2)上与DNA延伸基底(1)相对的一面为放置发光相关酶和发光辅助物的发光透明基片面(21)，在化学发光透明基片(2)的另一面的外部设有发光检测器(4)，发光检测器(4)的输出端接信号分析装置(5)；DNA延伸基底(1)的材料为玻璃、石英、金属、塑料、陶瓷、橡胶、凝胶、纸材、尼龙之一，或由它们中的多种组成的复合物，且DNA延伸基底(1)与化学发光透明基片(2)相对的一面为一水平面；在DNA延伸基底(1)上不与化学发光透明基片(2)相对的一面，放置温度调控装置；机械连接臂(3)上有两个卡(31)，它们分别用来固定DNA延伸基底(1)、化学发光透明基片(2)；这两个卡是其中一个卡固定在机械连接臂(3)上，另一个卡沿机械连接臂(3)上下滑动。1. A biochip sequencing device is characterized in that the device consists of a DNA extension substrate (1), a chemiluminescence transparent substrate (2), a mechanical link arm (3), a luminescence detector (4), a signal analysis device (5 ) constitutes; wherein, the DNA extension base (1) and the chemiluminescent transparent substrate (2) are respectively fixed on the mechanical connecting arm (3); the DNA extension base (1) is a solid material base, and the DNA extension base (1) The side opposite to the chemiluminescence transparent substrate (2) is the DNA extension base surface (11) for placing DNA and polymerase, and the side opposite to the DNA extension substrate (1) on the chemiluminescence transparent substrate (2) is the base surface for placing DNA and polymerase. The luminescent transparent substrate surface (21) of luminescent related enzymes and luminescent auxiliary substances is provided with a luminescent detector (4) outside the other side of the chemiluminescent transparent substrate (2), and the output terminal of the luminescent detector (4) is connected to a signal Analytical device (5); the material of the DNA extension substrate (1) is one of glass, quartz, metal, plastic, ceramics, rubber, gel, paper, nylon, or a composite composed of multiple of them, and The side opposite to the chemiluminescence transparent substrate (2) of the DNA extension substrate (1) is a horizontal plane; on the side of the DNA extension substrate (1) that is not opposite to the chemiluminescence transparent substrate (2), a temperature control device is placed; There are two cards (31) on the connecting arm (3), which are used to fix the DNA extension substrate (1) and the chemiluminescent transparent substrate (2) respectively; these two cards are one of the cards fixed on the mechanical connecting arm (3 ), the other card slides up and down along the mechanical linkage arm (3).2.根据权利要1求所述的生物芯片测序装置，其特征在于放置发光相关酶和发光辅助物的基片面(21)的材料为玻片、橡胶、石英、凝胶材料之一，或由它们中的多种组成的复合物。2. The biochip sequencing device according to claim 1, characterized in that the material of the substrate surface (21) on which luminescence-related enzymes and luminescence assistants are placed is one of glass slides, rubber, quartz, and gel materials, or made of A complex of many of them.3.根据权利要求1所述的生物芯片测序装置，其特征在于发光检测器(4)为光电耦合器件CCD、光电倍增管PMT和互补性氧化金属半导体CMOS中的一种。3. The biochip sequencing device according to claim 1, characterized in that the luminescence detector (4) is one of a photocoupler device CCD, a photomultiplier tube PMT and a complementary metal oxide semiconductor CMOS.4.一种如权利要求1所述的生物芯片测序装置的测序方法，其特征在于待测DNA模板和DNA聚合酶放置在DNA延伸基底面(11)上，发光相关的生物酶和发光辅助物则放置在发光透明基片面(21)上；在DNA延伸基底面(11)上加入dNTP后，将上述两部分面对面进行接触压印，延伸反应所产生的焦磷酸从DNA延伸基底面(11)转移到发光透明基片面(21)上，并在该发光透明基片面(21)上与发光相关生物酶和发光辅助物作用，放出有一定强度的光信号；该光信号由所述发光检测器(4)检测。4. A sequencing method of a biochip sequencing device as claimed in claim 1, characterized in that the DNA template to be tested and the DNA polymerase are placed on the DNA extension base surface (11), and the relevant biological enzymes and luminescent auxiliary substances of luminescence Then place it on the luminescent transparent substrate surface (21); after adding dNTPs on the DNA extension base surface (11), the above two parts are contacted and imprinted face to face, and the pyrophosphate generated by the extension reaction is released from the DNA extension base surface (11) Transfer to the luminescent transparent substrate surface (21), and act on the luminescent transparent substrate surface (21) with luminescence-related biological enzymes and luminescent auxiliary substances to emit a light signal with a certain intensity; the light signal is generated by the luminescence detector (4) Detection.5.根权利要求4所述的生物芯片测序装置的测序方法，其特征在于该方法中DNA延伸基底面(11)上放置的待测DNA模板(6)是PCR扩增产物，或是滚环扩增产物，或其它任何形式的待测序列DNA；该待测DNA模板，直接把双链DNA固定在基底的面上，再经处理获得单链DNA，然后每一单链DNA再与相应的测序引物杂交；或是将双链DNA先处理成单链DNA后，再将其固定于延伸部分基底面上，每一单链DNA再与相应的测序引物杂交。5. The sequencing method of the biochip sequencing device according to claim 4, is characterized in that the DNA template to be measured (6) placed on the DNA extension base surface (11) is a PCR amplification product, or a rolling circle Amplified products, or any other form of sequence DNA to be tested; the DNA template to be tested directly immobilizes double-stranded DNA on the surface of the substrate, and then is processed to obtain single-stranded DNA, and then each single-stranded DNA is combined with the corresponding Sequencing primer hybridization; or double-stranded DNA is processed into single-stranded DNA first, and then fixed on the base surface of the extension part, and each single-stranded DNA is hybridized with the corresponding sequencing primer.6.根据权利要求4所述的生物芯片测序装置的测序方法，其特征在于DNA延伸反应所需要dNTP的加入方法为通过微量喷雾的方法进行加样，或分别从四个不同的印章上转印加样。6. the sequencing method of biochip sequencing device according to claim 4, it is characterized in that the adding method of required dNTP of DNA extension reaction is to add sample by the method for microspray, or add respectively from four different seals. Sample.7.根据权利要求4所述的生物芯片测序装置的测序方法，其特征在于发光透明基片面(21)上的发光生物酶为荧光素酶，同时包括ATP硫酸化酶和腺苷三磷酸双磷酸降解酶中的一种或二种组成；发光辅助物为腺苷磷酰硫酸和荧光素。7. The sequencing method of the biochip sequencing device according to claim 4, characterized in that the luminescent bio-enzyme on the luminescent transparent substrate (21) is luciferase, including ATP sulfurylase and adenosine triphosphate Composition of one or two kinds of degrading enzymes; luminescent auxiliary substances are adenosine phosphoryl sulfate and fluorescein.8.根据权利要求4所述的生物芯片测序装置的测序方法，其特征在于接触压印是将DNA延伸基底(1)固定，用机械操作装置将化学发光透明基片面(21)压印于DNA延伸基底面(11)上；或反之将化学发光透明基片(2)固定，用机械操作装置将DNA延伸基底面(11)压印于化学发光透明基片面(21)上。8. The sequencing method of the biochip sequencing device according to claim 4, characterized in that the contact imprinting is to fix the DNA extension substrate (1), and the chemiluminescent transparent substrate surface (21) is imprinted on the DNA with a mechanical operating device. on the extended base surface (11); or conversely fix the chemiluminescent transparent substrate (2), and imprint the DNA extended basal surface (11) on the chemiluminescent transparent substrate surface (21) with a mechanical operating device.

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