WO2019161810A2 - 基于微孔阵列芯片的单分子文库pcr扩增的基因测序方法 - Google Patents

基于微孔阵列芯片的单分子文库pcr扩增的基因测序方法 Download PDF

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WO2019161810A2
WO2019161810A2 PCT/CN2019/082561 CN2019082561W WO2019161810A2 WO 2019161810 A2 WO2019161810 A2 WO 2019161810A2 CN 2019082561 W CN2019082561 W CN 2019082561W WO 2019161810 A2 WO2019161810 A2 WO 2019161810A2
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chip
sequencing
dna
microwell
micropore
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WO2019161810A3 (zh
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刘亚宝
岳耀斐
张志峰
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张家港万众一芯生物科技有限公司
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0663—Whole sensors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates

Definitions

  • the invention relates to a gene sequencing method for PCR amplification of a single molecule library based on a microwell array chip, and belongs to the technical field of gene sequencing.
  • Sequencing technology can be traced back to the 1950s.
  • early sequencing techniques such as the determination of polyribonucleotide sequences by chemical degradation methods such as Whitfeld; The deoxynucleotide end-termination method (Sanger gene sequencing method) and the chemical degradation method invented by Gilbert and others marked the birth of the first generation of sequencing technology; since then, in the development of more than 30 years, the second generation of sequencing technology has been successively produced.
  • the third-generation sequencing technology is suitable for genome-wide assays with a small initial amount, high throughput, and high degree of automation. Therefore, it is not applicable to the detection of a single genetic locus that is not demanding, such as genetic diagnosis of monogenic diseases. That is, the price/performance ratio is reduced. In addition, the third-generation sequencing technology still needs to reduce the background noise, improve the accuracy, and reduce the cost of sequencing. In addition, how to maintain the ductility of the DNA in the fixation of DNA without dimer structure There are also places to be solved and perfected.
  • Next-generation sequencing technologies such as Ion Torrent's semiconductor sequencing technology
  • semiconductor ion-sensitive field-effect sensors use semiconductor ion-sensitive field-effect sensors to continuously reduce sensor size, increase array size, increase sequencing throughput, and reduce costs by using the semiconductor industry's Moore's Law.
  • the process of Ion Torrent's semiconductor sequencing technology is too cumbersome, including library DNA by water-in-water microbead/single molecule library DNA amplification reaction by onetouch2 instrument, magnetic bead purification and DNA denaturation by ES instrument, and chip centrifugation.
  • the steps of sequencing the chip on the library DNA require a variety of devices, and the operation is complicated and the experiment time is long.
  • the present invention provides a gene sequencing method for single-molecule library PCR amplification based on micro-well array chip, which solves the cumbersome operation process of the sequencing technology in the prior art.
  • it is necessary to use a variety of equipment and supporting reagents, and the technical problems of complicated operation, long time and high cost.
  • the present invention provides a gene sequencing method for PCR amplification of a single molecule library based on a microwell array chip.
  • the method comprises the steps of:
  • Step one mixing a solution of the DNA sample to be tested with a composition of a PCR amplification solution containing DNA polymerase, dNTPs and primer S1, and dropping it onto a chip having a microwell array, so that each micropore on the chip Each contains a mixed solution; then covered with a sealing sheet on the surface of the chip and pressed tightly, so that the micropores on the chip are independent reaction spaces, and only one DNA sample fragment is fixed in the micropores;
  • Step two the sealed microchip array chip is subjected to PCR amplification on a PCR machine
  • Step 3 After the amplification is completed, the sealing tablet on the surface of the chip is removed, the solution in the micropore is removed, and then the double-stranded DNA in the micropore is denatured into single-stranded DNA, and then the cleaning solution is added to clean the micropores, and then Remove the cleaning solution from the micropores;
  • Step 4 adding a sequencing primer S2 to the micropore of the chip processed in the third step, and then using the annealing method to bind the sequencing primer S2 to the DNA sample to be tested in the micropore; removing the solution in the micropore and adding the sequencing enzyme Solution, for incubation;
  • Step 5 When sequencing, add deoxynucleotide triphosphate dNTPs to the micropores in the order of G, C, A, T. If the base pairing of the added dNTPs with the DNA template is successful, it will be under the action of the sequencing enzyme. Adding to the 3 ' end of the sequencing primer, releasing hydrogen ions and PPi ions of pyrophosphate, increasing the charge of the DNA sample itself, releasing the hydrogen ions or pyrophosphate PPI ions, causing the signal response of the sensor at the bottom of the microwell, recording the sensor Responding to the signal and processing the signal to obtain the genetic sequence information of the measured DNA;
  • Step 6 Repeat step 5 to serially sequence the DNA samples in the microwells.
  • the above method may further include at least one of the following additional technical features:
  • one and only one DNA sample fragment is immobilized in the microwell by: the inner wall of the microwell is modified with an oligonucleotide L1 at both ends of the DNA sample
  • the S linker and the L linker are respectively inserted, and the L linker is denatured and hybridized with the oligonucleotide L1 in the microwell to fix the DNA sample on the inner surface of the microwell.
  • the chip with the microwell array is a semiconductor sensor chip, the chip comprising: a semiconductor chip body having a plurality of micropores on the surface and an oligomer fixed on the inner wall of the microwell by a chemical modification method
  • the nucleotide L1 layer is provided with an ion-sensitive field effect sensor or a nanowire transistor sensor directly under the micropore.
  • the charge, hydrogen ion or pyrophosphate PPi ion generated by the base extension reaction triggers a current or voltage change of the sensor under the micropore, thereby converting the chemical signal into an electrical signal, and then Electrical signal processing to translate electrical signals into corresponding gene sequence information.
  • the semiconductor chip further includes a PCR adapter including a base that matches a PCR heating nest or a heating plate, a pressing sheet that seals the micropores, and an upper cover; the base A chip holder for placing a chip is disposed thereon, and the center of the chip holder, the tablet, the base and the upper cover are aligned; and the upper cover is provided with a buckle.
  • a PCR adapter including a base that matches a PCR heating nest or a heating plate, a pressing sheet that seals the micropores, and an upper cover; the base A chip holder for placing a chip is disposed thereon, and the center of the chip holder, the tablet, the base and the upper cover are aligned; and the upper cover is provided with a buckle.
  • the surface of the sensor is coated with a probe that is selective for focusing phosphoric acid ppi ions or phosphate ions or a film that selectively passes through phosphoric acid ppi ions or phosphate ions.
  • the annealing method is specifically: placing the chip on the PCR instrument, processing at 80-99 degrees for 0.5 to 10 minutes, then processing at 15 to 55 degrees for 0.5 to 10 minutes, and then sequencing.
  • Primer S2 is bound to the DNA sample in the microwell; then the solution in the microwell is removed, and the sequencing enzyme solution is added, and the mixture is incubated at 20 to 40 degrees for 0.5 to 30 minutes and then sequenced.
  • the method of removing the solution in the micropores is by vacuum pumping, microfluidic cleaning with a cleaning solution, or blowing with a stamping gas.
  • the preparation process of the chip is as follows:
  • the amino molecule is provided by APTES or AEAPTES; the amination modification is carried out by a vapor phase deposition method to obtain a dense monolayer on the inner surface of the micropores.
  • step 5 the electrical signal generated on the sensor is converted to obtain the genetic sequence information, and the specific process is:
  • the original electrical signal matrix is generated one by one through data acquisition, and four original electrical signal matrices are generated for each sequencing cycle;
  • the present invention realizes single-molecule library DNA amplification reaction on a semiconductor chip microarray, and then directly performs sequencing, removing onetouch2 instrument for water-in-oil microbead single molecule library DNA amplification reaction, and ES instrument magnetic beads Complex steps such as purification and DNA denaturation, sequencing chips on the library DNA of the chip centrifuge greatly simplify the operation process, shorten the experimental operation time and reduce the test cost.
  • the present invention simplifies the experimental procedure while also eliminating the use of corresponding reagents and reducing the experimental cost.
  • the amount of the sequencing reagent of the present invention is smaller than that of the prior art sequencing reagent, and the reagent cost in the PCR amplification and sequencing steps is also saved.
  • FIG. 1 is a schematic structural view of a microchip array of a semiconductor chip according to an embodiment of the present invention, wherein D is a schematic diagram of microporous amplification;
  • a is a schematic diagram of a structure when a PCR is used with a 96-well plate
  • b is a schematic view taken along line AA of a
  • c is a schematic structural view of an enlarged portion C of b
  • d is a schematic view of the PCR adapter
  • FIG. 3 is a schematic diagram showing PCR amplification of DNA to be detected in a microchip array of a semiconductor chip according to an embodiment of the present invention
  • FIG. 4 is a schematic diagram showing sequencing of DNA to be tested in a semiconductor microwell array according to an embodiment of the present invention
  • Figure 5 is a flow chart of a gene sequencing method according to an embodiment of the present invention.
  • FIG. 6 is a schematic diagram of a data conversion process according to an embodiment of the present invention.
  • Figure 7 is a schematic diagram showing the conversion of an analog signal of gene translation in a microwell of an embodiment of the present invention into an electrical signal
  • FIG. 8 is a schematic diagram showing specific process steps 2) to 3) of converting an electrical signal into gene sequence information after being processed in an embodiment of the present invention.
  • 1 semiconductor chip body 11 micropores; 12 oligonucleotide L1 layer; 13 pedestal; 14 sensor; 15 metal layer; 2 primer S1; 3 determination of DNA; 4 sealing plate; 5 primer S2; Plate; 7 PCR adapter; 71 base; 72 chip holder; 73 upper cover; 74 tablet; 75 snap.
  • the present invention also provides a gene sequencing method for PCR amplification of a single molecule library based on a microwell array chip, comprising the following steps:
  • the DNA sample solution to be tested at a concentration of 1-100 pmol/L is uniformly mixed with the PCR amplification solution containing DNA polymerase, dNTPs and primer S1, and the sequence of the primer S1 can be as shown in SEQ ID NO: 2.
  • 5'-CCATCTCATCCCTGCGTGTCTC SEQ ID NO: 2
  • the mixed solution is added dropwise to the microwell array chip, so that each micropore contains a mixed solution, and the DNA solution to be tested is placed during the sample loading process.
  • the number of fragments of DNA is less than or equal to the number of micropores so that there is only one DNA fragment in the micropore; then the surface of the semiconductor chip is covered with a sealing sheet or a flexible film and the sealing is pressed so that each of the semiconductor chips
  • the microwells are independent reaction spaces; and one or more DNA sample fragments in as many microwells as possible are immobilized on the surface of the microwell.
  • the gene sequence of the DNA to be tested is shown in SEQ ID NO: 4. 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG(NNNNNNNN) (10-40) TCACCGACTGCCCATAGAGAGG (SEQ ID NO: 4).
  • Step 2 the sealed semiconductor chip is subjected to PCR amplification on the PCR instrument, as shown in FIG. 3; during the amplification process, the semiconductor chip can be directly placed on the flat-bed PCR instrument, and the semiconductor chip can also be passed.
  • the PCR adapter is amplifying on a conventional PCR instrument, such as a 96-well plate. When amplifying, a plurality of sealed PCR adapters are placed in the holes of the 96-well plate.
  • the base can be made of metal or a heat conductive material for the semiconductor chip. Thermal conductivity can also be achieved by heating and cooling the sealing plate on the semiconductor chip.
  • the amplification conditions are shown in Table 1.
  • Step 3 After the amplification is completed, the sealing tablet on the surface of the chip is removed, the solution in the micropore is removed, and then the double-stranded DNA is denatured into single-stranded DNA, and the denaturation method can be treated with an alkaline solution such as NaOH or KOH or the like.
  • Step 4 adding a sequencing primer S2 to the microwell of the chip processed in the third step, and the gene sequence of the sequencing primer S2 is as shown in SEQ ID NO: 3.
  • the sequencing primer S2 is bound to the DNA sample to be tested in the microwell by annealing, and the chip is sealed by tableting, and then the sealed chip is placed on a flatbed PCR instrument at 80- 99 degree treatment for 0.5 to 10 minutes, 15 to 55 degrees for 0.5 to 10 minutes, or the same treatment in a 96-well plate by PCR adapter, then remove the solution in the micropore and add the sequencing enzyme solution, incubate at 20 to 40 degrees. ⁇ 30 minutes, sequencing was performed.
  • Step 5 When sequencing, add deoxynucleotide triphosphate dNTPs to the micropores in the order of G, C, A, T. If the base pairing of the added dNTPs with the DNA template is successful, it will be under the action of the sequencing enzyme. Add to the 3 ' end of the sequencing primer while releasing a hydrogen ion or a portion of pyrophosphate PPi ion; if the added dNTP is just able to base pair with the DNA template in the microwell, it will be under the action of the sequencing enzyme.
  • the processing device records the response signal of the sensor and processes the signal to obtain the genetic sequence information of the measured DNA.
  • the sensor signal response is represented by a change in voltage or current, and The specific process of converting a signal into gene sequence information after processing is:
  • the original electrical signal matrix is generated one by one through data acquisition, that is, four original electrical signal matrices are generated for each sequencing cycle;
  • the original data is a data point sampled 25 times to 60 times per second, and the analog signal translated by the gene in the micro hole is sampled and converted into a digital signal, that is, an electrical signal;
  • the raw data can be divided into the following types: 1.
  • the first type is 0, that is, there is no pulse signal, and there is no reaction for transmitting base amplification, that is, the current base does not match the DNA sample on the sensor.
  • the second type is 1, ie there is a pulse of hydrogen ions, ie the current base matches one base of the DNA sample on the sensor. 3.
  • the third type is 2 to 5, that is, 2 to more hydrogen ion pulses are generated, that is, 2 or more base matches of the current base and the DNA sample on the sensor, that is, the sample has multiple The appearance of a base.
  • FIG. 8 according to this method, four types of data bases corresponding to four bases A, T, G, and C are respectively generated in sequence; after completion, the original original data is deleted to save storage space. This step not only completed the primary analysis of the data, but also greatly reduced the data size.
  • the four classification data matrix determine the gene type of the DNA sample to be tested in each micropore in the sequencing cycle, and generate a DNA base information matrix; as shown in Fig. 8, four matrices (1, 1) Position, matrix A, T, G are all 0, matrix C is 1, that is, the base of the current sample (1,1) position can only be G, in order to pair with C. Similarly, the (2,4) position of the four matrices, only the matrix G is a non-zero value of 3, meaning that the base of the current sample (2,4) position can only be 3 bases C, ie CCC. This operation produces a gene signal matrix as an output. Finally, the base matrix is followed by the corresponding base matrix of the previous cycle to form a DNA matrix sequence.
  • All the base matrices are connected end to end, that is, a DNA matrix sequence is formed.
  • the senor generates a current or voltage signal by: 1.
  • the charge of the new base pair generated in the sequencing reaction will be permanently solidified on the text.
  • the generated signal will be a DC signal, and the serially sequenced signal will be a stepped electrical signal; 3.
  • the two pairs of the base pair's own charge, the released hydrogen ion or the phosphate ion in the sequencing reaction cause a double signal: For example, a stepped DC signal generated by the charge itself added by DNA and a pulse signal generated by a hydrogen ion or a phosphate ion (both ions diffuse out of the reaction micropore over time).
  • the base pairing which is judged to be successful by detecting both signals is generated, thereby reducing the error rate of the interference generated in the sequencing process, and improving the data quality and the sequencing accuracy.
  • Step 6 Repeat step 5 to perform the next deoxynucleotide triphosphate dNTP and sequence the DNA sample in the microwell.
  • the treatment method for removing the solution in the micropore is:
  • Vacuum pump is used to remove the reaction reagent to achieve the purpose of cleaning the reaction micropore; 2. Microfluidic method is used to rinse the micropores with the cleaning solution; 3. The reaction solution is blown away by using a stamping gas.
  • the above sensors are ion sensitive field effect transistors or semiconductor nanowire transistors.
  • the above step solution is added by a droplet dropping method, such as a droplet sequencer, or by an automatic pipette, or an inkjet printing method, etc., to reduce the amount of liquid used.
  • a droplet dropping method such as a droplet sequencer, or by an automatic pipette, or an inkjet printing method, etc.
  • the microwell array chip used in the above sequencing method is a semiconductor sensor chip, comprising a semiconductor chip body having a plurality of micropores on the surface thereof and an oligonucleotide L1 immobilized on the inner wall of the microwell by a chemical modification method.
  • an S linker and an L linker are respectively inserted at both ends of the DNA sample, and the L linker is denatured and hybridized with the oligonucleotide L1 in the microwell to fix the DNA sample on the inner surface of the microwell; the microwell Immediately below, an ion-sensitive field effect sensor or a nanowire transistor sensor is disposed; after the amplification liquid is added into the micropore, the surface of the chip body is covered with a sealing plate or a flexible film and pressed.
  • the semiconductor chip can be directly amplified on a flatbed PCR machine, and the semiconductor chip can be amplified by a PCR adapter on a conventional PCR instrument, such as a 96-well plate, as shown in FIG.
  • the PCR adapter includes a base matched with the PCR heating nest, a sealing sheet for sealing, and an upper cover, and the base is provided with a chip holder for placing a semiconductor chip, the chip holder, the pressing piece, the base and The center of the upper cover is aligned; the upper cover is provided with a buckle.
  • one or more sealed PCR adapters are placed in the holes of a 96-well plate.
  • the base can be made of metal or a heat conductive material to conduct heat conduction to the semiconductor chip, or by sealing the sheet above the semiconductor chip. Heating and cooling are performed to achieve PCR amplification.
  • the pyrophosphate ppi ion produced by the sequencing reaction is two phosphate ions generated after hydrolysis.
  • the surface of the sensor may be coated with a focused phosphate ion or a phosphate ion.
  • the probe, or the surface of the sensor is coated with a film that selectively passes through a phosphate or phosphate ion, and then a voltage change is produced by the Nernstein phenomenon.
  • the oligonucleotide L1 is immobilized on the inner wall of the micropore of the semiconductor chip by a chemical modification method.
  • a semiconductor chip is placed in an alcohol solution containing an amino molecule for amination modification to form an amino structure on the inner surface of the micropore of the semiconductor chip; the amino molecule is APTES or AEAPTES; and the APTES is a dense method obtained by vapor deposition. Sexual monolayer.
  • the aminated semiconductor chip is reacted with a glutaraldehyde solution, that is, the amino group is polymerized with the aldehyde group of glutaraldehyde to modify the surface of the micropore of the semiconductor chip;
  • oligonucleotide L1 sequence is modified with an amino group as shown in SEQ ID NO: 1.
  • 5'-NH2-CCTCTCTATGGGCAGTCGGTGA SEQ ID NO: 1;

Abstract

本发明提供一种基于微孔阵列芯片的单分子文库PCR扩增的基因测序方法，包括：步骤一，将PCR扩增液滴加到具有微孔阵列的芯片上，使得芯片上的微孔均是独立的反应空间，有且仅有一个DNA样本片段被固定在微孔内；步骤二，将密封好的微孔芯片于PCR仪上进行PCR扩增；步骤三，扩增结束后，将微孔内的双链DNA变性为单链DNA；步骤四，采用退火的方法，将测序引物S2结合在微孔中的待测DNA样本上；步骤五，测序时，向微孔内按G、C、A、T的顺序加入脱氧核苷酸三磷酸dNTP，配对成功后，DNA样本自身电荷的增加、氢离子或焦磷酸PPI离子的释放引起微孔底部的传感器的信号响应，记录传感器的响应信号并对信号进行处理，得到被测DNA的基因序列信息；步骤六，重复步骤五。

Description

基于微孔阵列芯片的单分子文库PCR扩增的基因测序方法
优先权信息
本申请请求2018年02月22日向中国国家知识产权局提交的、专利申请号为201810153345.9的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及一种基于微孔阵列芯片的单分子文库PCR扩增的基因测序方法，属于基因测序技术领域。
背景技术
快速和准确地获取生物体的遗传信息对于生命科学研究一直具有十分重要的意义。对于每个生物体来说，基因组包含了整个生物体的遗传信息。测序技术能够真实地反映基因组DNA上的遗传信息，进而比较全面地揭示基因组的复杂性和多样性，因而在生命科学研究中扮演了十分重要的角色。
测序技术最早可以追溯到20世纪50年代，早在1954年就已经出现了关于早期测序技术的报导，即Whitfeld等用化学降解的方法测定多聚核糖核苷酸序列；1977年Sanger等发明的双脱氧核苷酸末端终止法(Sanger基因测序法)和Gilbert等发明的化学降解法，标志着第一代测序技术的诞生；此后在三十几年的发展中陆续产生了第二代测序技术，包括Roche公司的454技术、Illumina公司的Solexa技术和ABI公司的SOLiD技术；近几年，Helicos公司的单分子测序技术、Pacific Biosciences公司的单分子实时(Single Molecule Real Time,SMRT)测序技术和Oxford Nanopore Technologies公司正在研究的纳米孔单分子测序技术被认为是第三代测序技术；同时以Illumina为代表的第二代测序技术及美国Ion Torrent公司开发的半导体基因测序技术持续取得突破，发展出新一代基因测序技术(NGS)。通过大规模传感器阵列实现高通量并行测序，极大降低了基因测序价格及测序时间，并将准确度提高到99％以上，测序技术正向着高通量、低成本、高准确率的方向发展。
但是，目前的测序方法存在诸多问题。第一代测序技术成本高，据估算，用该方法完成人类基因组计划，需花30亿美元；数据分析量大；自动化程度不高或需手工操作；一些聚合酶链式反应(PCR)产物不能被分析而需制备单克隆；且该法速度慢，测序时间长，据估算，用该法完成人类基因组的测序，至少需用时3年。第二代测序相对而言，工作量仍然比较 大，费用仍然比较高，不适合用于序列已知的单基因的突变检测；关键在于读长比较短，时间还不够快，所需模板用量还比较多，故无法在单细胞、单分子水平进行检测。第三代测序技术适用于起始量少、需要高通量、自动化程度很高的全基因组测定，因此对于要求不高的单个基因位点的检测如单基因病等的基因诊断反而不适用，即性价比反而降低；此外，第三代测序技术仍需要致力于背景噪音的减少，提高准确率，及降低测序费用；另外，在DNA的固定方面如何保持DNA的延展性而不出现二聚体结构，也有待解决和完善的地方。
新一代测序技术，比如Ion Torrent公司的半导体测序技术，由于采用了半导体离子敏感场效应传感器，可以借助半导体工业的摩尔定律持续减小传感器尺寸，增加阵列规模，提高测序的通量，降低费用。但是Ion Torrent公司的半导体测序技术的操作流程太过繁琐，其中包括文库DNA通过onetouch2仪器进行油包水的微珠/单分子文库DNA扩增反应、ES仪器进行磁珠纯化和DNA变性、芯片离心机进行文库DNA上测序芯片等步骤，需要用到多种设备，并且操作复杂，实验时间长。
发明内容
本发明为了解决现有技术中存在的上述缺陷和不足，提供了一种基于微孔阵列芯片的单分子文库PCR扩增的基因测序方法，解决了现有技术中测序技术的操作流程太过繁琐、同时需要利用多种设备及配套试剂、且操作复杂、时间长成本高的技术难题。
为解决上述技术问题，本发明提供一种基于微孔阵列芯片的单分子文库PCR扩增的基因测序方法。根据本发明的实施例，所述方法包括以下步骤：
步骤一，将待测DNA样本溶液与含有DNA聚合酶、dNTPs和引物S1的PCR扩增液的组成混合溶液，滴加到具有微孔阵列的芯片上，使所述芯片上的每个微孔内均含有混合溶液；然后用密封压片覆盖于芯片表面并压紧密封，使得芯片上的微孔均是独立的反应空间，有且仅有一个DNA样本片段被固定在微孔内；
步骤二，将密封好的具有微孔阵列的芯片于PCR仪上进行PCR扩增；
步骤三，扩增结束后，去除芯片表面的密封压片，将微孔内的溶液清除，然后将微孔内的双链DNA变性为单链DNA，再加入清洗液对微孔进行清洗，再去掉微孔内的清洗液；
步骤四，向经步骤三处理的芯片的微孔内加入测序引物S2，然后采用退火的方法，将测序引物S2结合在微孔中的待测DNA样本上；清除微孔内溶液后加入测序酶溶液，进行孵育；
步骤五，测序时，向微孔内按G、C、A、T的顺序加入脱氧核苷酸三磷酸dNTP，若加入的dNTP与DNA模板的碱基配对成功，则会在测序酶的作用下，添加到测序引物的3 ’末端，同时释放出氢离子及焦磷酸PPi离子，DNA样本自身电荷的增加、氢离子或焦磷酸PPI离子的释放引起微孔底部的传感器的信号响应，记录传感器的响应信号并对信号进行处理，得到被测DNA的基因序列信息；
步骤六，重复步骤五，对微孔中的DNA样本进行连续测序。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，有且仅有一个DNA样本片段被固定在微孔内是通过如下方式进行的：所述微孔的内壁上修饰有寡聚核苷酸L1，在DNA样本的两端分别接入S接头和L接头，L接头变性后与微孔内的寡聚核苷酸L1杂交，从而将DNA样本固定在微孔的内表面。
根据本发明的实施例，所述具有微孔阵列的芯片为半导体传感器芯片，所述芯片包括：表面设有若干微孔的半导体芯片本体以及采用化学修饰的方法固定在微孔内壁上的寡聚核苷酸L1层，所述微孔的正下方设有离子敏感场效应传感器或者纳米线晶体管传感器。
根据本发明的实施例，在测序时，碱基延伸反应产生的本身电荷、氢离子或焦磷酸PPi离子触发微孔下方的传感器的电流或电压变化，从而将化学信号转换为电信号，再对电信号处理，以将电信号翻译为对应的基因序列信息。
根据本发明的实施例，所述半导体芯片还包括PCR适配器，所述PCR适配器包括与PCR加热巢或加热板相配的底座、对微孔起到密封作用的压片和一个上盖；所述底座上设有用于放置芯片的芯片座，所述芯片座、压片、底座和上盖的中心对齐；所述上盖上设有卡扣。
根据本发明的实施例，所述传感器的表面涂有一层对焦磷酸ppi离子或磷酸根离子具有选择性的探针或者对焦磷酸ppi离子或磷酸根离子具有选择性通过的薄膜。
根据本发明的实施例，步骤四中，退火的方法具体为：将芯片置于PCR仪上，于80-99度处理0.5～10分钟、然后15～55度处理0.5～10分钟，再将测序引物S2结合在微孔中的DNA样本上；然后清除微孔内溶液后，加入测序酶溶液，20～40度孵育0.5～30分钟后进行测序。
根据本发明的实施例，清除微孔内溶液的方法为采用真空泵吸、采用微流控的方式利用清洗液冲洗或者采用冲压气体吹。
根据本发明的实施例，所述芯片的制备过程如下：
1.1)将具有微孔阵列的芯片置于含有氨基分子的有机溶液中进行修饰，使得微孔内表 面形成氨基结构；
1.2)将氨基化的芯片与戊二醛溶液在氰基硼氢化钠催化的条件下进行反应，使得芯片微孔内表面的氨基与戊二醛的醛基发生聚合反应，以便使得半导体芯片微孔内表面发生醛基化修饰；
1.3)将寡聚核苷酸L1序列的5’端进行氨基修饰；
1.4)在氰基硼氢化钠催化条件下，将5’端具有氨基修饰的寡聚核苷酸L1与醛基化的芯片进行聚合反应，以便使寡聚核苷酸L1偶联到芯片的微孔上。
根据本发明的实施例，所述氨基分子是以APTES或AEAPTES提供的；所述氨基化修饰是通过采用气相沉积法进行的，以便在所述微孔的内表面得到的致密性单分子层。
根据本发明的实施例，步骤五中，传感器上产生的电信号经过转换得到基因序列信息，具体过程为：
5.1)对每个A、T、G、C碱基进行测序后，通过数据采集，逐一产生原始的电信号矩阵，每个测序周期均产生四个原始的电信号矩阵；
5.2)对每个电信号矩阵进行分型，产生四个分型数据矩阵，并删除原始的电信号矩阵；
5.3)依据四个分型数据矩阵判断出测序周期中每个微孔内待测DNA样本的基因类型，并产生一个DNA碱基信息矩阵；
5.4)将所有的碱基矩阵首尾相接，形成DNA矩阵序列。
本发明所达到的有益技术效果：
1)本发明实现了在半导体芯片微阵列上进行单分子文库DNA扩增反应，之后直接进行测序，去除了onetouch2仪器进行油包水的微珠单分子文库DNA扩增反应、ES仪器的磁珠纯化和DNA变性、芯片离心机的文库DNA上测序芯片等复杂的步骤，大幅简化了操作流程，缩短了实验操作时间，降低了试验成本。
2)本发明简化了实验步骤，同时也省去了相应试剂的使用，实验成本降低。
3)由于使用了独立的微孔反应体系，本发明的测序试剂用量较现有技术的测序试剂用量少，也节约了PCR扩增和测序步骤中的试剂成本。
附图说明
图1本发明实施例的半导体芯片微孔阵列结构示意图，其中D为微孔放大的结构示意图；
图2本发明实施例的PCR适配器结构示意图，其中，a为PCR配合96孔板使用时结 构示意图，b为a中沿A-A线剖视示意图，c为b中放大部分C的结构示意图，d为PCR适配器俯视示意图；
图3本发明实施例的半导体芯片微孔阵列中待测DNA的PCR扩增示意图；
图4本发明实施例的半导体微孔阵列中待测DNA的测序示意图；
图5本发明实施例的基因测序方法的流程图；
图6本发明实施例的数据转换流程示意图；
图7本发明实施例的微孔中的基因翻译的模拟信号转换成电信号的示意图；
图8本发明实施例的电信号经处理后转换为基因序列信息的具体过程步骤2)-3)的示意图。
其中：1半导体芯片本体；11微孔；12寡聚核苷酸L1层；13基座；14传感器；15金属层；2引物S1；3测定DNA；4密封板；5引物S2；6 96孔板；7PCR适配器；71底座；72芯片座；73上盖；74压片；75卡扣。
发明详细描述
下面结合具体实施例对本发明作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
下面结合附图和实施例对本发明专利进一步说明。
如图5所示，本发明还提供一种基于微孔阵列芯片的单分子文库PCR扩增的基因测序方法，包括以下步骤：
步骤一，将浓度为1-100pmol/L的待测DNA样本溶液与含有DNA聚合酶、dNTPs和引物S1的PCR扩增液混合均匀，引物S1的序列可以如SEQ ID NO:2所示。5’-CCATCTCATCCCTGCGTGTCTC(SEQ ID NO:2)，并将混合后的溶液滴加到微孔阵列的芯片上，使每个微孔内均含有混合溶液，上样过程中，让待测DNA溶液中的DNA的片段数量小于或等于微孔的数量，使得微孔内有且仅有一个DNA片段；然后用密封压片或柔性膜覆盖于半导体芯片表面并压紧密封，使得半导体芯片上的每个微孔均是独立的反应空间；且争取尽量多的微孔中有且仅有一个DNA样本片段被固定在微孔表面。作为本发明的一个具体实施例，待测DNA的基因序列如SEQ ID NO:4所示。5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG(NNNNNNNNNN) (10-40)TCACCGACTGCCCATAGAGAGG(SEQ ID NO:4)。
步骤二，将密封好的半导体芯片于PCR仪上进行PCR扩增，如图3所示；在进行扩增 过程中，可以将半导体芯片置于平板PCR仪上直接进行，还可以将半导体芯片通过PCR适配器在传统的PCR仪上进行扩增，比如96孔板，扩增时，将一个多个密封的PCR适配器置入96孔板的孔洞内，底座可以为金属或导热材料，对半导体芯片进行导热，也可以通过对半导体芯片上的密封板进行加热和降温来实现PCR扩增，扩增条件如表1所示。
表1：PCR扩增条件
Figure PCTCN2019082561-appb-000001
步骤三，扩增结束后，去除芯片表面的密封压片，将微孔内的溶液清除，然后将双链DNA变性为单链DNA，变性方法可以采用NaOH或KOH等碱性溶液处理或者其他将双链DNA变性为单链DNA的方法，再将微孔内的溶液清除后加入清洗液对微孔进行清洗，再清除微孔内的清洗液。
步骤四，向经步骤三处理的芯片的微孔内加入测序引物S2，测序引物S2的基因序列如SEQ ID NO:3所示。
5’-CCATCTCATCCCTGCGTGTCTCCGAC(SEQ ID NO:3)。
如图4所示，采用退火的方法将测序引物S2结合在微孔中的待测DNA样本上，用压片对芯片进行密封处理，然后将密封后的芯片置于平板PCR仪上于80-99度处理0.5～10分钟、15～55度处理0.5～10分钟，或者通过PCR适配器于96孔板内作相同的处理，然后清除微孔内溶液后加入测序酶溶液，20～40度孵育0.5～30分钟，进行测序。
步骤五，测序时，向微孔内按G、C、A、T的顺序加入脱氧核苷酸三磷酸dNTP，若加入的dNTP与DNA模板的碱基配对成功，则会在测序酶的作用下，添加到测序引物的3 ’末端，同时释放出一份氢离子或一份焦磷酸PPi离子；如果加入的dNTP刚好能与微孔内 DNA模板的碱基配对，则会在测序酶的作用下，添加到测序引物的3 ’末端，同时释放出一份氢离子和焦磷酸PPi离子；如果加入的dNTP不能和微孔内的DNA模板的碱基配对，则上述反应不会发生；重复上述步骤直至找到可以与当前DNA模板的碱基成功配对的dNTP，并释放出氢离子和焦磷酸PPi离子；DNA样本自身电荷的增加、氢离子或焦磷酸PPi离子的释放引起微孔底部的传感器的信号响应，处理装置记录传感器的响应信号并对信号进行处理，得到被测DNA的基因序列信息，如图6所示，传感器信号响应表现为电压或电流的变化，电信号经处理后转换为基因序列信息的具体过程为：
1)对每个A、T、G、C碱基进行测序后，通过数据采集，逐一产生原始的电信号矩阵，即每个测序周期均产生四个原始的电信号矩阵；如图7所示的2×2四个传感器为例，原始数据是每秒采样25次到60次的数据点，将微孔中的基因翻译的模拟信号进行采样并转换成数字信号，即电信号；
2)对每个电信号矩阵进行分型，分别产生四个分型数据矩阵，并删除原始的电信号矩阵以节省存储空间；如图7所示，以pH的变化检测基因扩增的原理为例，原始数据可以分为如下几种类型：1.第一种类型是0，即没有脉冲信号，没有发送碱基扩增的反应，即当前的碱基和该传感器上的DNA样本不匹配。2.第二种类型是1，即有一份氢离子脉冲，即当前的碱基和该传感器上的DNA样本的一份碱基匹配。3.第三种类型是2到5，即产生了2份到多份氢离子脉冲，即当前的碱基和该传感器上的DNA样本的2份或多份碱基匹配，即该样本有多重碱基的出现。如图8所示，按这个方法，依次分别产生A、T、G、C四种碱基对应的四个分型数据矩阵；完成后删除原来的原始数据以节省存储空间。这个步骤不仅完成了数据的初级分析，还大量压缩了数据规模。
3)依据四个分型数据矩阵判断出测序周期中每个微孔内待测DNA样本的基因类型，并产生一个DNA碱基信息矩阵；如图8所示，4个矩阵的(1，1)位置，矩阵A、T、G都是0，矩阵C是1，即当前样本(1，1)位置的碱基只能是G，才能和C配对。同理，4个矩阵的(2，4)位置，只有矩阵G是非零值3，意味着当前样本(2，4)位置的碱基只能是3重碱基C，即CCC。这一操作，即产生了基因信号矩阵，作为输出。最后，这个碱基矩阵再接在上一周期的对应碱基矩阵的后面，形成DNA矩阵序列。
4)将所有的碱基矩阵首尾相接，即形成DNA矩阵序列。
其中，传感器产生电流或电压信号是通过以下方式实现的：1.由测序反应中产生的焦磷酸ppi离子经水解后产生的2个磷酸根离子引起的，为了实现这个功能，在传感器表面涂有对焦磷酸离子或磷酸根离子有选择性的探针，或者传感器表面涂有对焦磷酸离子或磷酸根 离子有选择性通过的薄膜，然后通过能斯特Nernstein现象产生电压变化。由于焦磷酸的电荷数量是氢离子的四倍且扩散速度更慢，因而信号更大，信号更稳定；2.测序反应中产生的新的碱基对的本身电荷将永久性的固化在文本上，产生的信号将是直流信号，连续测序的信号将是阶梯状的电信号；3.测序反应中碱基对的本身电荷、释放出来的氢离子或磷酸根离子中的两种引起双信号：比如DNA增加的本身电荷产生的台阶式直流信号和氢离子或磷酸根离子产生的脉冲信号(两种离子都会随时间扩散出反应微孔)。实际测序时，通过检测两种信号都产生才判断为成功的碱基配对，因而可降低测序过程中干扰物产生的错误率，提高数据质量和测序准确率。
步骤六，重复步骤五，进行下一种脱氧核苷酸三磷酸dNTP，对微孔中DNA样本进行测序。
上述过程中，对微孔内溶液进行清除的处理方法为：
1.采用真空泵吸走反应试剂达到清洗反应微孔的目的；2.采用微流控的方式利用清洗液冲洗微孔；3.采用冲压气体吹走反应溶液。
上述传感器为离子敏感场效应晶体管或者半导体纳米线晶体管。
上述步骤溶液加入是采用液滴滴加法，比如液滴测序仪，或采用自动移液器，或喷墨打印方法等液滴控制法来完成，降低液体使用量。
如图1所示，上述测序方法中所用微孔阵列芯片为半导体传感器芯片，包括表面设有若干微孔的半导体芯片本体以及采用化学修饰的方法固定在微孔内壁上的寡聚核苷酸L1层，在DNA样本的两端分别接入S接头和L接头，L接头变性后与微孔内的寡聚核苷酸L1杂交,从而将DNA样本固定在微孔的内表面；所述微孔的正下方设有离子敏感场效应传感器或者纳米线晶体管传感器；在所述微孔内加入扩增液后，使用密封板或柔性膜覆盖于芯片本体表面并压紧。
在进行扩增过程中，可以将半导体芯片置于平板PCR仪上直接进行扩增，还可以将半导体芯片通过PCR适配器在传统的PCR仪上进行扩增，比如96孔板，如图2所示，所述PCR适配器包括与PCR加热巢相配的底座、起到密封作用的压片和一个上盖，所述底座上设有用于放置半导体芯片的芯片座，所述芯片座、压片、底座和上盖的中心对齐；所述上盖上设有卡扣。扩增时，将一个或多个密封的PCR适配器置入96孔板的孔洞内，底座可以为金属或导热材料做成，对半导体芯片进行导热，也可以通过对半导体芯片上方的密封压片进行加热和降温来实现PCR扩增。
由测序反应中产生的焦磷酸ppi离子是水解后产生的2个磷酸根离子，为了实现将焦磷 酸ppi离子信号转变为电信号，可在传感器表面涂有对焦磷酸离子或磷酸根离子有选择性的探针，或者传感器表面涂有对焦磷酸离子或磷酸根离子有选择性通过的薄膜，然后通过能斯特Nernstein现象产生电压变化。
半导体传感器芯片的制备：采用化学修饰的方法将寡聚核苷酸L1固定在半导体芯片微孔的内壁上。
具体过程如下：
1.1)将半导体芯片置于含有氨基分子的酒精溶液中进行氨基化修饰，使得半导体芯片微孔内表面形成氨基结构；所述氨基分子为APTES或AEAPTES；所述APTES为采用气相沉积法得到的致密性单分子层。
1.2)在氰基硼氢化钠催化作用下，将氨基化的半导体芯片与戊二醛溶液反应，即氨基与戊二醛的醛基发生聚合反应，使得半导体芯片微孔表面内醛基化修饰；
1.3)将寡聚核苷酸L1序列的5’端修饰上氨基，序列如SEQ ID NO:1所示。5’-NH2-CCTCTCTATGGGCAGTCGGTGA(SEQ ID NO:1)；
1.4)在氰基硼氢化钠NaBH3CN催化条件下，将5’端修饰有氨基的寡聚核苷酸L1与醛基化的半导体芯片进行聚合反应，以便使寡聚核苷酸L1偶联到半导体芯片的微孔上。
以上以较佳实施例公布了本发明，然其并非用以限制本发明，凡采取等同替换或等效变换的方案所获得的技术方案，均落在本发明的保护范围内。

Claims (11)

  1. 一种基于微孔阵列芯片的单分子文库PCR扩增的基因测序方法，其特征在于，包括以下步骤：
    步骤一，将待测DNA样本溶液与含有DNA聚合酶、dNTPs和引物S1的PCR扩增液组成的混合溶液，滴加到具有微孔阵列的芯片上，使所述芯片上的每个微孔内均含有混合溶液；然后用密封压片覆盖于芯片表面并压紧密封，使得芯片上的微孔均是独立的反应空间，有且仅有一个DNA样本片段被固定在微孔内；
    步骤二，将密封好的具有微孔阵列的芯片于PCR仪上进行PCR扩增；
    步骤三，扩增结束后，去除芯片表面的密封压片，将微孔内的溶液清除，然后将微孔内的双链DNA变性为单链DNA，再加入清洗液对微孔进行清洗，再去掉微孔内的清洗液；
    步骤四，向经步骤三处理的芯片的微孔内加入测序引物S2，然后采用退火的方法，将测序引物S2结合在微孔中的待测DNA样本上；清除微孔内溶液后加入测序酶溶液，进行孵育；
    步骤五，测序时，向微孔内按G、C、A、T的顺序加入脱氧核苷酸三磷酸dNTP，若加入的dNTP与DNA模板的碱基配对成功，则会在测序酶的作用下，添加到测序引物的3 ，末端，同时释放出氢离子及焦磷酸PPi离子，DNA样本自身电荷的增加、氢离子或焦磷酸PPI离子的释放引起微孔底部的传感器的信号响应，记录传感器的响应信号并对信号进行处理，得到被测DNA的基因序列信息；
    步骤六，重复步骤五，对微孔中的DNA样本进行连续测序。
  2. 根据权利要求1所述的基因测序方法，其特征在于，有且仅有一个DNA样本片段被固定在微孔内是通过如下方式进行的：所述微孔的内壁上修饰有寡聚核苷酸L1，在DNA样本的两端分别接入S接头和L接头，L接头变性后与微孔内的寡聚核苷酸L1杂交，从而将DNA样本固定在微孔的内表面。
  3. 根据权利要求1所述的基因测序方法，其特征在于，所述具有微孔阵列的芯片为半导体传感器芯片，所述芯片包括：表面设有若干微孔的半导体芯片本体以及采用化学修饰的方法固定在微孔内壁上的寡聚核苷酸L1层，所述微孔的正下方设有离子敏感场效应传感器或者纳米线晶体管传感器。
  4. 根据权利要求3所述的基因测序方法，其特征在于，在测序时，碱基延伸反应产生的本身电荷、氢离子或焦磷酸PPi离子触发微孔下方的传感器的电流或电压变化，从而将化学 信号转换为电信号，再对电信号处理，以将电信号翻译为对应的基因序列信息。
  5. 根据权利要求3所述的基因测序方法，其特征在于，所述半导体芯片还包括PCR适配器，所述PCR适配器包括与PCR加热巢或加热板相配的底座、对微孔起到密封作用的压片和一个上盖；所述底座上设有用于放置芯片的芯片座，所述芯片座、压片、底座和上盖的中心对齐；所述上盖上设有卡扣。
  6. 根据权利要求3所述的基因测序方法，其特征在于：所述传感器的表面涂有一层对焦磷酸ppi离子或磷酸根离子具有选择性的探针或者对焦磷酸ppi离子或磷酸根离子具有选择性通过的薄膜。
  7. 根据权利要求1所述的基因测序方法，其特征在于：步骤四中，退火的方法具体为：将芯片置于PCR仪上，于80-99度处理0.5～10分钟、然后15～55度处理0.5～10分钟，再将测序引物S2结合在微孔中的DNA样本上；然后清除微孔内溶液后，加入测序酶溶液，20～40度孵育0.5～30分钟后进行测序。
  8. 根据权利要求1所述的基因测序的方法，其特征在于，清除微孔内溶液的方法为采用真空泵吸、采用微流控的方式利用清洗液冲洗或者采用冲压气体吹。
  9. 根据权利要求2所述的基因测序方法，其特征在于，所述芯片的制备过程如下：
    1.1)将具有微孔阵列的芯片置于含有氨基分子的有机溶液中进行氨基化修饰，使得微孔内表面形成氨基结构；
    1.2)将氨基化的芯片与戊二醛溶液在氰基硼氢化钠催化的条件下进行反应，使得芯片微孔内表面的氨基与戊二醛的醛基发生聚合反应，以便使得半导体芯片微孔内表面发生醛基化修饰；
    1.3)将寡聚核苷酸L1序列的5’端进行氨基修饰；
    1.4)在氰基硼氢化钠催化条件下，将5’端具有氨基修饰的寡聚核苷酸L1与醛基化的芯片进行聚合反应，以便使寡聚核苷酸L1偶联到芯片的微孔上。
  10. 根据权利要求9所述的基因测序的方法，其特征在于，所述氨基分子是以APTES或AEAPTES提供的；所述氨基化修饰是通过气相沉积法进行的，以便在所述微孔的内表面得到的致密性单分子层。
  11. 根据权利要求4所述的基因测序的方法，其特征在于，步骤五中，传感器上产生的电信号经过转换得到基因序列信息，具体过程为：
    5.1)对每个A、T、G、C碱基进行测序后，通过数据采集，逐一产生原始的电信号矩阵，每个测序周期均产生四个原始的电信号矩阵；
    5.2)对每个电信号矩阵进行分型，产生四个分型数据矩阵，并删除原始的电信号矩阵；
    5.3)依据四个分型数据矩阵判断出测序周期中每个微孔内待测DNA样本的基因类型，并产生一个DNA碱基信息矩阵；
    5.4)将所有的碱基矩阵首尾相接，形成DNA矩阵序列。
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