WO2009140802A1 - 检测探针、通用寡核苷酸芯片及核酸检测方法及其用途 - Google Patents
检测探针、通用寡核苷酸芯片及核酸检测方法及其用途 Download PDFInfo
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- WO2009140802A1 WO2009140802A1 PCT/CN2008/001695 CN2008001695W WO2009140802A1 WO 2009140802 A1 WO2009140802 A1 WO 2009140802A1 CN 2008001695 W CN2008001695 W CN 2008001695W WO 2009140802 A1 WO2009140802 A1 WO 2009140802A1
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
Definitions
- the invention relates to a detection probe, a universal oligonucleotide chip and a nucleic acid detection method, in particular to a nucleic acid detection method for nucleic acid detection and sequence analysis, and a detection probe and a universal oligonucleotide used thereby chip.
- Gene mutation refers to the change of base pair composition or arrangement order of genomic DNA molecules, including base substitution and deletion or insertion of small fragments, which is one of the important causes of genotype diseases.
- Polymorphism refers to DNA sequence changes that have accumulated in the population during evolution. Gene mutations and polymorphism analysis play a very important role in biomedical research, especially in the diagnosis and pathological study of genotype diseases. With the development of the overall sequencing of the human genome, the task of exploring gene mutations and polymorphisms has become very urgent.
- PCR-RFLP polymerase chain restriction length polymorphism analysis
- STR short tandem repeat length polymorphism
- Methods for detecting unknown mutations include single strand conformation polymorphism (SSCP), heteroduplex analysis (HPA), MALDI-TOF mass spectrometry (matrix) Assisted laser desorpti onion Ization time 1 of flight mass spectrometry), denaturing gradient gel electrophoresis (DGGE), enzymatic cleavage mismatch (EMC), dideoxy fingerprinting (ddF), and DNA sequencing, etc. Many principles and applications have been reported.
- SSCP single strand conformation polymorphism
- HPA heteroduplex analysis
- MALDI-TOF mass spectrometry matrix
- DGGE denaturing gradient gel electrophoresis
- EMC enzymatic cleavage mismatch
- ddF dideoxy fingerprinting
- DNA sequencing is the most fundamental method for detecting mutations. Although it can accurately determine the location and nature of mutations, the sequencing technology based on gel electrophoresis is time-consuming, and the cost of sequencing of automatic sequencers is high, which limits its actuality. Application in .
- Bio-microarray (or biochip) technology is a fast, high-throughput detection tool that has emerged in recent years, using microarray technology to align thousands of biological components (cells, proteins, DNA, etc.) On the phase substrate, the detected component in the biological sample reacts with a specific substance on the substrate, and then a corresponding signal (such as fluorescence) is introduced to achieve the purpose of analyzing the biological sample.
- Bio-microarray technology enables some traditional biological analysis methods to be performed as quickly as possible in as small a space as possible.
- biochip technology is rapidly developing, including chip, protein chip, tissue chip, cell chip, chip lab and other chip technologies have been used by researchers for large-scale mutation detection and polymorphism screening.
- SNP flux such as Beckman Analysis System (SNPStream assay (Orchid Cellmark/Beckman) Coulter)), Illumina's GoldenGate genotyping assay (Illumina), GeneChip Human Mapping assays (Affymetrix) , Infinity genotyping assay (Illumina), Targeted Genotyping System for drug metabolizing enzymes and transports (DMETs) Analysis (Affymetrix)) and other mutations (such as SNPs) detection platform, and related gene mutation detection (such as SNPs detection) in related patent technologies such as US 5427930, US 6479242, W09300447, US 5871921, US 6858412, US 5866337, etc. ) and nucleic acid sequencing analysis are also introduced.
- the main object of the present invention is to overcome the defects of the existing gene mutation detection and nucleic acid sequencing analysis methods, and to provide a new detection probe, a universal oligonucleotide chip and a nucleic acid detection method, and the technical problem to be solved is It is very suitable for practical use because it can achieve low cost, high specificity and high sensitivity.
- a detection probe according to the present invention characterized in that it is a detection probe for each site to be tested For P1, P2, PI and P2 contain sequences HI and H2 complementary to the sequences flanking the base site to be tested, respectively, and each contains a related universal oligonucleotide sequence H3 and H4, and at P1 or P2 also contains at least a specific Tag sequence, and the detection probes are connected to P1 and P2 as one probe by the following method: The detection probe is annealed to the test nucleic acid sample by ⁇ and P2, and passed through HI.
- H2 is complementary to the sequence on both sides of the base site to be tested, and a gap corresponding to the base at the base site to be detected is formed between the probes P1 and P2, and the annealed reaction system is divided into A, Four reaction systems of T, G, and C, adding dATR to the A system, adding dTTR to the T system, adding dGTR to the G system, adding dCTP to the C system, and a base complementary to the base at the base site to be tested.
- the gap is filled by the action of DNA polymerase and ligase, and the detection probes are connected to P1 and P2 as one probe.
- the object of the present invention and solving the technical problems thereof can be further achieved by the following technical measures.
- the aforementioned detection probe wherein the detection probe pair further comprises another probe P2 ', and the probe contains a sequence HI or H2 complementary to the sequence on the side of the base site to be tested, a related universal Oligonucleotide sequence H3 or H4, a specific Tag sequence and a sequence H5 complementary to the inserted sequence of the insert site of the sample to be tested.
- the aforementioned detection probe further comprises at least a biotin-labeled molecule on the probe P1 or P2, and the biotin-labeled molecule further comprises a connecting arm between the nucleotide sequence of the probe.
- the aforementioned detecting probe wherein the connecting arm is a carbon connecting arm, or a connecting arm of a non-carbon type.
- the aforementioned detection probe, wherein the non-carbon type linking arms are polyethylene glycol, P olyA, polyT.
- a universal oligonucleotide chip according to the present invention comprising four identical regions divided into A, T, G, and C, each of which corresponds to a respective point on a corresponding detection probe The same sequence of oligonucleotide sequences as the Tag sequence.
- the aforementioned universal oligonucleotide chip can be applied to the field of resequencing analysis of a target nucleic acid sequence, detection and analysis of mutation sites and insertion/deletion sites in known nucleic acid sequences, and typing detection of pathogenic microorganisms.
- a nucleic acid detection method based on a universal oligonucleotide microarray comprises the following steps: 1 preparation of a nucleic acid sample to be tested; 2 preparation of a detection probe, wherein the probe is P1 and P2 are composed of detection probe pairs for each site to be tested, and P1 and P2 respectively contain sequences HI and H2 complementary to sequences on both sides of the base site to be tested, and each contains a correlation-specific oligo Nucleotide sequences H3 and H4, and at least one specific Tag sequence on P1 or P2; 3 annealing and hybridizing P1 and P2 to the nucleic acid sample, forming a base to be detected between the probes The corresponding base of the point is notched; 4 The annealing reaction system is divided into four reaction systems: G, T, and C.
- DATP is added to the A system
- dTTP is added to the T system
- dGTP is added to the G system.
- adding dCTP to the C system under the action of DNA polymerase and ligase, the base complementary to the base at the base site to be tested fills the gap, and the detection probe is connected to P1 and P2.
- each chip is divided into four hybrid regions of A, T, G, C, each bit corresponding to each region Pointing on the same oligonucleotide sequence as the corresponding Tag sequence on the corresponding detection probe P1 or P2; 8 using the four hybrid regions A, T, G, C of the above oligonucleotide chip for respectively
- the probes in the four reaction systems after amplification were hybridized; 9 pairs of chips were analyzed for results.
- the preparation step of the detection probe further includes another probe P2 ', which contains a sequence HI or H2 complementary to the sequence on the side of the base site to be tested, A related universal oligonucleotide sequence H3 or H4, a specific Tag sequence and a sequence H5 complementary to the inserted sequence of the insert site of the sample to be tested.
- nucleic acid detecting method described above, wherein the nucleic acid sample to be tested is an animal or plant chromosomal DNA, a target nucleic acid PCR amplification product, mitochondrial DNA, cDNA, bacteria or viral DNA or RNA.
- the nucleic acid detecting method wherein the end of the detecting probe P1 or P2 that does not undergo a ligation reaction further contains at least a biotin-labeled molecule, and the biotin-labeled molecule and the nucleotide sequence of the probe There is also a connecting arm between the columns to facilitate subsequent probe purification and amplification.
- the connecting arm is a carbon linking arm, polyethylene glycol, polyA or polyT.
- nucleic acid detecting method described above, wherein the purifying step of the probe is carried out by using a streptavidin-coated carrier medium.
- the carrier medium is magnetic microparticles or polystyrene microspheres.
- the aforementioned nucleic acid detecting method wherein the amplification step of the probe is symmetric or asymmetric PCR amplification using a universal primer.
- the aforementioned nucleic acid detection method if conventional PCR amplification is carried out using a universal primer, contains a molecular marker at at least 5' of the universal primer. If a universal primer is used for asymmetric PCR amplification, at least one of the universal primers is a restriction primer and the other is a non-limiting primer, and at least the molecular marker is included at the 5' end of the non-limiting primer.
- nucleic acid detecting method described above, wherein the molecular marker is a non-isotopic label or a radioisotope label.
- the non-isotopic label is a fluorescent substance label such as a fluorescein molecule (cy3, cy5, FITC), a metal label such as Hg, a hapten label such as digoxin, and an enzyme such as horseradish peroxide. Enzyme (HRP), galactosidase, alkaline phosphatase, and the like.
- the aforementioned nucleic acid detecting method wherein the radioisotope label is 32 P or 35 S.
- the step of performing detection and analysis on the chip is to perform monochrome fluorescent scanning on the chip and determine the detection result according to the occurrence of the fluorescent signal on the corresponding position of the four partitions on the chip, or perform the detection on the chip.
- the method of enzyme color development determines the detection result.
- the aforementioned nucleic acid detection method can be applied to the field of resequencing analysis of a target nucleic acid sequence, detection and analysis of a mutation site and an insertion/deletion site in a known nucleic acid sequence, and typing detection of pathogenic microorganisms.
- the present invention has significant advantages and advantageous effects over the prior art.
- the nucleic acid detection method based on the universal oligonucleotide microarray proposed by the invention uses the detection probe to pair P1 and P2 to the nucleic acid sequence A certain base site in the column is detected, and the sequences of the 5' end and the 3' end of the two probes are complementary to the nucleic acid sequences on the left and right sides of the base site to be tested, respectively, when the probe and the template are annealed and hybridized.
- the present invention is based on the general-purpose chip technology, and the chip is divided into four regions, and the tag sequences at the specific sites in each region on the chip are the same.
- the amplification system in the four reaction tubes is independently hybridized with the four regions on the chip, and finally the chip is scanned by a monochrome fluorescent scanner, and the fluorescence is corresponding to the corresponding sites in the four regions on the chip.
- the presence or absence of the signal for the analysis of the test results is very simple. Simultaneous selection of universal Tag sequences on microarrays can rule out the selection and optimization of conditions for sequence-specific probes and microarray hybridization.
- a novel detection probe, a universal oligonucleotide chip and a nucleic acid detection method of the invention have the following advantages and beneficial effects:
- the cost of nucleic acid detection is reduced from the following aspects: First, a pair of universal primers can be used to amplify all the detection probes, thereby saving the multiplex PCR amplification according to Target sequence design and optimization of the cost of a large number of amplification primers; Secondly, the method is based on the general oligonucleotide chip technology, using Tag sequences present on the detection probe and point-specific sites on the chip for testing Positioning analysis of the locus avoids the selection and optimization of the conditions for sequence-specific probe and microarray hybridization; again, the method performs fluorescein cy3, cy5 or FITC labeling at the 5' end of the universal primer P3 or P4. The detection of the base site to be detected by the introduction of a single-color fluorescent label during probe amplification further reduces the cost of the scanning and result analysis equipment required for detection.
- the detection probe specifically recognizes the sample of the nucleic acid sample to be tested for P1 and P2, and fills and connects the gap formed by the P1 and P2 probes in the separated four reaction systems, Magnetic particle purification of the attached probe, these steps make the method Has a high specificity.
- a related sequence of a pair of universal primers P3 and P4 is introduced into all detection probes during probe design.
- P3 and P4 are universal primers for asymmetric PCR amplification, which not only forms a single strand that facilitates hybridization with the chip, but also overcomes the multiplex PCR-based method.
- the uncontrolled cross-reactivity caused by the increase in the target sequence further enhances the sensitivity and specificity of the assay.
- the design method, amplification system and chip design method of the detection probe unique to the nucleic acid detection method of the present invention have high-throughput detection capability, and the new chip design system is advantageous for the new detection site to be detected. It can also be easily added to the chip array.
- the present invention achieves high throughput detection of bases or mutations to be tested by designing linear detection probes for P1 and P2 pairs. After the probe and the template are annealed and hybridized, they are divided into four reaction systems of A, G, T and C, respectively, and a corresponding deoxyribonucleotide-dATP, dTTP, dGTP, dCTP is added, followed by DNA polymerase. Under the action of ligase, the corresponding detection probes are connected to P1 and P2 into a probe in four reaction systems, and the linked probes are purified by streptavidin magnetic particles, and then monochromatic.
- Asymmetric PCR amplification of a fluorescently labeled pair of universal nucleic acid sequences allows amplification of the signal for hybridization to the chip.
- the chip is designed to independently correspond to the four reaction systems A, G, T, and C, and the four partitions A, G, T, and C are independently hybridized with the final amplification products in the four reaction systems. Washing, scanning with a monochrome fluorescent scanner and performing signal analysis based on the presence or absence of signals on the corresponding sites on the four regions of the chip to determine the base type or mutation site genotype of the site to be tested.
- the invention has the above-mentioned many advantages and practical values, and has great improvement in methods and functions, has significant advances in technology, and has produced useful and practical effects, and is more sensitive than the existing gene mutation detection.
- the nucleic acid sequencing analysis method has an enhanced and outstanding multiple functions, which is more suitable for practical use, and is a novel, progressive and practical new design.
- FIGS. 1A-1B are schematic views of a detection probe of an embodiment of a nucleic acid detection method for a general oligonucleotide microarray of the present invention.
- 2A-2F are schematic flow diagrams showing a method for detecting a nucleic acid of a universal oligonucleotide microarray of the present invention.
- 3 is a schematic diagram of a chip of a nucleic acid detection method of a universal oligonucleotide microarray of the present invention.
- Fig. 4 is a schematic diagram showing the analysis of the results of the nucleic acid detection method for the nucleic acid sequence re-sequencing of the universal oligonucleotide microarray of the present invention.
- Fig. 5 is a schematic diagram showing the analysis of the results of the nucleic acid detection method of the universal oligonucleotide microarray of the present invention for SNP site detection.
- 6A-6C are schematic views of a detection probe of another embodiment of the nucleic acid detection method of the universal oligonucleotide microarray of the present invention.
- FIGS. 7A-7D are schematic flow diagrams showing a nucleic acid detecting method of another embodiment of the nucleic acid detecting method of the universal oligonucleotide microarray of the present invention.
- Fig. 8 is a schematic diagram showing the analysis of the results of the chip for the detection of insertion/deletion sites by the nucleic acid detection method of the universal oligonucleotide microarray of the present invention.
- Fig. 9A - Fig. 9C are verification electrophoresis patterns of the detection results of the nucleic acid detection method of the universal oligonucleotide microarray of the present invention for the mutation site present on the cloned human genome P450 drug metabolizing enzyme gene fragment.
- Fig. 10 is a scanning result of the method of nucleic acid detection of the universal oligonucleotide microarray of the present invention for chip hybridization of a mutation site present on a cloned human genome P450 drug metabolizing enzyme gene fragment.
- the detection probe, the universal oligonucleotide chip and the nucleic acid detection method according to the present invention are combined with the accompanying drawings and preferred embodiments. Its specific implementation, structure, characteristics and efficacy are described in detail below.
- the principle of the present invention is to design a linear test based on a universal oligonucleotide gene chip.
- the probe pairs the PI and P2 pairs to achieve high throughput detection of the base or mutation site to be tested.
- the reaction is divided into four systems: A, G, T, and C.
- the connection between probes P1 and P2 is completed by DNA polymerase and ligase, and purified and amplified and fluorescent signals are obtained.
- it hybridizes with the corresponding four hybrid regions of A, G, T, and C on the chip.
- the corresponding corresponding sites in the four regions on the chip are made with the corresponding Tag sequences on the probe.
- the chip is cleaned, scanned with a monochrome fluorescent scanner, and signal analysis is performed according to the presence or absence of signals on the corresponding sites on the four regions of the chip to determine the base type or mutation site genotype of the site to be tested.
- FIGS. 1A-1B are schematic diagrams showing a detection probe of an embodiment of a nucleic acid detection method for a general oligonucleotide microarray of the present invention.
- a design method for detecting a probe is : It was detected by two linear probes P1 and P2, and the designed oligonucleotide sequence is described in detail below.
- the oligonucleotide sequence of the probe P1 designed by the present invention is composed of three parts: ⁇ 1, H3 and a spacer, wherein the H3 at the 5' end of the probe P1 is A 20 nt universal oligonucleotide sequence for symmetrical or asymmetric PCR amplification of the probe; HI at the 3' end of probe P1 is about 20 nt and the base site to be detected on the template a sequence complementary to the nucleic acid sequence of one side; in addition, in order to purify the probe for subsequent operation, there is a biotin-labeled molecule at the 5' end of the probe P1; in order to facilitate purification and PCR amplification of the probe, The biotin-labeled molecule and the 5'-end nucleotide of the probe P1 are designed with a connecting arm, which may be a carbon connecting arm or other types of connecting arms.
- a connecting arm which may be a carbon connecting arm or other types of connecting arms.
- the oligonucleotide sequence of the probe P2 designed in the present invention is composed of three parts: H2, H4 and tag, wherein the sequence of about 20 nt at the 5' end of the probe P2 is to be tested. a sequence complementary to the nucleic acid sequence on the other side of the base site; a sequence of about 20 nt at the 3' end of the probe P2 is a sequence H4 complementary to another universal primer P4, and 5 of the universal primer P4 sequence
- the fluorescein molecule cy3 is labeled at the end; in addition, a specific Tag sequence is contained in the probe P2 for hybridization and recognition with a specific site on the chip.
- FIGS. 2A-2F are schematic flow diagrams showing a method for detecting a nucleic acid of a universal oligonucleotide microarray of the present invention. Please refer to Figure 2A for the probe and nucleic acid samples required for detection under suitable temperature conditions.
- a base gap is formed at the 3' end of the probe P1 and the 5' end of the P2, so that the base at the gap is just at the base of the base to be tested. Bases correspond.
- a base gap is formed at the 3' end of each pair of probe PI and the 5' end of P2, so that the gap corresponds to the base at the base site to be tested, and then in A , 'T, G, C four reaction tubes were respectively added with a corresponding deoxyribonucleotide-dATP, dTTP, dGTP, dCTP and DNA polymerase required for the reaction, ligase.
- FIG. 2C-2D shows the process of purifying the probe after completion of ligation with streptavidin magnetic particles. The specific process is as follows: Gap-filling and ligation are completed in each pair of probes P1 and P2 required for detection.
- Figure 2E-2F shows the amplification process of the probe, which is a template that is connected by a gap filling and ligation process and is purified by streptavidin-coated magnetic particles.
- the sequence H3 and P4 are used as a pair.
- the primers were subjected to symmetric or asymmetric PCR amplification. DNA polymerase and reaction buffer were added, and sequence H3 was used as a restriction primer, P4 was a non-limiting primer for asymmetric amplification, and P4 had a fluorescent molecule labeled cy3 at the 5th end.
- the labeled single-stranded probe is then magnetically separated and the supernatant is removed for hybridization to the corresponding region of the chip. Due to the specific Tag sequence contained in the probe P2, the cytosolic cy3-labeled amplification product sequence contains a complementary Anti-Tag sequence after completion of PCR amplification, and the amplification products and points in the four systems are used. Hybridization is performed on corresponding regions on the chip with the Tag sequence, and the detection results are determined by washing and scanning analysis.
- FIG. 3 is a schematic diagram of a chip of a nucleic acid detection method of a universal oligonucleotide microarray of the present invention. On one chip, it is divided into four regions, A, T, G, and C, which are used to hybridize with the probes in the four reaction systems after symmetric or asymmetric PCR amplification, respectively.
- a sequence identical to the Tag sequence on the probe P2 is separately designated for identifying and locating the specific base site to be tested, and the four regions are respectively used to join the system of the corresponding base.
- the amplified product is subjected to hybridization. As shown in Fig.
- the 15 tag sequences are used as an example to illustrate the partitioning of the chip, and the corresponding sites of the 15 tag sequences can detect 15 base sites to be tested, and along with each region.
- the detection throughput of the present technique increases, so that the technique can achieve a relatively large detection throughput.
- the universal oligonucleotide microarray proposed by the present invention can be used for resequencing analysis of a target nucleic acid sequence.
- a schematic diagram of the chip result analysis of the nucleic acid sequence resequencing is shown in FIG. 4 . If a fluorescent signal appears in the A region of the A region on the chip, it indicates that the HI and .H2 sequences of the detection probe corresponding to the 1-site Tag sequence are present in the target nucleic acid sequence, and the sites in the target nucleic acid sequence are detected.
- the base is! 1 .
- a fluorescent signal is present at the G site 2 site on the chip, it indicates that the HI and H2 sequences of the detection probe corresponding to the 2-site Tag sequence are present in the target nucleic acid sequence, and the site is detected in the target nucleic acid sequence.
- the base is (:. If a fluorescent signal appears at the 3 position of the T region on the chip, it indicates that the HI and H2 sequences of the detection probe corresponding to the 3-site Tag sequence are present in the target nucleic acid sequence, and the target nucleic acid sequence
- the base at the site tested is A.
- the universal oligonucleotide microarray proposed by the present invention can also be used for detecting mutations of known nucleic acid sequences, such as detection of SNPs mutation sites.
- a schematic diagram of the chip result analysis of the SNP site detection is shown in FIG. 5.
- the 1 site, the 2 site and the 3 site correspond to the three mutation sites of SNP1, SNP2 and SNP3, respectively. If the result of the chip scan is as shown in the figure, that is, the A region and the G region of the 1 site have The fluorescence signal appears, the fluorescence signal appears in the C region of the 2 site, and the fluorescent signal appears in the C and T regions of the 3 site.
- the genotype of the SNP1 site is T/C heterozygous, and the genotype of the SNP2 site. For the G/G homozygous type, the genotype of the SNP3 locus is G/A heterozygous.
- the universal oligonucleotide microarray proposed by the present invention can also be used for insertion/deletion sites for detection analysis.
- Pl For (one or a few bases) to a short segment of an insertion / deletion polymorphism genotyping sites, preclude the use of three probes Pl is, P2? P2, and its type, the design of the probe sequence Please refer to FIG. 6A - FIG. 6C.
- the oligonucleotide sequence of probe P1 shown in Fig. 6A is composed of three parts of H1, H3 and a tether, and is additionally labeled with biotin at its 5' end.
- the oligonucleotide sequence of probe P2 shown in Figure 6C consists of three parts, H2, H4 and tag'.
- HI is a sequence complementary to the sequence of the insertion/deletion mutation site on the template
- H5 is the probe P2
- the sequence of the 5' end complementary to the insertion type polymorphic site is H2 is inserted into the template/
- the sequence complementary to the sequence on the other side of the deletion site H5 is a sequence complementary to the sequence of one or several bases of the insertion type polymorphism site, which exists only at the 5' end of the probe P2', but The 5th end of the probe P2 is not.
- H3 and H4 are a pair of related universal oligonucleotide sequences used to amplify the probe.
- Each of the probes P2' and P2 also contains a specific Tag sequence for hybridization and recognition to a specific site on the chip.
- 7A to 7D are schematic flow charts showing the nucleic acid detecting method of this embodiment.
- 7A and 7B show the process of annealing and hybridizing the probe and template required for detection at a suitable temperature, and using one base adjacent to the insertion/deletion mutation site on the template as a probe and template annealing. The base corresponding to the gap formed later. After the probe and template annealing hybridization, a gap of one base is formed at the 3' end of the probe P1 and the 5' end of P2 or P2.
- Figures 7C and 7D show the DNA polymerase and ligase after the probe and template annealing hybridization, if the base and probe added to the reaction system are complementary to the base formed by the template after annealing.
- the probes P1 and P2 or P2' are connected into one probe by action. The purification, amplification and hybridization process of the probe is then identical to the detection procedure described above.
- FIG. 1 A schematic diagram of the chip result analysis of the insertion/deletion site detection is shown in FIG.
- FIG. For insert / When a deletion site is detected, an insertion/deletion site is detected using two sites in each region on the chip. That is, if an insertion/deletion site X is detected with 4 sites and 5 sites on the chip, and the tag sequence ordered by the site corresponds to the tag sequence on the probe P2' for the insertion site detection.
- the tag' sequence ordered by the 5 site corresponds to the tag' sequence on the probe P2 for the deletion site detection; the corresponding base of the gap formed by annealing the probe and the template is G; Point and 7 sites detect another insertion/deletion site Y, and the tag sequence ordered by the 6 site corresponds to the tag sequence on the probe P2' for the insertion site detection, and the 7 site is The tag' sequence corresponds to the tag sequence on the probe P2 for the deletion site detection; when the base corresponding to the gap formed by annealing the probe and the template is T, as shown in Fig. 8, if the site is 4 And the 5 sites have fluorescence signals in the C region, then the genotype of the site X is an insertion/deletion hybrid genotype. If only 7 sites have fluorescent signals in the A region and no fluorescent signals appear in the 6 sites, Then the genotype of locus Y is a homozygous deletion/deletion genotype.
- probes P1 and P2 were prepared as follows: probes P1 and P2 each took 10 fmol, and the template took 1 to 5 ng. 0.1 ⁇ ⁇ of DNA polymerase and DNA Ligase were added, and 10 X reaction buffer was added thereto, and divided into four reaction tubes, and 0.1 ⁇ M of dATP, dTTP, dGTP, and dCTP were added to each reaction tube. Ultrapure water complements 20 1 . The reaction was carried out under the following reaction conditions: 94 ° C, 5 min; 94 ° C, 1 min, 57. C, 2 min; 30 cycles, 57 ° C, 5 min.
- the amplification system is as follows, taking 1 ⁇ ⁇ resuspended magnetic particles as a template, each taking 0. 1 ⁇ ⁇ of the universal primers ⁇ 3 and ⁇ 4, adding 7. 5 ⁇ ⁇ 2 X Taq PCR mastermix and 6. 5 ⁇ l of dH20 make the reaction system 15 ⁇ l, and perform PCR amplification under the following conditions: 94 ° C, 5 min; 94 ° C, 1 min, 55 ° C, 1 min, 72 ° C, 1 min; 20 cycles, 72 V, 5 min, 4 ° C storage. After the reaction is completed, the four systems are magnetically separated, and the supernatant is removed for use.
- oligonucleotide chip An aldolylated tablet was prepared, and divided into four hybridization regions according to the requirements of the claims, and corresponding Tag sequences were prepared in each region. Dry and spare.
- Hybridization of the probe The PCR amplification product of the step (5) is mixed with the hybridization buffer, and hybridized with the corresponding chip region at 60 4 for 4 h. After completion, it is washed and dried.
- Chip scanning and result analysis The chip was scanned with a monochrome fluorescent scanner, and the result is shown in Fig. 5D. The results were analyzed based on the scan.
- Fig. 9A - Fig. 9C are verification electrophoresis patterns of the detection results of the mutation sites present on the cloned human genome P450 drug-metabolizing enzyme gene fragment by the nucleic acid detection method of the universal oligonucleotide microarray of the present invention.
- Figure 9A shows the results of detection of the wild type (T) template of the CYP3A4*17, c. 566T>C (F189S) mutation present on the cloned human genome P450 drug-metabolizing enzyme gene fragment.
- the four reaction systems were subjected to probe purification and PCR amplification, and the amplified products were subjected to 1.5% agarose gel electrophoresis.
- Lane M is DNA Marker2000.
- Lanes A, C, G, and T are the lanes in which the four reaction systems are located, respectively. As can be seen from the figure, only a strip with a length of more than lOOnt appears in the lane, and no corresponding strip appears in the other three lanes. This is the same as expected for the experiment.
- the band that has appeared is recovered by gel, and the recovered product is amplified by PCR with universal primers P3 and P4.
- the gel was subjected to 1.5% agarose gel electrophoresis, and the results are shown in Fig. 9B.
- a strip also appears at the same position as lane A of Fig. 9A, which also proves that the strip appearing in lane A of Fig. 9A is indeed after the probes P1 and P2 are connected,
- the template was subjected to PCR amplification using the universal primers P3 and P4 as primers.
- Figure 9C shows the results of a mixture of wild type (T) and mutant (C) templates for CYP3A4*17, c. 566T>C (F189S) mutations present on the cloned human genome P450 drug-metabolizing enzyme gene fragment.
- the four reaction systems were subjected to probe purification and PCR amplification, and the amplified products were subjected to 12% PAGE gel electrophoresis.
- Lane M is DNA Marker2000.
- Lanes A, C, G, and T are the lanes in which the four reaction systems are located. As can be seen from the figure, a band of more than lOOnt in length appears in lanes A and C, and no corresponding bands appear in the other two lanes. This is the same expected result for the detection of wild type (T) and mutant (C) mixed templates in the experiment.
- Figure 10 shows the results of hybridization of the wild type (T) and mutant (C) templates for the CYP3A4*17, c. 566T>C (F189S) mutations present on the cloned human genome P450 drug-metabolizing enzyme gene fragment. Scan the map.
- each area on the chip is divided into 6 points, two blank control points, and four points of the Tag sequence.
- the four reaction systems of A, T, G, and C are subjected to PCR amplification after probe purification, and hybridization scan results of 6 ⁇ l of the amplified product are taken.
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WO2009140802A8 (zh) | 2010-12-09 |
CN101586150A (zh) | 2009-11-25 |
US8501459B2 (en) | 2013-08-06 |
CN101586150B (zh) | 2016-09-28 |
US20110218115A1 (en) | 2011-09-08 |
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