CN102010911B - Method for detecting target DNA sequence, gene chip adopting same and application of gene chip - Google Patents
Method for detecting target DNA sequence, gene chip adopting same and application of gene chip Download PDFInfo
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- CN102010911B CN102010911B CN 201010563900 CN201010563900A CN102010911B CN 102010911 B CN102010911 B CN 102010911B CN 201010563900 CN201010563900 CN 201010563900 CN 201010563900 A CN201010563900 A CN 201010563900A CN 102010911 B CN102010911 B CN 102010911B
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Abstract
The invention discloses a method for detecting a target DNA sequence. The method comprises the following steps: 1) an RNA probe contacts with a target DNA to be annealed, wherein one end of the RNA probe is fixed on a substrate, the RNA probe contains a detection sequence close to the fixed end and a marking sequence close to the free end and the target DNA has a segment which is complimentary to the detection sequence in the RNA probe; 2) ribonuclease is used to hydrolyze the obtained DNA after annealling, wherein the RNA component in the RNA heteroduplex releases the target DNA and the marking sequence of the RNA probe; and 3) the free target DNA and marking sequence are removed through washing, the marking DNA with a marker is bound with the RNA probe of the detection sequence to perform sequence analysis on the target DNA, wherein the RNA probe is not hydrolyzed and is fixed on the substrate, and the marking DNA is complimentary to the marking sequence of the RNA probe. By adopting the method to detect the target DNA, the target gene does not require PCR amplification and the target DNA is directly detected, thus avoiding adopting the false negative and false positive results of the PCR amplification and increasing the detection accuracy rate.
Description
Technical field
The present invention relates to a kind of molecular Biological Detection method of gene order, and utilize this method to carry out the gene chip that gene order detects, and the application of this method.
Background technology
At present to genetic background genes involved (Disease-causing gene, tumour aberrant gene); The detection of the transgene component of bacterium, virus, genetically modified animals and plants is mainly through the design primer and corresponding D NA fragment carried out pcr amplification, realizes the purpose that detects through follow-up detection platform then.Detect through pcr amplification, cause false negative (pcr amplification failure) or false positive results such as (pcr amplification pollutions) easily, thereby influence exactness and efficient that gene order detects.In addition, the amount of the needed target gene of pcr amplification is bigger, and under the rare situation of sample, difficulty satisfies the detection demand.
Summary of the invention
The purpose of this invention is to provide a kind of pcr amplification that need not, can directly detect, avoid the method for the detection target DNA sequence of false positive and false negative result appearance target gene; Providing a kind of in addition is the gene chip that carries out the target DNA sequential detection of using this method; And the application of this method.
Be to realize that above-mentioned purpose, the method that the present invention detects the target DNA sequence comprise the steps,
Step 1: rna probe is contacted with target DNA and anneals; One end of described rna probe is fixed on the substrate; Rna probe comprises near the detection sequence of inboardend with near the mark sequence of free end, described target DNA have with rna probe in detect sequence complementary section;
Step 2:, discharge the mark sequence in target DNA and this rna probe with RNA part in the DNA:RNA heteroduplex of Yeast Nucleic Acid enzymic hydrolysis annealing back formation;
Step 3: free target DNA, mark sequence and rnase are removed in washing; Combine to detect the on-chip rna probe that is fixed on that sequence is not hydrolyzed with the mark DNA of tape label; The mark sequence of described mark DNA and rna probe is complementary, thereby target DNA is carried out sequential analysis.
Wherein, the rnase in the step 2 is a ribonuclease H; Be labeled as biotin labeling, fluorescent mark or isotopic labeling on the mark DNA.
Target DNA of the present invention is single stranded DNA or double-stranded DNA, and the sex change before step 1 annealing of described double-stranded DNA forms single stranded DNA, on the target DNA with rna probe in detection sequence complementary sequence can only be a fragment on the target DNA.
In the target DNA that discharges in the step 2 of the present invention and this system on other rna probes complementary detect sequence annealed once more; Form the DNA:RNA heteroduplex, the process of the step 2 that moves in circles all rna probes that contains the complementary detection sequence in system all are hydrolyzed.
A kind of gene chip; One end of rna probe 1 is fixed on the chip carrier; Rna probe comprises near the detection sequence of chip carrier inboardend with near the mark sequence of free end, and the array sampling point is set on the chip carrier, rna probe according to the different fixing that detects sequence in different sampling points.
During use, in each sampling point, add sample DNA, after operating by method of the present invention, detect each sampling point internal labeling, sample DNA contain with chip on do not detect the detection sequence complementary sequence of rna probe in the sampling point of mark.
Sample DNA of the present invention is generally the unknown sample of user censorship, wherein possibly contain with rna probe in detect sequence complementary target DNA sequence, also possibly be the unknown dna sequence dna that does not contain the target DNA sequence.
Adopt method of the present invention to can be used for detecting hereditary genetic background genes involved (Disease-causing gene, tumour aberrant gene).
Adopt method of the present invention to can be used for the gene of bacterial detection or virus.
Adopt method of the present invention to can be used for detecting the transgene component of genetically modified animals and plants.
Use detection method of the present invention that target DNA is detected; Need not target gene is carried out pcr amplification; Directly target DNA is detected; Thereby false negative and the false positive results of having avoided PCR to occur in detecting have improved the detection accuracy, have reduced the laboratory simultaneously greatly because pcr amplification and pollution of nucleic acid that subsequent detection caused; In addition, this detection method only needs the target DNA molecule of minute quantity just can realize testing goal.
Description of drawings
Fig. 1 detects the synoptic diagram of the method for target DNA sequence for the present invention;
Fig. 2 detects synoptic diagram for the result of test sample DNA gene chip of the present invention.
Embodiment
Further explain in the face of the part term that occurs among the present invention down,
Term among this paper " ribonuclease H " is also referred to as RNase H; Refer to a kind of endoribonuclease; The phosphodiester bond of RNA chain on the specificity hydrolysis DNA:RNA hybridization chain; Produce 5 ' Nucleotide, this enzyme is to the not effect of nucleic acid (single stranded DNA or single stranded RNA), double-stranded DNA or double-stranded RNA of strand.
Term among this paper " annealing " refers to two strand polynucleotide form duplex molecule through the hydrogen bond between the complementary base process.Can take place between the nucleic acid chains, can form double chain DNA molecule, double-stranded RNA or DNA-RNA hybrid molecule.
Term among this paper " sex change " refers to nucleic acid molecule and is loosened by stable double-spiral structure and be the phenomenon of random linear structure.Keep the hydrogen bond rupture of duplex stability during sex change, the accumulation force between base is destroyed, but does not relate to the change of its primary structure.
By specifying technology contents of the present invention, structural attitude, realized purpose and effect, give explanation below in conjunction with embodiment and conjunction with figs. are detailed.
As shown in Figure 1, the method for detection target DNA sequence of the present invention comprises the steps,
Step 1: rna probe 1 is contacted with target DNA 2 and anneals; One end of described rna probe 1 is fixed on the substrate; Rna probe 1 comprises near the detection sequence 11 of inboardend with near the mark sequence 12 of free end, described target DNA 2 have with rna probe 1 in detect sequence 11 complementary sections;
Step 2:, discharge the mark sequence 12 in target DNA 2 and this rna probe 1 with RNA part in the DNA:RNA heteroduplex 4 of rnase 3 hydrolysis annealing back formation;
Step 3: free target DNA 2, mark sequence 12 and rnase 3 are removed in washing; Combine to detect the on-chip rna probe 1 that is fixed on that sequence 11 is not hydrolyzed with the mark DNA5 of tape label; Mark sequence 12 complementations of described mark DNA5 and rna probe 1, thus target DNA 2 is carried out sequential analysis.
Wherein, the rnase in the step 23 is a ribonuclease H; Be labeled as biotin labeling, fluorescent mark or isotopic labeling on the mark DNA5, perhaps other any existing feasible mark modes.
Target DNA 2 of the present invention is single stranded DNA or double-stranded DNA, and the sex change before step 1 annealing of described double-stranded DNA forms single stranded DNA.
In the target DNA that discharges in the step 2 of the present invention 2 and this system on other rna probes 1 complementary detect sequence 11 annealed once more; Form DNA:RNA heteroduplex 4, the process of the step 2 that moves in circles all rna probes that contains complementary detection sequence 11 1 in system all are hydrolyzed.
As shown in Figure 2, adopt the gene chip of method test sample DNA of the present invention, an end of rna probe 1 is fixed on the chip carrier 6, and rna probe 1 comprises near the detection sequence 11 of chip carrier 6 inboardends with near the mark sequence 12 of free end.Array sampling point 61 is set on the chip carrier 6, rna probe 1 according to the different fixing that detects sequence 11 in different sampling point 61.
During use, in each sampling point 61, add sample DNA, after operating by method of the present invention, detect each sampling point 61 internal labelings, sample DNA contain with chip on do not detect the detection sequence 11 complementary sequences of rna probes 1 in the sampling point 61 of mark.
The concrete detection step and the principle of this gene chip are following:
The mixed solution reaction that in the chip sampling point of having fixed rna probe, adds sample DNA and ribonuclease H; Target dna fragment will combine with the detection sequence of rna probe; At this moment, ribonuclease H is with the RNA chain portion of the formed DNA:RNA heteroduplex part of specific recognition and this heteroduplex of selective hydrolysis.Target dna fragment and rna probe mark sequence discharge subsequently; Target dna fragment continues to detect sequences with other interior rna probes of chip sampling point and combines; At this moment, ribonuclease H is incited somebody to action once more the RNA chain portion of the formed DNA:RNA heteroduplex part of specific recognition and this heteroduplex of selective hydrolysis.The process of this " target dna fragment combination-enzymic hydrolysis-target dna fragment and rna probe mark sequence discharge " will continue to carry out, and contain the rna probe molecule that detects gene with the target DNA complementary up to all and be hydrolyzed.Subsequently, free target DNA, mark sequence and rnase are removed in washing.Then, add mark DNA, mark DNA will combine with the mark sequence-specific of rna probe on staying chip.Mark DNA has carried out biotin labeling or fluorescent mark or isotopic labeling or other any feasible marks, through mark DNA is detected, can realize the analysis of target DNA sequence.Not containing the rna probe that detects sequence with the target DNA complementary can not be degraded, and is retained in chip surface, can be detected; And contain detection sequence and the target DNA specific combination that detects the rna probe of sequence with the target DNA complementary; Formed the DNA:RNA heteroduplex; By the degraded of Yeast Nucleic Acid enzyme spcificity, after dissociating out, the mark sequence on it is washed removal, therefore can't be detected.So just can judgement sample DNA contain with chip on do not detect the detection sequence complementary sequence of rna probe in the sampling point of mark.
Adopt method of the present invention can be used to detect the genetic background genes involved, like Disease-causing gene, tumour aberrant gene.
Adopt method of the present invention can be used for the gene of bacterial detection or virus.
Adopt method of the present invention can be used to detect the transgene component of genetically modified animals and plants.
The above is merely embodiments of the invention; Be not so limit claim of the present invention; Every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to be done; Or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Embodiment 1:
Present embodiment uses gene chip of the present invention that Shigellae is detected, wherein:
Target DNA is double-stranded DNA from the Shigellae genome, is made up of SEQ ID NO:1;
Rna probe is made up of SEQ ID NO:2 synthetic, and the sequence shown in the 1-22 position of SEQ ID NO:2 is for detecting sequence, and the sequence shown in the 23-42 position is the mark sequence; Sequence shown in the 478-499 position of the detection sequence of rna probe and SEQ ID NO:1 is identical, and is complementary with the complementary strand of this sequence;
Mark DNA is made up of SEQ ID NO:3 synthetic, and wherein 5 ' end has carried out biotin labeling;
Concrete experimental technique is following:
1, extracting genome DNA;
Get the 1.5ml culture and change in the centrifuge tube, the centrifugal 3min of 12,000 * g;
Abandon supernatant, the bacterial precipitation thing is suspended in 100 μ L ddH2O, with the abundant mixing of turbine mixer;
Live centrifuge tube with the boom frame, place boiling water bath, 10min;
Centrifugal at once 10min after the taking-up, supernatant is prepared genomic dna solution.
2, gene chips detects:
Reagent:
Coating buffer: 0.05mol/L disodium phosphate soln (pH8.5); Confining liquid: 3% bovine serum albumin or 0.5% casein solution: washings: PBST; Stop buffer: 2M H2SO4
Step: use rna probe (0.1 μ M) the 100 μ L through the coating buffer dilution to encapsulate the nucleic acid board, 4 ℃ are spent the night.Next day, seal 60min in 37 ℃ with confining liquid 200 μ L.With washings washing 3 times, subsequent use.The every hole on the plate of encapsulating after confining liquid washing adds the mixed solution 25 μ L of sample DNA and ribonuclease H (DNA amount 10-1000 copies; Rnase total amount 5U), add hybridization solution 100 μ L again.Behind the mixing, 37 ℃ of reaction 60min are with washings washing 5 times (removing free target DNA, mark sequence and rnase); Add mark DNA (0.01 μ M) 100 μ L in every hole, behind the mixing, 37 ℃ of reaction 60min are with washings washing 5 times (removing free mark DNA).The horseradish peroxidase 100 μ L that add the streptavidin mark in every hole, 37 ℃ of reaction 30min are with washings washing 5 times.Every hole adds colour developing liquid 100 μ L, 37 ℃ of colour developing 10min; Every hole adds stop buffer 100 μ L termination reactions.With 630nm is reference wavelength, measures absorbancy in wavelength 492nm place.
Wherein, each hole adds following sample:
Negative control sample: ddH2O;
Positive control sample: positive control sample is one section synthetic oligonucleotide DNA sequence, and the detection sequence of this sequence and rna probe is complementary, and this positive control sequence is SEQID NO:4 among this embodiment.This positive control concentration is 0.01 μ M, and every hole adds 1 μ L, adds ribonuclease H in addition, and TV is 25 μ L.
Sample 1 contains the target DNA of 10 copies; Sample 2 contains the target DNA of 100 copies; Sample 3 contains the target DNA of 1000 copies;
Detected result:
The absorbance of the negative contrast of deduction value reduces 0.2.
The negative control absorbance should be not less than 1.5.
Positive control should be positive, and absorbance should not be higher than 0.2, as less than 0.05 by 0.05.
| Point sample hole (row) | 1 | 2 | 3 | On average |
| The negative control sample | 2.317 | 2.409 | 2.415 | 2.380 |
| Sample 1 (10 copy) | 0.735 | 0.793 | 0.812 | 0.780 |
| Sample 2 (100 copy) | 0.693 | 0.717 | 0.659 | 0.670 |
| Sample 3 (1000 copy) | 0.627 | 0.651 | 0.690 | 0.658 |
| Positive control sample | 0.056 | 0.069 | 0.053 | 0.059 |
Method of the present invention is described and is adopted the gene chip of this method can be quick, sensitive detects the number that whether contains target DNA in the sample and reflect its content.It can be used for the gene of bacterial detection or virus.
Embodiment 2:
Present embodiment uses gene chip of the present invention that the DNA of the transgene component Cry1A (b) of transgenic corns Bt11 is detected, wherein:
Target DNA is double-stranded DNA from transgenic corns Bt11, is made up of SEQ ID NO:5;
Rna probe is made up of SEQ ID NO:6 synthetic, and the sequence shown in the 1-21 position of SEQ ID NO:6 is for detecting sequence, and the sequence shown in the 22-41 position is the mark sequence; Sequence shown in the 214-234 position of the detection sequence of rna probe and SEQ ID NO:1 is identical, and is complementary with the complementary strand of this sequence;
Mark DNA is made up of SEQ ID NO:7 synthetic, and wherein 5 ' end has carried out biotin labeling;
Concrete experimental technique is following:
1, extracting genome DNA: (extracting solution composition: Tris-HCl pH7.5,150mM; NaCl 100mM; EDTA pH8.0,15mM; CTAB 1.5%; β-ME 1.5% (adding before the use)).
Take by weighing a certain amount of leaf of Semen Maydis, liquid nitrogen pulverization, every gram material add the CTAB extracting solution 2mL and the 5 μ L beta-mercaptoethanols of 95 ℃ of preheatings, after stirring fast, and 65 ℃ of water-bath 60-90min.Add the equal-volume chloroform.Fine rotation half a hour is until layering.Room temperature 800rpm, centrifugal 10min, supernatant add 2/3 volume isopropanol precipitating DNA.Thick DNA dissolves through TE, RNA enzymic digestion RNA, phenol/chloroform, chloroform purifying, last ethanol precipitated dna.
2, gene chips detects:
Reagent:
Coating buffer: 0.05mol/L disodium phosphate soln (pH8.5); Confining liquid: 3% bovine serum albumin or 0.5% casein solution: washings: PBST; Stop buffer: 2M H2SO4
Step: use rna probe (0.1 μ M) the 100 μ L through the coating buffer dilution to encapsulate the nucleic acid board, 4 ℃ are spent the night.Next day, seal 60min in 37 ℃ with confining liquid 200 μ L.With washings washing 3 times, subsequent use.The every hole on the plate of encapsulating after confining liquid washing adds the mixed solution 25 μ L of sample DNA and ribonuclease H (DNA amount 10-1000 copies; Rnase total amount 5U), add hybridization solution 100 μ L again.Behind the mixing, 37 ℃ of reaction 60min are with washings washing 5 times (removing free target DNA, mark sequence and rnase); Add mark DNA (0.01 μ M) 100 μ L in every hole, behind the mixing, 37 ℃ of reaction 60min are with washings washing 5 times (removing free mark DNA).The horseradish peroxidase 100 μ L that add the streptavidin mark in every hole, 37 ℃ of reaction 30min are with washings washing 5 times.Every hole adds colour developing liquid 100 μ L, 37 ℃ of colour developing 10min; Every hole adds stop buffer 100 μ L termination reactions.With 630nm is reference wavelength, measures absorbancy in wavelength 492nm place.
Wherein, each hole adds following sample:
Negative control sample: ddH2O;
Positive control sample: positive control sample is one section synthetic oligonucleotide DNA sequence, and the detection sequence of this sequence and rna probe is complementary, and this positive control sequence is SEQID NO:8 among this embodiment.This positive control concentration is 0.01 μ M, and every hole adds 1 μ L, adds ribonuclease H in addition, and TV is 25 μ L.
Sample 1 contains the target DNA of 10 copies; Sample 2 contains the target DNA of 100 copies; Sample 3 contains the target DNA of 1000 copies;
Detected result:
The absorbance of the negative contrast of deduction value reduces 0.2.
The negative control absorbance should be not less than 1.5.
Positive control should be positive, and absorbance should not be higher than 0.2, as less than 0.05 by 0.05.
| Point sample hole (row) | 1 | 2 | 3 | On average |
| The negative control sample | 2.512 | 2.489 | 2.469 | 2.490 |
| Sample 1 (10 copy) | 0.892 | 0.905 | 0.874 | 0.890 |
| Sample 2 (100 copy) | 0.746 | 0.719 | 0.733 | 0.733 |
| Sample 3 (1000 copy) | 0.701 | 0.720 | 0.693 | 0.705 |
| Positive control sample | 0.073 | 0.064 | 0.061 | 0.066 |
Method of the present invention is described and is adopted the gene chip of this method can be quick, sensitive detects the number that whether contains target DNA in the sample and reflect its content.It can be used for detecting the transgene component of genetically modified animals and plants.
Further, can know method of the present invention and adopt the gene chip of this method can be used in and detect genetic background genes involved (Disease-causing gene, tumour aberrant gene) by embodiment 1,2.
Claims (9)
1. the method for the detection target DNA sequence of non-medical diagnosis on disease purpose is characterized in that: it comprises the steps,
Step 1: rna probe is contacted with target DNA and anneals; One end of described rna probe is fixed on the substrate; Rna probe comprises near the detection sequence of inboardend with near the mark sequence of free end, described target DNA have with rna probe in detect sequence complementary section;
Step 2:, discharge the mark sequence in target DNA and this rna probe with RNA part in the DNA:RNA heteroduplex of Yeast Nucleic Acid enzymic hydrolysis annealing back formation;
Step 3: free target DNA, mark sequence and rnase are removed in washing; Combine to detect the on-chip rna probe that is fixed on that sequence is not hydrolyzed with the mark DNA of tape label; The mark sequence of described mark DNA and rna probe is complementary, thereby target DNA is carried out sequential analysis.
2. the method for detection target DNA sequence according to claim 1 is characterized in that: the rnase in the step 2 is a ribonuclease H.
3. the method for detection target DNA sequence according to claim 1 is characterized in that: be labeled as biotin labeling, fluorescent mark or isotopic labeling on the mark DNA in the step 3.
4. the method for detection target DNA sequence according to claim 1 is characterized in that: described target DNA is single stranded DNA or double-stranded DNA, and the sex change before step 1 annealing of described double-stranded DNA forms single stranded DNA.
5. according to the method for any one described detection target DNA sequence in the claim 1 to 4; It is characterized in that: in the target DNA that discharges in the described step 2 and this system on other rna probes complementary detect sequence annealed once more; Form the DNA:RNA heteroduplex, the process of the step 2 that moves in circles all rna probes that contains the complementary detection sequence in system all are hydrolyzed.
6. a gene chip is characterized in that, an end of the rna probe on the said gene chip is fixed on the chip carrier, and rna probe comprises near the detection sequence of chip carrier inboardend with near the mark sequence of free end.
7. gene chip according to claim 6 is characterized in that: the array sampling point is set on the described chip carrier, rna probe according to the different fixing that detects sequence in different sampling points.
8. gene chip according to claim 7; It is characterized in that: in each sampling point, add sample DNA; After any one described method is operated in the claim 1 to 5; Detect each sampling point internal labeling, sample DNA contain with chip on do not detect the detection sequence complementary sequence of rna probe in the sampling point of mark.
9. the application of the non-medical diagnosis on disease purpose of the method for any described detection target DNA sequence in the claim 1 to 5 is characterized in that: this method is used to detect the transgene component that the genetic background genes involved perhaps is used for the gene of bacterial detection or virus or is used to detect genetically modified animals and plants.
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| KR20040041529A (en) * | 2001-09-28 | 2004-05-17 | 산코 준야쿠 가부시키가이샤 | Signal amplification method for dna chip |
| CN101260432A (en) * | 2008-04-10 | 2008-09-10 | 上海交通大学 | RNA quantitative determination method utilizing S1 enzyme cutting single-chain nucleic acid characteristic |
| CN101760527B (en) * | 2008-12-26 | 2014-04-16 | 上海透景生命科技有限公司 | PCR amplification-free and detecting instrument-free nucleic acid analysis method |
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