CN1580782A - Transgenic product low-density gene chip detecting method - Google Patents
Transgenic product low-density gene chip detecting method Download PDFInfo
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Abstract
The low-density gene chip detection method of transgenic product relates to a detection method for transgenic product by uisng low-density gene chip. Said method includes the following steps: selecting primer and probe; processng chip carrier, preparing chip; dissolving probein ultrapure water; adding sample application liquid; fixing the probeon the treated chip carrier, sampling; multigene PCR amplification; labeling MPCR product; chip hybridization experiment of MPCR product; scanning analysis and data processing. As compared with traditional detection method said invention has the characteristics of quick seep, high efficiency and high automation level, and can simultaneously make detection and analysis of common 9 exogenous DNA sequences of promoter, terminator, screening marker gene, weedicide-resisting gene and insect-resisting gene, etc. in transgenic products.
Description
Technical field
The present invention relates to a kind of detection method of transgenic product, especially adopt low density gene chip to be used for the detection method of transgenic product.
Background technology
Fast development along with transgenic crop; the Environmental security of transgenic product and edible safety have caused the national governments and the public's common concern (Anklam E; Gadani F; Heinze P; et al; Analytical methods for detection anddetermination of genetically modified organisms in agricultural crops and plant-derived food products.Eur.Food Res.Technol.; 2002; 214; 3-26) right to know and right to choose in order to protect the consumer; ensure people's dining table safety; guarantee the sound development of International Agricultural Trade; countries in the world have all been strengthened in the detection of transgenic product and the research input aspect the safety evaluation, China from requirement on March 20 in 2002 transgenic product is detected and sign (the State Council of the PRC. " agriculture genetically modified organism safety management regulations " .2001 May 23).Compare with the fast development of transgenic product, the development of transgenic product detection method relatively lags behind, relevant international standard is not put into effect yet, detection method commonly used at present mainly contains following several (Markus L, Peter B, Klaus P, et al.IUPAC collaborative trialstudy of a method to detect genetically modified soy beans and maize in dried powder.Journal of AOAC International, 1999,82:923～928):
1. immunological method: the immunological method that with the expressing protein is target is to react according to antigen and antibody specific, design at a certain specific transgenic product, (enzyme-linked immunosorbent assay is ELISA) with test strips method (lateral flow strip) comprising Western blot (western blot), enzyme linked immunosorbent assay.At present reported that some reliable and stable immunological methods can detect a few transgenic product, detected RoundupReady as the ELISA method
TMThe expressing protein cp4epsps of soybean, Flavor Savr
TM(Stave J W such as the expressing protein nptII of tomato, Magin K, Schimmel H, et al.AACC collaborative study of a protein method for detection ofgenetically modified corn.Cereal Foods World, 2000,45:497～501).
Estimate: immunological method has that specificity is good, easy and simple to handle, expense is low, to instrument and the less demanding characteristics of personnel, is applicable to raw material and semi-manufacture analysis.But only at a kind of differential protein, coverage rate is low for these class methods; For processed food, be subjected to the sex change of target protein in the conformation of target protein and the restriction of content, the especially deep-processed food, influence the specific recognition of antigen-antibody, and the uncertainty of detection is increased; Some gene is modified in new host, only express or do not express at some position, also just be difficult to detect (Anklam E, Gadani F, Heinze P, et al with these class methods, Analyticalmethods for detection and determination of genetically modified organisms in agricultural crops andplant-derived food products.Eur.Food Res.Technol, 2002,214,3-26).
2. qualitative PCR method: with the foreign DNA be target Detection comprise DNA hybrid method (southern blot) and PCR method (polymerase chain reaction, PCR).The DNA hybrid method accurately and reliably, but there are shortcoming (Anklam E such as sensitivity is low, complex operation, Gadani F, Heinze P, et al, Analytical methods for detectionand determination of genetically modified organisms in agricultural crops and plant-derived foodproducts.Eur.Food Res.Technol, 2002,214,3-26).The PCR method is meant that the dna replication dna mode is in the technology of external certain special area of selective amplification DNA in the analogue body, by the exogenous DNA array in the pcr amplification transgenic product, using the agarose gel electrophoresis assay products then, is present the most frequently used transgenic detection method.
Estimate: not high to the equipment reagent requirement, cost is low, and is fast and convenient, can be used as shaker test.Electrophoretic analysis is rough, easily not enough (Wenjin Su such as large scale analysis need be verified, be difficult to quantitatively, be not suitable for to pollution, testing result but exist, Siyang Song, Minnan Long, and Guangming Liu, Multiplex polymerase chain reaction/membrane hybridization assayfor detection of genetically modified organisms.Journal of Biotechnology, 2003,105:227-233).
3. competitive quantifying PCR method (competitive quantitative PCR): with foreign DNA to be measured and competitor dna (with disappearance or insert the plasmid that the exogenous DNA array of tens bp makes up) as the substrate of vying each other, competition primer and enzyme etc. in same PCR reaction system, its PCR product that differs tens bp can be distinguished with quantitative by the gel imaging analysis system, make the typical curve of " target DNA concentration-target DNA product amount/competitor dna product amount " with serial transgenosis standard items DNA, can try to achieve gm content (the Hardegger M in the sample according to " DNA product amount to be measured/competitor dna product amount ", Brodmann P, Herrmann A.Quantitative detection of the 35S promoter andthe NOS terminator using quantitative competitive PCR.Eur Food Res Technol, 1999,209:83-87).
Estimate: competitive PCR method in a reaction tube, increase simultaneously testing sample and competitive template, eliminated the interference of various variable elements in the experiment, obtained result comparatively accurately, but quantitative test, cost is lower.But there are complex operation, technical sophistication (needing to make up competitive DNA), require not enough (Ke LD such as electrophoretic analysis, easy pollution, Chen Z, Yung WK.A reliability test of standard-based quantitative PCR:exogenous vs endogenousstandards.Mol.Cell Probes, 2000,14:127-135).
4. quantitative fluorescent PCR (fluorophore quantitative PCR, FQPCR) method: in the PCR reaction system, add fluorescence labeling probe, variation by fluorescence signal in the photoelectricity conducting system direct detection pcr amplification process is to obtain quantitative result, so have the high specific of DNA hybridization and the high precision of spectral technique.The collection of data and analysis can be finished automatically, simplified experimentation, reduced and polluted and false-positive chance (Becker K., D.Pan and C.B.Whitely.Real-time quantitative polymerase chain reaction to assess gene transfer.Hum.GeneTher, 1999,10:2559-2566).
Estimate: above-mentioned each the method height of fluorescence quantifying PCR method remolding sensitivity, accuracy and good reproducibility, but and quantitative measurement (Whitcombe D, Browie J, Gillard H L, et al.A homogeneous fluorescence assay for PCRamplicons:its application to real-time, single-tube genotyping.Clin Chem, 1998,44:918～923).But fluorescent quantitative PCR technique cost height commonly used at present, inconvenience popularization and application (Li Q, Luan G, Guo Q, et al.A new class of homogeneous nucleic acid probes based on specific displacementhybridization.Nucleic Acids Res., 2002,30 (2): E5-5).
5. (multiplex PCR, MPCR) method: along with development of molecular biology, the transgenosis detection technique is just developed to polygenes PCR direction by single-gene PCR polygenes PCR.The single-gene round pcr is simple to operate, but it can only detect a certain special genes composition, if transgene component is not clear or contain multiple transgene component in the sample to be checked, adopt different PCR that the transgene component that may exist is repeatedly increased with regard to needing, this moment, round pcr just became loaded down with trivial details time-consuming, and the false negative result that omission causes may occur.
Estimate: MPCR is a kind of special P CR technology, more faster, more economical than single-gene PCR reaction, can in a reaction system, detect multiple possible transgene component, remedied some shortcomings (the Minunni M of single-gene PCR, Tombelli S, Mariotti E, et al.Biosensors as new analytical tool for DNA detection.Fresenius J Anal Chem, 2001,369:589-593).At present, traditional gel electrophoresis analysis method is still continued to use in the detection of polygenes PCR product, should method is simple, but lack specificity and sensitivity, easily human and environment is produced harm simultaneously.
Summary of the invention
The present invention aim to provide a kind of high flux, rapidly and efficiently, the low density gene chip detection method of automaticity and the transgenic product that sensitivity is higher and cost is low.
Another object of the present invention is to provide a kind of can be simultaneously to 9 methods that exogenous DNA array carries out check and analysis such as promoter commonly used in the transgenic product, terminator, selection markers gene, anti-herbicide gene, anti insect genes.
Another object of the present invention is to provide a kind of method that can detect multiple transgenic product.
Step of the present invention is:
1, selects primer and probe;
2, process chip carrier: select chip carrier, through clean, drying, silanization again with 1, soaks the chip carrier of silanization in 4-benzene diisothiocyanic acid salt (POC) solution, dry;
3, preparation chip: probe is dissolved in ultrapure water, adds sampling liquid, on the chip carrier of having handled, point sample is placed, and cleans with probe stationary, dries, and drying, standby;
4, MPCR (polygenes PCR) amplification: adding DMSO (dimethyl sulfoxide (DMSO)) is resuspended with Cy3 (single function fluorescent dye) dry powder, and the dyestuff branch that suspends is again installed in the tubule, with the DNA concentration systems it is drained, and drying is carried out polygenes PCR reaction;
5, MPCR product mark: get the sex change of polygenes PCR product, carry out the fluorescence labeling amplification, in reaction tube, add damping fluid, sex change PCR product, every pipe adds the absolute ethyl alcohol precipitate nucleic acids, and is centrifugal, and NaHCO is used in washing
3Dissolving is with NaHCO
3The polygenes PCR product of dissolving joins in the Cy3 dyestuff tubule of packing, and dyestuff dry powder is resuspended, places, and hatches, and uses PCR product purification kit, and purifying, freeze-drying are concentrated into volume required;
6, the chip hybridization of MPCR product test: each reaction tube adds EDTA (ethylenediamine tetraacetic acid) solution, the mark PCR product that adds purifying again, sex change is taken out and is positioned on ice the adding hybridization solution, make the hybridization mixed liquor, the chip of preparation is dripped the hybridization mixed liquor, put to hybridize in the hybrid heater and spend the night, the chip after the hybridization is through washing, centrifugal, dry;
7, scanning analysis and data processing: the chip behind the hybridization reaction is inserted in the detection hole of scanner, carry out result's scanning and analyzing and processing.
Said chip carrier is selected from slide, nylon membrane or cellulose membrane etc.
Since biochip technology is the nineties in 20th century, along with human and other biological gene database sharply enlarge and a kind of extensive, the robotization of being born, the technique of gene detection of high reliability, fields such as various pathogen detection, genetic diseases diagnosis, environmental monitoring have been applied to.The point sample density of current chip technology has reached tens thousand of probe/cm
2, provide a strong tool for transgenosis detects research; Owing to adopted modern electronics and ray machine power technology, genetic chip all is robotization control from being prepared into interpretation of result; Parallel laboratory test is set in chip technology and various contrast is very easy, the collection of data and analyze and finish by computer, testing result is objective reliable; Therefore, high flux, quick, sensitive biochip technology are the inevitable directions that transgenic product detects development.
The low density gene chip detection method (LD-CHIP) of transgenic product of the present invention is fixed in present general special oligonucleotide fragment makes detection chip on the slide, extract from sample to be checked that DNA carries out that polygenes increases simultaneously, hybridize with chip behind the mark, hybridization signal is analyzed judgement by scanner and computing machine.
The present invention has at first established the amino silaneization and the POC handling procedure of homemade slide, make the amino on the slide be converted into isothiocyanic acid base with reactivity, the amino covalence of mark combines on the latter and the oligonucleotide probe, thereby makes probe be incorporated into surface of glass slide securely.Consider that probe is combined with certain loss on slide, in chip preparation test, adopted the oligonucleotide probe of higher concentration (2 μ g/ μ L), apparently higher than document (Takahashi Y, Ishii Y, Nagata T, et al.Clinicalapplication of oligonucleotide probe array for full-length gene sequencing of TP53 in coloncancer.Oncology, 2003,64:54-60) the concentration standard of Tui Jianing (500ng/ μ L), guaranteed like this at point sample, in rinsing and the crossover process, probe still can be kept certain concentration.
The present invention has determined that transgenosis detects the top condition of oligonucleotide chip preparation: adopt amido modified and add the oligonucleotide probe of polyT (polythymidylic acid) arm, modify slide as medium with the isothiocyanic acid base, the chip room temperature is placed behind the point sample, ammoniacal liquor with 1%, 0.2% SDS (lauryl sodium sulfate) and pure water clean, and room temperature is dried.
The present invention selects suitable hybridization conditions to make to react between the probe on polygenes PCR product and the chip to be in the optimum, reduces the mispairing rate between the dna molecular.The factor that influences the formation of allos heteroduplex comprises target concentration, concentration and probe concentration, hybridization both sides' sequence composition, salinity and temperature etc.The present invention has determined hybridization temperature and the hybridization time that LD-CHIP detects.
The present invention judges to be transgenic product by detecting general gene locis such as promoter, terminator and marker gene, judge it is any transgenic product, the transgenic product of China's approved whether by detecting antiweed, the specific genes of interest of each species such as pest-resistant, this method can be applicable to comprise the detection of multiple transgenic product such as soybean, corn and potato, increases, hybridizes to result's scanning from template extraction, target sequence and can finish in two day time.
The DNA chip has characteristics such as high flux, robotization in transgenic product detects, demonstrate distinctive superiority and potential use greatly.
Genetic chip is a kind of high flux parallel analysis technology, is the matrix by a large amount of DNA or oligonucleotide probe dense arrangement, can detect hundreds and thousands of kinds of transgene components simultaneously, has solved the efficiency during transgenosis detects well.At first, by a graded with the oligonucleotide probe point sample on chip, sample DNA polygenes PCR product and this chip with mark carries out hybridization reaction then, hybridization signal carries out scanning analysis.Biochip technology is compared with traditional detection method, has rapidly and efficiently characteristics such as automaticity height.The low density gene chip (LD-CHIP) that the present invention sets up can carry out check and analysis to 9 exogenous DNA arrays such as promoter commonly used in the transgenic product, terminator, selection markers gene, anti-herbicide gene, anti insect genes simultaneously.
Description of drawings
Fig. 1 is the sample application array that is used for the low density gene chip of transgenic product detection.
Fig. 2 detects the scanning result of GM (transgenosis) soybean standard items (A) and GM Standard for Maize product (B) for LD-CHIP hybridization.
Fig. 3 detects scanning result for sample LD-CHIP hybridization.In Fig. 3, and A (0.3% genetically engineered soybean standard items and B (S1): 18S rRNA, CaMV 35S, Tnos, lectin and cp4epsps-1/-2 be the positive as a result; C (0.15% transgenic corns standard items) and D (M1): 18S rRNA, CaMV 35S, Tnos, nptII, invertase and cryIAb-1/-2 arepositive; E (S-P8) and F (P3): 18S rRNA, CaMV 35S, Tnos, nptII, and cryIIIA is the positive as a result.
Embodiment
Following examples will the present invention is further illustrated in conjunction with the accompanying drawings.
Embodiment 1
Selecting suitable primer and probe is that the key that DNA analysis detects is carried out in success.Sequence at the most frequently used 9 foreign DNAs and crt gene (as 18S rRNA, soybean lectin endogenous gene and corn invertase endogenous gene) in the transgenic product, adjust parameters by the combination of repeated multiple times ground, through software Primer Express
TMWith OLIGO 4.0 searching analysis, and carry out the comparison of each oligonucleotides, therefrom select satisfactory primer and probe.Primer and the relevant information selected for use see Table 1, and used probe and relevant information see Table 2.
Table 1
| The primer numbering | Primer sequence (5 '-3 ') | Target dna |
| 18S-1 | TAA?TAG?AGC?AAT?GAA?CAG?TCG?G | |
| 18S-2 | CTT?TCA?ACA?ACG?GAT?CTCTT?G?G | The 18S rRNA |
| lec-1 | GTG?CTA?CTG?ACC?AGC?AAG?GCA?AAC?TCA?GCG | |
| lec-2 | GAG?GGT?TTT?GGG?GTG?CCG?TTT?TCG?TCA?AC | Soybean lectin endogenous gene |
| inv-1 | GGC?CGG?ATC?GTC?ATG?CTC?TAC?A | |
| inv-2 | TTG?GCG?TCC?GAC?TTG?ACC?CAC?T | Corn invertase endogenous gene |
| 35S-3 | GAT?TCC?ATT?GCC?CAG?CTA?TCT?G | |
| 35S-4 | TAG?AGG?AAG?GGT?CTT?GCG?AAG?G | The CaMV 35S promoter |
| fmv-1 | AGT?CCA?AAG?CCT?CAA?CAA?GGT?C | |
| fmv-2 | CAT?TAG?TGA?GTG?GTC?TGT?CAG?G | The FMV 35S promoter |
| nos-3 | GAA?TCC?TGT?TGC?CGG?TCT?TG | |
| nos-4 | CGA?ATT?CCC?GAT?CTA?GTA?AC | The T-nos terminator |
| npt-1 | TTT?CTC?GGC?AGG?AGC?AAG?G | |
| npt-2 | ACT?GGG?CAC?AAC?AGA?CAA?TC | The NPT-II selection markers |
| bar-1 | ACA?AGC?ACG?GTC?AAC?TTC?C | |
| bar-2 | ACT?CGG?CCG?TCC?AGT?CGT?A | Grass fourth phosphinothricin acetyl transferase gene |
| epsps-1 | TGT?GCT?GTA?GCC?ACT?GAT?GC | |
| epsps-2 | TGA?TGT?GAT?ATC?TCC?ACT?GAC?G | Shikimic acid-3-phosphate synthase gene |
| epsps-3 | CGA?ATA?TCC?GAT?TCT?CGC?TGT?C | |
| epsps-4 | CGC?CCT?CAT?CGC?AAT?CCA?C | Wild type CP4-EPSPS |
| epsps-5 | CGT?CTT?GAA?GGT?CGT?GGT?A | |
| epsps-6 | GCA?ACA?GCG?AGA?ATT?GGA?TA | Modification type CP4-EPSPS |
| cry-1 | TCG?ACA?TCA?GCC?TGA?GCC?TG | |
| cry-2 | TGG?TTG?ATC?AGC?TGC?TCG?ATC | Desinsection toxalbumin CryIAb gene |
| cry-3 | AGG?CTG?CCA?ACC?TGC?ACT?T | |
| cry-4 | TCA?AAG?CCC?CAC?CGT?TGT | Desinsection toxalbumin CryIAc gene |
| cry-5 | TCT?GGA?TAC?GAG?GTT?CTA | |
| cry-6 | AAA?GAC?GCT?CAA?ATT?TAT?GG | Desinsection toxalbumin CryIIIA gene |
Table 2
| The probe numbering | Probe sequence (5 '-3 ') | Target dna |
| 18s-P | TTC?AGA?CCT?CCA?CCC?TTG?AAT | The 18s rRNA |
| lec-P | CTT?CAC?CTT?CTA?TGC?CCC?TGA?CAC | Soybean lectin endogenous gene |
| inv-P | CGA?GCA?GAC?CGC?CGT?GTA?CTT?CTA?CC | Corn invertase endogenous gene |
| 35S-P3 | GGC?CAT?CGT?TGA?AGA?TGC?CTC?TGC?C | The CaMV 35S promoter |
| fmv-P | TAA?TCT?TGT?CAA?CAT?CGA?GCA | The FMV 35S promoter |
| nos-P3 | AAT?GTA?TAA?TTG?CGG?GAC?TCT?AAT?C | The T-nos terminator |
| npt-P | ACT?TCG?CCC?AAT?AGC?AGC?CAG?TCC?CTT?C | The NPT-II selection markers |
| bar-P | AGC?GCT?ATC?CCT?GGC?TCG?T | Grass fourth phosphinothricin acetyl transferase gene |
| epsps-P1 | TTC?ATG?TTC?GGC?GGT?CTC?GCG | Shikimic acid-3-phosphate synthase gene |
| epsps-P3 | GTC?TGG?AAG?AAC?TCC?GCG?TCA?AGG?AAA?GC | Wild type CP4-EPSPS |
| epsps-P5 | TGG?CTG?ACT?TGC?GTG?TTC?GTT?CTT?CTA?CTT?TG | Modification type CP4-EPSPS |
| cry-P1 | ATG?TCC?ACC?AGG?CCC?AGC?ACG | Desinsection toxalbumin CryIAb gene |
| cry-P3 | TCG?GTG?CTC?CGC?GAT?GTC?TCC | Desinsection toxalbumin CryIAc gene |
| cry-P5 | AAC?ATA?TGC?ACA?AGC?TGC?CAA | Desinsection toxalbumin CryIIIA gene |
Remarks: probe 5 ' end adds ten T and mark NH
4
Selection microscope slide (homemade sailing boat board) is a chip carrier, cleans with the absorbent cotton wiping, soaks with the dichromic acid washing lotion, with impurity such as removal surface organic matters, uses distilled water (ddH then
2O) clean; Soak 14h again in 25% ammoniacal liquor, ultrapure water cleans.With the NaOH-70% alcohol immersion 2h of 2mol/L, ultrapure water cleans, oven dry, is cooled to room temperature in the exsiccator.Slide immerses 20min in 95% ethanol (regulating pH to 4.5 with glacial acetic acid) contain aminopropyl-trimethoxy silane (APS) of 1%, 95% ethanol ultrasonic cleaning 5min, pure water ultrasonic cleaning 5min, oven dry.Slide 1 of 2g/L with silanization soaks 2h in 4-benzene diisothiocyanic acid salt (POC) solution; Methyl alcohol soaks 7min, and acetone soaks 5min, and room temperature is dried.Synthetic oligonucleotide probe (5OD) is dissolved in 40 μ L ultrapure waters earlier, adds 40 μ L, 2 * sampling liquid again, and DNA concentration is 2 μ g/ μ L.Utilize automatic point sample instrument with probe stationary on the slide of having handled, according to array pattern (16 * 4) point sample of AxSys software design, each probe repeats 4 points, the point sample volume is 10nl, diameter is 0.25 μ m, dot spacing is 0.5mm * 0.5mm.The chip room temperature that point sample finishes is placed, and 1% ammoniacal liquor cleans 5min; 0.2% SDS (lauryl sodium sulfate) cleans 5min; Pure water cleans 5min, and room temperature is dried, and 4 ℃ of preservations are standby in the exsiccator.
It is resuspended with the Cy3 dyestuff dry powder of Amersham to add 12 μ L DMSO, and the dyestuff that suspends the again amount branch with 2 μ L/ pipes is installed in the tubule, with the DNA concentration systems it is drained under the room temperature condition; Dried packing dyestuff places lighttight box, and 4 ℃ of preservations are standby in the exsiccator.Polygenes PCR reaction is by reaction system [10 * PCR damping fluid, 5 μ L, 10mmol/Ld ATP (deoxyadenosine triphosphate), dGTP (deoxidation crow guanosine triphosphate), each 0.5 μ L of dCTP (deoxycytidine triphosphate), 20mmol/L dUTP (deoxyuridine triphosphate) 0.5 μ L, 1U/ μ L AmpErase
UNG enzyme (DNA glycosylase) 1.0 μ L, 25mmol/L MgCl
25 μ L, 10 μ mol/L primer (primer), 2 * n μ L, DMSO 2 μ L, 50ng/ μ L dna profiling 1 * n μ L, 5U/ μ L AmpliTaq
TMGold archaeal dna polymerase 1 μ L adds ddH
2O to 50 μ L] and (94 ℃/5min of response procedures; 94 ℃/30s, 55 ℃/30s, 40 circulations; 72 ℃/5min) carry out.
Get polygenes PCR product in 99 ℃ of sex change 3min; According to Ready-To-Go
TMDNA labeling Beads operational manual carries out the fluorescence labeling amplification, adds 1 μ L aa-dUTP, 4 μ L TE (Trisaminomethane/ethylenediamine tetraacetic acid) damping fluids, 45 μ L sex change PCR products, 37 ℃ of incubation 1h in new reaction tube.Every pipe adds 2.5 times of volume absolute ethyl alcohol 125 μ L precipitate nucleic acids, the centrifugal 5min of 14000r; With 500 μ L, 70% ethanol washing 2 times, with 10 μ L 0.1mol/L NaHCO
3Dissolving.With NaHCO
3The polygenes PCR product of dissolving joins in the Cy3 dyestuff tubule of packing, and piping and druming is fully resuspended with dyestuff dry powder repeatedly; Open in dark place under the room temperature, hatch 60min.Use the PCR purification kit, purifying, freeze-drying is concentrated into volume required.
Each reaction tube adds 4 μ L 10mmol/L EDTA (ethylenediamine tetraacetic acid) solution, adds the mark PCR product of 1.33 μ L purifying again, and 95 ℃ of sex change 10min take out immediately and are positioned over 2min on ice, add 13.3 μ L hybridization solutions, make the hybridization mixed liquor.The chip of preparation is kept flat, drip 13 μ L hybridization mixed liquor (contain and detect template), carefully add cover glass, put in the special-purpose hybrid heater and spend the night in 55 ℃ of constant temperature lucifuge hybridization.Chip after the hybridization is used 1 * SSC (sodium chloride/sodium citrate damping fluid), 0.2 * SSC, 0.1 * SSC washing successively through the washing under the stringent condition, and each 3min is not hybridized residue to remove, the centrifugal slide 5min of 500r/m, and room temperature is dried.
Chip behind the hybridization reaction is inserted Scan Array
TMIn the detection hole of lite scanner, utilize ScanArrayExpress software to carry out result's scanning (scan intensity 50%～60%, modified value gets-5, and resolution is 10 μ m) and analyzing and processing.
Embodiment 2
Utilize PixSys
TMThe full-automatic point sample instrument of 5500 types is designed to 16 * 4 arrays, and comprise 14 and detect target-probe, 2 negative controls, every kind of probe repeats 4 points (referring to Fig. 1).One section human gene that does not almost have homology with detector probe is set as negative control on the chip, to get rid of the interference of non-specific hybridization in the crossover process.
Embodiment 3
Chip hybridization is meant that the probe on fluorescently-labeled sample and the chip reacts and produce the process of a series of information.Influence the double-stranded factor that forms of chip hybridization and comprise target concentration, hybridization solution composition and hybridization temperature etc., select the great majority in the appropriate condition energy enabling hybridization reaction to be in the optimum, that is to say correct counter pair as much as possible is not omitted, have the hybridization of mispairing to reduce to minimum.Listed hybridization program (comprising reaction conditionss such as probe modification, target concentration, hybridization temperature, hybridization time and slide washing) all has been optimized, for example probe 5 ' end carries out the amination modification, and after connecting the linking arm of 10 thymidines (T) composition, can obviously improve the fixed efficiency of probe on slide, and improve crossbreeding effect; Hybridization temperature select 55 ℃ (being lower than probe Tm value) and hybridization time selection spend the night (＞13h) can obtain desirable hybridization signal; Chip after the hybridization (is used 1 * SSC, 0.2 * SSC, 0.1 * SSC) successively, can be removed not most residues of hybridization through 3 times under stringent condition washings.According to the test condition after optimizing, use known positive criteria product and carried out the sensitivity test that LD-CHIP detects, result's demonstration, the LD-CHIP method can detect the MPCR product (referring to Fig. 2) of the GM maize dna of 0.1% GM soy bean DNA and 0.1%.
Using the LD-CHIP method of setting up detects 6 parts of known standard samples (0% and 0.3% soy meal, 0% and 0.15% corn flour, and potato S-P7 and S-P8).The result shows that 0% soy meal, 0% corn flour and potato S-P7 have only occurred corresponding interior with reference to gene recombination detection signal (as 18S rRNA, lectin or invertase); And 0.3% soy meal, 0.15% corn flour, and potato S-P8 has not only obtained interior hybridization detection signal with reference to gene, obtained special foreign gene hybridization detection signal simultaneously, be that soy meal detects CaMV 35S, Tnos and cp4epsps gene, corn flour detects CaMV 35S, Tnos, nptII and cryIAb gene, and potato detects CaMV 35S, Tnos, nptII and cryIIIA gene.
(be numbered P1～P6), (be numbered S1～S9), (be numbered unknown sample such as M1～M6) detects the LD-CHIP method of use setting up for 6 parts of corn class samples for 9 parts big beans samples to 6 parts of potato class samples respectively.The result shows, 5 parts big beans samples (being numbered S1, S2, S6, S7, S9) detect 18S rRNA, lectin, CaMV35S, Tnos and cp4epsps gene, 3 parts of corn class samples (being numbered M1, M3, M6) detect 18S rRNA, invertase, CaMV 35S, Tnos, nptII and cryIAb gene, and 2 parts of potato class samples (being numbered P3, P5) detect 18S rRNA, CaMV 35S, Tnos, nptII and cryIIIA gene (referring to Fig. 3); All the other 11 duplicate samples have only corresponding interior with reference to the gene recombination detection signal.In Fig. 3, A (0.3% genetically engineered soybean standard items); B (S1): 18SrRNA, CaMV 35S, Tnos, lectin and cp4epsps-1/-2 be the positive as a result; C (0.15% transgenic corns standard items); D (M1): 18S rRNA, CaMV 35S, Tnos, nptII, invertase and cryIAb-1/-2 be the positive as a result; E (S-P8) and F (P3): 18S rRNA, CaMV 35S, Tnos, nptII and cryIIIA be anode as a result.
Claims (3)
1, the low density gene chip detection method of transgenic product is characterized in that the steps include:
1) selects primer and probe;
2) process chip carrier: select chip carrier, through clean, drying, silanization again with 1, soaks the chip carrier of silanization in the 4-benzene diisothiocyanic acid salt solusion, dry;
3) preparation chip: probe is dissolved in ultrapure water, adds sampling liquid, on the chip carrier of having handled, point sample is placed, and cleans with probe stationary, dries, and drying, standby;
4) polygenes pcr amplification: the adding dimethyl sulfoxide (DMSO) is resuspended with single function fluorescent dye Cy3 dry powder, and the dyestuff branch that suspends is again installed in the tubule, with the DNA concentration systems it is drained, and drying is carried out polygenes PCR reaction;
5) MPCR product mark: get the sex change of polygenes PCR product, carry out the fluorescence labeling amplification, in reaction tube, add damping fluid, sex change PCR product, every pipe adds the absolute ethyl alcohol precipitate nucleic acids, and is centrifugal, and NaHCO is used in washing
3Dissolving is with NaHCO
3The polygenes PCR product of dissolving joins in the Cy3 dyestuff tubule of packing, and dyestuff dry powder is resuspended, places, and hatches, and uses the PCR purification kit, and purifying, freeze-drying are concentrated into volume required;
6) chip hybridization of MPCR product test: each reaction tube adds edta solution, the mark PCR product that adds purifying again, sex change is taken out and is positioned on ice the adding hybridization solution, make the hybridization mixed liquor, the chip of preparation is dripped the hybridization mixed liquor, put to hybridize in the hybrid heater and spend the night, the chip after the hybridization is through washing, centrifugal, dry;
7) scanning analysis and data processing: the chip behind the hybridization reaction is inserted in the detection hole of scanner, carry out result's scanning and analyzing and processing.
2, the low density gene chip detection method of transgenic product as claimed in claim 1 is characterized in that said primer selects primer sequence (5 '-3 ') for use:
TAA?TAG?AGC?AAT?GAA?CAG?TCG?G
CTT?TCA?ACA?ACG?GAT?CTC?TTG?G
GTG?CTA?CTG?ACC?AGC?AAG?GCA?AAC?TCA?GCG
GAG?GGT?TTT?GGG?GTG?CCG?TTT?TCG?TCA?AC
GGC?CGG?ATC?GTC?ATG?CTC?TAC?A
TTG?GCG?TCC?GAC?TTG?ACC?CAC?T
GAT?TCC?ATT?GCC?CAG?CTA?TCT?G
TAG?AGG?AAG?GGT?CTT?GCG?AAG?G
AGT?CCA?AAG?CCT?CAA?CAA?GGT?C
CAT?TAG?TGA?GTG?GTC?TGT?CAG?G
GAA?TCC?TGT?TGC?CGG?TCT?TG
CGA?ATT?CCC?GAT?CTA?GTA?AC
TTT?CTC?GGC?AGG?AGC?AAG?G
ACT?GGG?CAC?AAC?AGA?CAA?TC
ACA?AGC?ACG?GTC?AAC?TTC?C
ACT?CGG?CCG?TCC?AGT?CGT?A
TGT?GCT?GTA?GCC?ACT?GAT?GC
TGA?TGT?GAT?ATC?TCC?ACT?GAC?G
CGA?ATA?TCC?GAT?TCT?CGC?TGT?C
CGC?CCT?CAT?CGC?AAT?CCA?C
CGT?CTT?GAA?GGT?CGT?GGT?A
GCA?ACA?GCG?AGA?ATT?GGA?TA
TCG?ACA?TCA?GCC?TGA?GCC?TG
TGG?TTG?ATC?AGC?TGC?TCG?ATC
AGG?CTG?CCA?ACC?TGC?ACT?T
TCA?AAG?CCC?CAC?CGT?TGT
TCT?GGA?TAC?GAG?GTT?CTA
AAA?GAC?GCT?CAA?ATT?TAT?GG
3, the low density gene chip detection method of transgenic product as claimed in claim 1 is characterized in that said probe selects probe sequence (5 '-3 ') for use:
TTC?AGA?CCT?CCA?CCC?TTG?AAT
CTT?CAC?CTT?CTA?TGC?CCC?TGA?CAC
CGA?GCA?GAC?CGC?CGT?GTA?CTT?CTA?CC
GGC?CAT?CGT?TGA?AGA?TGC?CTC?TGC?C
TAA?TCT?TGT?CAA?CAT?CGA?GCA
AAT?GTA?TAA?TTG?CGG?GAC?TCT?AAT?C
ACT?TCG?CCC?AAT?AGC?AGC?CAG?TCC?CTT?C
AGC?GCT?ATC?CCT?GGC?TCG?T
TTC?ATG?TTC?GGC?GGT?CTC?GCG
GTC?TGG?AAG?AAC?TCC?GCG?TCA?AGG?AAA?GC
TGG?CTG?ACT?TGC?GTG?TTC?GTT?CTT?CTA?CTT?TG
ATG?TCC?ACC?AGG?CCC?AGC?ACG
TCG?GTG?CTC?CGC?GAT?GTC?TCC
AAC?ATA?TGC?ACA?AGC?TGC?CAA
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITRM20080673A1 (en) * | 2008-12-17 | 2010-06-18 | Fond Parco Tecnologico Padano | MICROARRAY FOR THE IDENTIFICATION OF THE PRESENCE OR ABSENCE OF GMOs IN SAMPLES INCLUDING VEGETABLE MATERIAL. |
| CN102879574A (en) * | 2012-10-23 | 2013-01-16 | 吉林省农业科学院 | Bar/pat gene transfer plant and rapid test strip for derivative product of bar/pat transgenic plant |
| CN109182370A (en) * | 2018-08-03 | 2019-01-11 | 浙江大学 | A kind of plant polygene expression vector, transformant and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1099596C (en) * | 1998-05-08 | 2003-01-22 | 上海市儿童医院上海医学遗传研究所 | Method for detecting exogenous origin gene integrator animal embryon |
| WO2003027283A1 (en) * | 2001-09-21 | 2003-04-03 | National Food Research Institute | Competitive nucleic acid fragment, kit for quantifying recombinant gene and method of quantifying recombinant gene using the same |
| CN1247796C (en) * | 2002-09-04 | 2006-03-29 | 中华人民共和国上海出入境检验检疫局 | Quantitative measuring transgene component in transgene rapeseed and processed product |
| CN1485435A (en) * | 2002-09-24 | 2004-03-31 | 深圳市匹基生物工程股份有限公司 | Probe sequence for quantitatively detecting transgenic corn containing CrylA(b) gene using fluorescence PCR and reagent case |
| CN1420181A (en) * | 2002-10-11 | 2003-05-28 | 覃文 | Probe sequence and kit for real time fluorescence PCR detection of transgenic rapeseed |
| CN1438328A (en) * | 2003-03-08 | 2003-08-27 | 杭州博日科技有限公司 | Quantitative detection method for transgenic farm product and food mixture |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITRM20080673A1 (en) * | 2008-12-17 | 2010-06-18 | Fond Parco Tecnologico Padano | MICROARRAY FOR THE IDENTIFICATION OF THE PRESENCE OR ABSENCE OF GMOs IN SAMPLES INCLUDING VEGETABLE MATERIAL. |
| WO2010070462A1 (en) * | 2008-12-17 | 2010-06-24 | Fondazione Parco Tecnologico Padano | Microarray for the identification of the presence or absence of gmo in samples comprehensive of vegetable material |
| CN102879574A (en) * | 2012-10-23 | 2013-01-16 | 吉林省农业科学院 | Bar/pat gene transfer plant and rapid test strip for derivative product of bar/pat transgenic plant |
| CN109182370A (en) * | 2018-08-03 | 2019-01-11 | 浙江大学 | A kind of plant polygene expression vector, transformant and its application |
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