CN1584049A - Preparing method for tr-gene products detecting oligonucleotides chip and use thereof - Google Patents

Abstract

A method for preparing oligonucleotide chip for transgene product detection and use are disclosed. It includes: designing specific oligonucleotide probe and related primer by heterogeneric inserting gene, species internal standard gene, specific boundary sequence in transgene product gene set, fixing probe on glass slide to form transgene product detecting chip, amplifying DNA of plant to be tested by related primmer, marking by fluorescence, and hybridizing with chip. It can be use to acquire variable information.

Description

Transgenic product detects the making method and the application thereof of oligonucleotide chip

Technical field

The present invention relates to a kind of nucleic acid detection technique, particularly relate to a kind of making method and application thereof that is used for the oligonucleotide chip of transgenic product detection.

Background technology

Fast development along with biotechnology, by heterologous gene being imported the genome of natural phant, realization is to the transformation of its original gene, has the new variety of plant that high nutrition, high resistance to cold and diseases etc. are optimized proterties in the hope of obtaining, and and then to improve with it be the quality of the food and/or the feed of raw material, be at present to study widely and use.

Use consultative centre (ISAAA) statistics according to international Agricultural biotechnologies, calendar year 2001, transgenic product cultivated area in the whole world was 5,260 ten thousand hectares, existing more than ten country in the whole world planted transgenic product, wherein 99% transgenic product is planted in the U.S., Argentina, Canada and Chinese, and the business-like transgenic product of approved has soybean, corn, rape, cotton, tomato, potato etc.According to estimates, the food whole world with these transgenic product process for processing has nearly ten thousand kinds.

Meanwhile, the plant that this class obtains by transgenic technology and/or be that the food of raw material is to the health of humans and animals and the influence of ecotope with it, since transgenic technology occurs, just be the focal issue that international organizations such as countries in the world and United Nations are concerned about always.

United Nations had passed through " Biosafety Protocol " in 2000, required member states that all transgenic product of passing in and out are tested.The various laws and regulations relevant with transgenic product have been put into effect in now existing 36 countries and regions, the whole world.The Chinese government also pays much attention to the detection of transgenic product, the Ministry of Science and Technology had ratified State General Administration for Quality Supervision's " foundation of national transgenic product test experience chamber and transgenic product detection method system " special project in 2000, was devoted to the research of transgenic product detection method and instrument.

Import and export the detection of transgenic product, need to solve the problem of following several respects: 1. whether be transgenic product; 2. whether be business-like transgenic product of certain state's approved or strain (EVENT, promptly by which kind of transgenic product of which manufacturers produce) if 3. satisfy above-mentioned two simultaneously, whether the content that then also needs further to measure transgenosis composition in this product is in " threshold value " (threshold criteria of various countries differs).If though this product is not at the row of the business-like transgenic product of the Government's approved or belong to approved products but content overproof, then import prohibition or sale in principle.

Scan the detection technique of countries in the world transgenic product, can be divided into following three classes according to the difference of object to be checked: 1. the allos that detects in the transgenic product is inserted gene; 2. detect expression of heterologous genes thing (RNA or protein); 3. detect to insert heterologous gene to the influence of receptor biological genetic expression (mainly detect heterologous gene inserts near the influence of the genetic expression site to it and to the influence of whole receptor biological meta-bolites).Because detection cost height, the length consuming time of the third method, so use preceding two kinds of methods at present in real work, its detected object comprises: DNA, RNA and protein more.Wherein, proteinic detection is mainly by serological method, because some transgenic product not marking protein or expression amount instability, so serological method only can be used in the detection of transgenic product individually; The detection of DNA and/or RNA is then mainly adopted gene amplification diagnostic techniquess such as PCR or direct nucleic acid hybridization.

The development of gene amplification diagnostic techniques can be divided into following a few class so far: PCR/ electrophoresis detection, conventional hybridization or order-checking, PCR-ELISA, real-time fluorescence PCR etc.Aforesaid method all is to detect term single gene, and the detection of transgenic product, particularly some biased samples such as mixing raw material, food and feed etc. often need to detect simultaneously several, tens even up to a hundred genes, and aforesaid method obviously can't satisfy the demands.

Gene chip is an emerging technology that occurs in the U.S. late 1980s.Its principle is similar to Southern hybridization, promptly in advance particular procedure is carried out on upholder (substrate) surface and makes its lotus that becomes positively charged, and oligonucleotide fragment or cDNA are sprayed with high-precision point sample instrument or puts substrate surface and makes gene chip (6.5cm ²Can hold 100000 nucleic acid sites in the scope).Use fluorescently-labeled target nucleic acid to be checked and chip hybridization again, carry out the detection of hybridization signal at last with special high precision scanner.

According to the difference of fixed probe length on the gene chip, it can be divided into oligonucleotide chip (oligonucleotide probe array) and cDNA chip two classes.The former with oligonucleotide fragment as probe, the latter then with long PCR product as probe.Different purposes according to gene chip can be divided into it chip of expression spectrum and diagnostic detection chip, and chip of expression spectrum is mainly used in differential expression, the new gene of searching and the research gene function that detects gene; The diagnostic detection chip then is widely used in the detection of detection, transgenation and the single nucleotide polymorphism of various specific gene sequence, and human is also arranged, and it carries out gene sequencing.

Compare with common hybridizing method, the advantage of gene chip is: by using the point sample instrument and the scanner of pinpoint accuracy, once experiment just can detect tens simultaneously, hundreds of even thousands of genes, promptly have the characteristics of " high-throughput "; Improved the sensitivity and the tolerance range that detect, the result judges more science; Improve detection speed and level of automation, reduced experimental cost.

China utility model patent ZL 01214517.3 discloses a kind of transgenic product genes identified detection chip that is used for, promptly on the plane of flush type prop carrier, be coated with the rete of positively charged material, be placed with the heterologous gene probe of judging usefulness for transgenic product in the microarray mode on the surface of this rete.But do not provide the information of any related detection probe sequence and target gene amplimer to be checked, so can't be applied in the real work at its specification sheets at all.

Therefore, develop a kind of sensitivity, it is accurate, easy and simple to handle that to be used for oligonucleotide chip that transgenic product detects be to solve the task of top priority that domestic at present and even international transgenic product is identified a difficult problem.

Summary of the invention

One of the object of the invention is to fill up the blank of prior art, provides a kind of and can be used for the oligonucleotide chip (oligonucleotide probe array) that multiple transgenic product detects;

Another object of the present invention provides the making method of said chip;

Another object of the present invention provides the corresponding specificity amplification primer of target gene to be checked with above-mentioned various plants;

Another object of the present invention provides the application of said chip in detecting transgenic product.

Design of the present invention is as follows:

1. the gene constructed figure and the corresponding allos of collecting multiple commercial transgenic product by nucleic acid databases such as government and international organization website, GENEBANK, patent database is inserted gene, and replenishes and verify by clone and order-checking.These allos are inserted genes can be divided into two classes: 1. promotor, terminator and marker gene; 2. character gene (target gene can change the allos nucleotide fragments of plant trait).Insert corresponding probe of gene design and primer according to these allos.

2. collect the specificity species internal standard gene of five kinds of crops such as cotton, soybean, rape, corn, potato, design corresponding oligonucleotide probe and primer in view of the above.

3. even the transgenic product of producing in view of different vendor contains identical allos insertion gene, it inserts the site and also has nothing in common with each other, so the insertion site (detect these and insert segmental specificity border sequence) by contained heterologous gene in the detection transgenic product genome can identify the product which manufacturer is this transgenic product be (strain, EVENT).The inventor has measured the specificity border sequence of 8 kinds of genetically modified crops such as Bt9, Bt176, Bt11, Mon810, T25, GA21, RRS (Roundup-ready soybean), " luxuriant No. one of China " then, and designs corresponding oligonucleotide probe and primer in view of the above.

4. be separately fixed at above-mentioned all kinds of probes on the different slides or be fixed in the different zones of a slide, just can reach purpose multiple transgenic product is screened, kind detects and strain is identified.

The following allos of using in this specification sheets and claims is inserted the abbreviation of gene title and is had following implication unless stated otherwise:

CaMV35s terminator: cauliflower mosaic virus (CaMV) 35S terminator;

CaMV35S promotor: cauliflower mosaic virus (CaMV) 35S promoter;

CaMV CP: cauliflower mosaic virus (CaMV) glutelin;

EPSPS: derive from edaphic bacillus Agrobacterium, its coded product be the rare pure pyruvoyl oxalic acid of mutant 5--3-phosphate synthase (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS);

Cry I A (b): Bacillus thuringiensis toxoprotein gene IA (b) derives from Bacillus thuringiensis Bacillus thuringiensis subsp.Kurstaki;

Cry I A (c): Bacillus thuringiensis toxoprotein gene IA (c) derives from Bacillus thuringiensis Bacillus thuringiensis subsp.Kurstaki;

Cry9C: Bacillus thuringiensis toxoprotein gene 9C derives from Bacillus thuringiensis Bacillus thuringiensis subsp.Kurstaki;

CryIIIA: Bacillus thuringiensis toxoprotein gene IIIA derives from Bacillus thuringiensis Bacillus thuringiensis subsp.Kurstaki;

Nos promotor: Ti cigarette fat alkali synthase promoter;

Nos terminator: Ti cigarette fat alkali synthetic enzyme terminator;

Bar: derive from streptomycete Streptomyces viridochromogenes, its coded product is careless fourth phosphorus Transacetylase;

Barnase: derive from bacillus amyloliquefaciens Bacillus amyloliquefaciens, its coded product is a male sterile albumen;

Barstar: derive from bacillus amyloliquefaciens Bacillus amyloliquefaciens, its coded product is a fertility restorer albumen;

FMV35S promotor: radix scrophulariae class plant 35S promoter (35S promoter from a modified figwort mosaicvins);

Gox: derive from bacterium Ochrobactrum anthropi, its coded product is the glyphosate oxydo-reductase that head is modified;

Npt II: derive from intestinal bacteria E.coli, its coded product is a neomycin phosphotransferase;

PAT: derive from streptomycete Streptomyces viridochromogenes, its coded product is careless fourth phosphorus Transacetylase;

Rbcl: its coded product is 1, the big subunit of 5-Diphosphonate carboxylase;

Lectin: its coded product is a phytohaemagglutinin;

Fbp: its coded product is a chloroplast(id) fructose-1, the 6-Diphosphonate;

Zein: its coded product is a zein;

Wherein, the allos to be checked that is used for transgenic product " general sieve " (judging whether this plant is transgenic product) is inserted gene and is comprised: CaMV35S promotor, Nos terminator, Nos promotor, CAMV35S terminator, FMV35S promotor, Bar, PAT, Npt II, CaMV-CP.Wherein, CaMV-CP is used to judge whether the CaMV35s promotor positive is because sample to be checked has been subjected to due to the cauliflower mosaic virus pollution, in case false-positive appearance.

The allos insertion gene to be checked and the species internal standard gene that are used for transgenic product " kind detection " comprise:

Genetically engineered soybean: Lectin, CaMV35S promotor, Nos terminator, EPSPS (soybean);

Transgenic corns: Zein, CaMV35S promotor, CaMV35s terminator, Nos terminator, Bar, Cry9C, PAT, CryIA (b) 1;

Transgene cotton: CaMV35S promotor, Nos terminator, NptII, CryI A (c);

Transgene rape: Fbp, CaMV35S promotor, Nos terminator, FMV35S promotor, EPSPS (rape), GOX, Bar, PAT, Barnase, Barstar.

The specificity border sequence that is used for transgenic product " strain evaluation " is as follows: BT9

GTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTC

GCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGTCATT

TCTCTATTACTTCAGCCATAACAAAAGAACTCTTTTCTCTOTCTTATAAACCAAAAACC

ATGGCTGACTACCTGCAGATGACCGACGAGGACTACACCGACAGCTAC??<210>94

Partly for inserting the fragment 35S promoter, another partly is Bt9 genome sequence (Petunia CTP Leadingsequence) to underscore.

BT176

TTTCCTCCCGATACGCCCCTCCATCTCTCGCCGTTCATGTCCGTGGCTGGCTGCCCTCC

GTGGGAGCAGGCTGGCCGCACTCGTTCCCCGCCGCAGCCGGATCCAACA ATGGACAA

CAACCCCAACATCAACGAGTGCATCCCCTACAACTGCCTGAGCAATCCCGAGGTGGA

GGTGCTGGGCGGCGAGCGCATCGAGACCGGCTACACCCCCATCGACATCAGCCTGAG

CCTGACCCAGTTCCTGCTGAGCGAGTTCGTGCCCGGCGCCGGCTTCGTGCTGGGCCTG

GTGGACATCATCTGGGGCATCTTCGGCCCCAGCCAGTGGGACGCCTTCCTGGTGCAG

ATCGAGCAGCTGATCAACCAGCGCATCGAGGAGTT<210>95

Partly for inserting fragment Cry I A (b), another partly is a BT176 genome sequence (P-CDPK) to underscore.

GA21

ATGGTGGCTCCGTTCACCGGCCTTAAGTCCAACGCCGCCTTCCCCACCACCAAGAAGG

CTAACGACTTCTCCACCCTTCCCAGCAACGGTGGAAGAGTTCAATGTATGCAGGTGTG

GCCGGCCTACGGCAACAAGAAGTTCGAGACGCTGTCGTACCTGCCGCCGCTGTCTAT

GGCGCCCACCGTGATGATGGCCTCGTCGGCCACCGCCGTCGCTCCGTTCCAGGGGCTC

AAGTCCACCGCCAGCCTCCCCGTCGCCCGCCGCTCCTCCAGAAGCCTCGGCAACGTCA

CGCAACGGCGGAAGGATCCGGTGC ATGGCCGGCGCCGAGGAGATCGTGCTGCAGCCC

ATCAAGGAGATCTCCGCGGCACCGTCAAGCTGCCGGGGTCCAAGTCGCTTTCCAACCGG

ATCCTCCTACTCGCCGCCCTGTCCGAGGGGACAACAGTGGGTTGATAACCTGCTCGAAC

<210>96

Partly for inserting fragment EPSPS (Corn EPSPS), another partly is GA21 genome sequence (Corn Ctp) to underscore.Mon810

TCGAAGGACGAAGGACTCTAACGTTTAACATCCTTTG CCATTGCCCAGCTATCTGTCAC

TTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATCCCATCATTGCGATAA

AGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCC

ACCCACGAGGAGCATCGTGGA<210>97

Underscore partly is an insertion sequence, and another part is a MON810 corn gene group sequence.BT11

GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG

AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTG

CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA

ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA

AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGA

ACGAAAACTCACGTTAAGGGATTTTGGTCATGGAAGTCGATGATA????<210>98

Underscore partly is an insertion sequence, and another part is a BT11 corn gene group sequence.

T25

AAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACG

TCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATC

CCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGA

CAGGGTACCCGGGGATCCTCTAGAGTCGACATGTCTCCGGAGAGGAGACCAGTTGAGA

TTAGGCCAGCTACAGCAGCTGATAGGCCGCGGTTTGTGATATCGTTAACCATTACATTG

AGACGTCTACAGTGAACTTTAGGACAGAGCCACAAACACCACAAGAGTGGATTGATG

ATCTAGAGAGGTTGCAAGATAGATACCCTTGGTTGGTTGCTGAGGTGGAGGGTGTTGT

GGGCTGGTATTGCTTACGCTGGGC?????<210>99

Underscore partly is a 35S promoter, and another partly is PAT.

RRS(Roundup?Ready?soy)

GATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCT

TTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGTGCTCCTCGTGGGTGGGGGTCCA

TCTTTGGGACCACTGTCGGCAGAGGCATCTTCAACGATGGCCTTTCCTTTATCGCAATG

ATGGCATTTGTAGGAGCCACCTTCCTTTTCCATTTGGGTTCCCTATGTTTATTTAACCT

GTATGTATGATCTTATTTTGAATGAAATGCAATAAGTTATTTCTAGTAAAAAAAAATAAA

CATTTGATAGAAACAAATTAAAGCATGCAAAAATAACTCATTAGCATCGGTTAAATTGA

AGGGTTTGAATAATTTGCACAAGGTTCTGAATTC????<210>100

Underscore partly is a 35S promoter, and another part is the RRS genome sequence.

Luxuriant No. one of China

CAGAGACTAAAAGGAAGACCATTGATATTCTTGCAAACTGCATTTAAGGAGGCAATAA

AGTAGGGACTTTACACTTGCATATTCTTTAAAAACAGACAAAAAT AGGCGGGAAACGA

CAATCTGATCATGAGCGGAGAATTAAGGGAGTCACGTTATGACCCCCGCCGATGACGC

GGGACAAGCCGTTTTACGTTTGGAACTGACAGAACCGCAACGTTGAAGGAGCCACTC

AGCCGCGGGTTTCTGGAGTTTAATGAGCTAAGCACATACGTCAGAAACCATTATTGCGC

GTTCAAAAGTCGCCTAAGGTCACTATCAGCTAGCAAATATTTCTTGTCAAAAATGCTCC

ACTGACGTTCCATAAATTCCCCTCGGTATCCAATTAGAGTCTCATATTCACTCTCAATCC

AAATAATCTGCACCGGATCTGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCA

GGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAA

<210>101

Underscore partly is an insertion sequence, and another partly is tomato " luxuriant No. one of a China " genome sequence.

Technical scheme of the present invention is as follows:

The present invention's transgenic product detects oligonucleotide chip and comes down to a kind of oligonucleotide probe array, according to the poly-probe of the different 15-35 of the different designs of plant target gene to be checked, and makes the Tm value (melting temperature(Tm)) of these probes basic identical.These probes include but not limited to:

Allos is inserted gene	Probe	Length	???Tm
				The CaMV35S promotor	????TGCTCCACCATGTTGACGAAG<210>1	???21	??61.6
The FMV35S promotor	????AAAACCAAGAAGGAACTCCCATC<210>4	???23	??60.7
				The CAMV35 terminator	??GAAACCCTTAGTATGTATTTGTATTTGTAA<210>7	???30	??60.0
The Nos promotor	????GACCTTAGGCGACTTTTGAACG<210>10	???22	??60.9
				The Nos terminator	????AATCCTGTTGCCGGTCTTGC<210>13	???22	??62.3
?????NptII	????GCTCCTGCCGAGAAAGTATCC<210>16	???21	??60.6
				?????Bar	????CTGTGCCTCCAGGGACTTCA<210>19	???20	??60.5
?????PAT	????GCCACAACACCCTCAACCTCA<210>22	???21	??62.8
				????Barnase	????GTCTGAAGATATCCGCAACCC<210>25	???22	??60.4
????Barstar	????ACCTGGACGCTTTATGGGATT<210>28	???21	??60.3
				EPSPS (soybean)	????GGAAAGGCCAGAGGATTTGC<210>31	???20	??61.1
EPSPS (rape)	????GGGTCTTGTTGGTGTTTACGATT<210>34	???23	??60.1
				??Cry?I?A(b)	?????CAGCACGGGGTTGGTGTAGAT<210>37	???21	??62.1
??Cry?I?A(c)	?????TCTGGTAGATGTGGATGGGAAGT<210>40	???23	??60.1
				???CryIIIA	?????AAGCTGCCAACACCCACTTG<210>43	???20	??60.8
????Cry9C	???CAAGTACACCAACTACTGCGAGACC<210>46	???25	??62.3
				????GOX	????GTTCTTCAGAACTGGCAGGAGC<210>49	???22	??60.7
????CaMV-CP	????TATGACAACTACCGACGACTCGA<210>52	???23	??60.0
				The plant species internal standard gene	Probe	Length	???Tm
????Lectin	?????TCTATCAGATCCATCAAAACGACG<210>55	???24	??61.1
				????Zein	?????TTCTGGCTCTAACTTGGTTCCG<210>58	???22	??61.3
????Fbp	?????TGCTACTCGTTTCCCGCTCA<210>61	???20	??61.7

Rbcl (cotton)	????GGTGGGCTTGATTTTACCAAAG<210>64	???22	???60.7
				Rbcl (potato)	????TGTCTTCGAGGTGGACTTGATTT<210>67	???23	???60.5
Plant distinguished boundary sequence	Probe	Length	????Tm
				?????Bt9c	????AGAGAAATGACCCCGTGTTCTC<210>70	???22	???60.0
?????Bt176	?????GGGGTTGTTGTCCATTGTTGG<210>73	???21	???62.2
				?????GA21	???????GATCCGGTGCATGGCCG<210>76	???17	???63.2
?????Mon810	?????GGGCAATGGCAAAGGATGTT<210>79	???20	???62.1
				?????BT11	????GACGCTCAGTGGAACGAAAACT<210>82	???22	???61.0
?????BT25	????CTCCGGAGACATGTCGACTCTAG<210>85	???23	???60.6
				?????RRS	???AAAATAAACATAGGGAACCCAAATG<210>88	???25	???60.5
Luxuriant No. one of China	?????GATTACGAATTCCCATGGAGTC<210>91	???22	???60.0

By point sample instrument above-mentioned probe is transferred on the slide that chemical group is modified, handled and make through SDS, pure water.The slide modification group can be selected from one of aldehyde radical, isothiocyano or sulfydryl.

In addition, in order to monitor the non-specific hybridization in the hybridization, ad hoc upright negative control, this negative control are one section oligonucleotide fragment (probe) that has nothing to do with target gene to be checked; This oligonucleotide fragment can be selected from the genome sequence of the mankind, microorganism etc.If no signal is detected in the hybridization back, show normal; If signal is arranged, show the hybridization failure.

In order to monitor false-negative appearance in the hybridization, ad hoc upright positive control, this positive control be one section with 18SrRNAPCR product paired oligonucleotide fragment (probe) mutually; Simultaneously, this oligonucleotide fragment is used for determining the position of probe array hybridization region also as the locating point of probe array; If the hybridization back is detected signal is arranged, show normal; If no signal shows the hybridization failure.

In order to reduce the sterically hindered of hybridization, when synthetic, add one section polyT (connecting arm) at 5 ' end of oligonucleotide fragment, the length of connecting arm is 5-20 when poly-, crossbreeding effect is good.At last, for oligonucleotide fragment can be fixed on the slide.5 ' end at oligonucleotide fragment must carry out the chemical group modification.When the slide modification group is isothiocyano or aldehyde radical, adopt amido modified; When the slide modification group is sulfydryl, adopt sulfydryl modification.

Above-mentioned synthetic good probe is dissolved in the Na of 0.1M pH9.0 ₂CO ₃Make the sampling liquid that final concentration is 10 μ M in the/NaHCO3 solution; Set up the sampling liquid that does not contain oligonucleotide fragment simultaneously,, be used to monitor the background after the hybridization as blank.If no signal is detected in the hybridization back, show normal; If signal is arranged, show that sampling liquid is contaminated.

Sampling liquid is added in the point template, becomes desired matrix with the point sample instrument point, place 1-2h for 37 ℃, 1% ammoniacal liquor washes once, dries standby.

The making method of transgenic product detection chip of the present invention is as follows:

1. the modification of slide glass;

2. probe design reaches synthetic;

3. probe is fixing.

Concrete steps comprise:

1. the modification of slide glass

According to the difference of modification group, can adopt three kinds of methods to prepare slide:

1) the PDC legal system is equipped with slide

Clean slide is dipped in 2min in 95% acetone soln that contains 1%3-aminopropyl trimethyl silane, acetone embathes 5min, repeat 6 times, 110 ℃ of baking 45min, again slide is dipped in and contains 0.2%1,2h in 10% pyridine of 4-phenylene diisocyanate/DMF solution, acetone soln washes once, room temperature is dried, be put in 4 ℃ standby;

2) glutaraldehyde method prepares slide

Clean slide is dipped in to boil in the 1N NaOH solution carried out the hydroxylation pre-treatment in 10 minutes.Pretreated slide is dipped in 2min in 95% acetone soln that contains 1%3-aminopropyl trimethyl silane, and acetone embathes 5min, repeats 6 times, and 110 ℃ of baking 45min are dipped in slide in 5% glutaraldehyde water solution and spend the night under 50 ℃ of conditions.Take out slide, with 0.2%SDS washing 5 minutes, repeat 3 times, room temperature is dried standby;

3) the disulfide linkage legal system is equipped with slide

With 25% ammoniacal liquor soaked overnight, the MilliQ water logging is given a baby a bath on the third day after its birth minute with clean slide, and dehydrated alcohol washes once, under the room temperature slide is immersed in 1%MPTS, 95% ethanol, the 16mM acetic acid solution 30 minutes, and 95% ethanol, 16mM acetic acid solution wash once N ₂Jar in 6～8 hours.

2. probe design reaches synthetic

The poly-oligonucleotide probe of design 15-35 at difference target gene to be checked, and will adjust to basic identical at the Tm value (melting temperature(Tm)) of different probe as far as possible.

In addition, in order to monitor the non-specific hybridization in the hybridization, ad hoc upright negative control, this negative control are one section oligonucleotide fragment (probe) that has nothing to do with target gene to be checked; This oligonucleotide fragment can be selected from the genome sequence of the mankind, microorganism etc.;

In order to monitor false-negative appearance in the hybridization, ad hoc upright positive control, this positive control be one section with 18S rRNAPCR product paired oligonucleotide fragment (probe) mutually; Simultaneously, this oligonucleotide fragment is used for determining the position of probe array hybridization region also as the locating point of probe array;

In order to reduce the sterically hindered of when hybridization, when synthetic, add the preceding paragraph connecting arm (polyT) at 5 ' end of oligonucleotide fragment, the length that experiment showed, connecting arm is 5-20 when poly-, crossbreeding effect is good.In addition, can be fixed on the slide, also need to carry out chemical group and modify at its 5 ' end in order to make oligonucleotide probe.When the slide modification group is isothiocyano or aldehyde radical, adopt amido modified; When the slide modification group is sulfydryl, adopt sulfydryl modification.

3. probe is fixing

Synthetic above-mentioned probe (synthetic) by TaKaRa company, press institute to the nmol number, add the stock solution that TE (PH7.5) is made into 100uM, get 10ul with 5 times of TE (PH7.5) dilutions, be made into 20uM concentration 50ul (after the most handy GeneQuant instrument is quantitative concentration being adjusted unanimity), therefrom get 20ul and 20ul 0.1M PH9.0 Na ₂CO ₃/ NaHCO ₃It is the sampling liquid 40ul of 10uM that the damping fluid two-fold dilution becomes final concentration.Setting up the sampling liquid that does not contain oligonucleotide fragment simultaneously is blank, is used to monitor the background after the hybridization.

Get each 8ul of above-mentioned sampling liquid in 384 orifice plates, become desired matrix with the point sample instrument point.Each bar probe repeats point sample 5 times, dot spacing 500um.Place 1-2h for 37 ℃, 1% ammoniacal liquor washes once, dries standby.

The application method of transgenic product detection chip of the present invention is as follows:

1. design corresponding pcr amplification primer according to target gene to be checked, be respectively:

The target gene title	Primer sequence (5 '-3 ')	???Tm	Product length (bp)
				CaMV 35S promotor	???AGACTGGCGAACAGTTCATACAGA<210>2	??60.8	????188
?????GCAATGGAATCCGAGGAGGT<210>3	??61.1
		The FMV35S promotor	????CAGCATTCCAGATTGGGTTCA<210>5		??60.7	????172
????CTTTTGGCTAATGGTTTGGAGAC<210>6	??60.1
			The CaMV35s terminator	????CCCAGATAAGGGAATTAGGGTTC<210>8	??60.5		????134
????CCCTGGATTTTGGTTTTAGGAAT<210>9	??60.9
		The Nos terminator		????TGAATCCTGTTGCCGGTCTT<210>13	??60.2	????138
???AAATGTATAATTGCGGGACTCTAATC<210>14	??60.1
			???NptII	?????GGAAGGGACTGGCTGCTATTG<210>17	??61.3		????157
?????GATGTTTCGCTTGGTGGTCG<210>18	??61.1
		The Nos promotor		?????GAACCGCAACGATTGAAGGA<210>11	??60.8	????177
????TGGATACCGAGGGGAATTTATG<210>12	??60.7

??Cry?I?A(b)	??????TGTTCCCCAACTACGACTCCA<210>38	??60.2	???151
				??????AGGTGCGGGCTCCTGATGCT<210>39	??66.5
	??Cry?I?A(c)	?????AGACTCAACAGCAGTGGAAATAACA<210>41				??60.2	???123
?????GTGAATAGGGGTCACAGAAGCATA<210>42			??60.3
		????Cry9C		???????CTGCTGCTGCTGAAGGATGC<210>47	??61.7	???199
???????CCCTGCGGAACTGGTGGTAG<210>48	??62.6
			??CryIIIA	?????GGTTCTTTTCCTCACTACCTATGCTC<210>44	??61.6		???101
??????CCTCTTTCTCGTATCCCCACTCTT<210>45	??62.3
		????GOX		???????AGTTCGCTGGTCTCACTGCTG<210>50	??60.3	???133
???????GGACGGAAACCCATCCACTT<210>51	??60.9
			EPSPS (soybean)	??????AGAGCCGTGGATAGATTAAGGAAG<210>32	??60.6		???149
????????AGACCGCCGAACATGAAGGA<210>33	??62.6
		EPSPS (rape)		???????GGGTCTTGTTGGTGTTTACGATT<210>35	??60.1	???182
????????TGTAGGTGATTGGCGTTGGAG<210>36	??60.9
			???Barnase	???????TCCGTTTGTTTACTGGTGCTTG<210>26	??60.5		???192
???????GAGGGCTTGTGCTTCTGATTTT<210>27	??60.4
		???Barstar		?????AACAAATCAGAAGTATCAGCGACCT<210>29	??60.9	???145
???????AACTGCCTCCATTCCAAAACG<210>30	??61.6
			????Bar	???????GCTCCACGCTCTACACCCAC<210>20	??60.2		???181
???????AAACCCACGTCATGCCAGTT<210>21	??60.0
		????PAT		???????GACAGAGCCACAAACACCACAA<210>23	??60.7	???144
???????CAATCGTAAGCGTTCCTAGCCT<210>24	??60.8
			???CaMV-CP	?????GGGACCAAATTATTGATCTAACCTCT<210>53	??61.0		???148
???????TCTCCTGAATCGCTTTCGTCTT<210>54	??60.9
		????Lectin		????????CAAGTCGTCGCTGTTGAGTTTG<210>56	??61.0	???165
????????GCTGGTGGAGGCATCATAGGT<210>57	??61.3
			????Fbp	?????????CCTCACCTCAGCCACGAATC<210>62	??60.1		???169
??????GTCAGTGTTTGAAGCTCATACCCA<210>63	??61.0
		????Zein		??????CCAAGAACTGGATGAACGGTTAG<210>59	??60.7	???184
??????GAGAAGCCGGTCGAGGTAGATAG<210>60	??61.5
			Rbcl (cotton)	????GATTATCCGCTAAGAATTACGGTAGA<210>65	??60.4		???134
??????TTCGGCACAAAATAGGAAACG<210>66	??60.6
		Rbcl (potato)		???GAGCGGGATAAATTGAACAAGTATG<210>68	??61.7	???198
?????TTCGGCACAAAACAAGAAACG<210>69	??61.4
			???18s?RNA	?????GAGAAACGGCTACCACATCCA<210>103	??60.0		???254
??????CGTGCCATCCCAAAGTCCAA<210>104	??60.8
		????Bt9c		?????CCTTCGCAAGACCCTTCCTCTATA<210>71	??63.3	???190
??????GTAGCTGTCGGTGTAGTCCTCGT<210>72	??60.3

????Bt176	????CTCTCGCCGTTCATGTCCGT<210>74	??60.0	????213
				????GGTCAGGCTCAGGCTGATGT<210>75	??60.0
	????GA21	????ACGGTGGAAGAGTTCAATGTATG<210>77				??60.0	????270
????TCTCCTTGATGGGCTGCA<210>78			??59.6
		???Mon810		????TCGAAGGACGAAGGACTCTAACG<210>80	??62.7	????170
????TCCATCTTTGGGACCACTGTC<210>81	??61.4
			????Bt11	????GCTGAAGCCAGTTACCTTCGG<210>83	??61.6		????212
????TATCATCGACTTCCATGACCA<210>84	??60.0
		????T25		???CCTTCGCAAGACCCTTCCTCTATA<210>86	??62.1	????231
???AGATCATCAATCCACTCTTGTGGTG<210>87	??60.6
			????RRS	????TGTAGGAGCCACCTTCCTTTTC<210>89	??61.2		????177
????AACCCTTCAATTTAACCGATGC<210>90	??60.2
		Luxuriant No. one of China		????TACGGCGAGTTCTGTTAGGTCC<210>92	??62.0	????146
????CCAGGCTTTACACTTTATGCTTC<210>93	??62.3

2. genome DNA extraction:

The DNA extraction agent box that can adopt CTAB method or other companies to produce is existing known technology.

3.PCR reaction marking

Target gene to be checked is carried out fluorescent mark can adopt labeled primer method, fluorescence to mix method or TDT enzyme addition.Specific as follows:

Labeled primer method reaction system adopts the 25ul reaction system.Each composition dosage is 10 * PCR damping fluid (containing Mg2+) 2.5ul, dNTP (10mM) 0.5ul, and each primer final concentration 0.3uM, template DNA 100ng, Taq enzyme 0.5ul, ddH2O supplies volume to 25ul.PCR response procedures: 94 ℃ of 3min sex change; 94 ℃ of 15S, 55 ℃ of 15S, 72 ℃ of 15S, 35 circulations; 72 ℃ of 10min.Experimentation is noted lucifuge.

Mix the method reaction system and adopt the 25ul reaction system.Each composition dosage is 10 * PCR damping fluid (containing Mg2+) 2.5ul, d (ATG) TP (10mM) 0.5ul, dCTP (1mM) 0.5ul, Cy5-dCTP (0.1mM) 0.5ul, each primer final concentration 0.3uM, template DNA 100ng, Taq enzyme 0.5ul, ddH ₂O supplies volume to 25ul.PCR response procedures: 94 ℃ of 3min sex change; 94 ℃ of 15S, 55 ℃ of 15S, 72 ℃ of 15S, 35 circulations; 72 ℃ of 10min.Experimentation is noted lucifuge.

TDT enzyme addition reaction system adopts the 25ul reaction system.Each composition dosage is 10 * PCR damping fluid (containing Mg2+) 2.5ul, dNTP (10mM) 0.5ul, and each primer final concentration 0.3uM, template DNA 100ng, Taq enzyme 0.5ul, ddH2O supplies volume to 25ul.PCR response procedures: 94 ℃ of 3min sex change; 94 ℃ of 15S, 55 ℃ of 15S, 72 ℃ of 15S, 35 circulations; 72 ℃ of 10min.Experimentation is noted lucifuge.

Get PCR reaction product 1ul, 5 * damping fluid 5ul, TDT enzyme 2U, ddH ₂O supplies volume to 25ul.37℃?30min。

4. hybridization

Get 6ul PCR reaction solution, add the hybridization solution 6 μ l through 55 ℃ of preheatings, 95 ℃ 3 minutes, 0 ℃ point sample zone of all transferring to chip after 5 minutes behind the mixing adds cover glass (noting not having bubble); Hybridization adds several dripping in the cabin, to keep humidity; Chip is put into the hybridization cabin, and sealing hybridization cabin is put into then in 55 ℃ of water-baths and is incubated 1 hour.

5. develop a film

Open the hybridization cabin, take out chip, wash out cover glass, then chip is put into the staining jar that fills 0.2%SDS, placed 5 minutes, use ddH with 0.2%SDS ₂O flushing two times.Room temperature lucifuge drying.

6. sweep sheet

Slide is put into laser confocal scanning instrument (GMS418 scanner) scanning, and by software kit analyzing and testing result.

The present invention's the oligonucleotide chip that is used for the transgenic product detection can detect the allos of multiple commercial transgenic product simultaneously and insert gene; The multiple target genes such as specificity border sequence of 8 kinds of genetically modified crops such as the specificity species internal standard gene of five kinds of crops such as cotton, soybean, rape, corn, potato and Bt9, Bt176, Bt11, Mon810, T25, GA21, RRS (Roundup-ready soybean), " luxuriant No. one of China " are " ' general sieve ', ' the kind detection ' and ' strain evaluation ' etc. that integrate transgenic product are all multi-functional ".

Wherein, a certain genetically modified crops " kind detection " contained multiple allos insertion sequences such as the promotor that may contain in this crop, terminator, marker gene, character gene, reached the purpose of mutual checking; This chip has also been set up negative and positive contrast and blank system in addition, so fundamentally guaranteed the specificity of detected result, has efficient, sensitive, characteristic of accurate, has effectively filled up the blank of prior art.

Description of drawings

Fig. 1 is making and the operating process synoptic diagram that transgenic product of the present invention detects oligonucleotide chip;

Fig. 2 is a kind of structural representation that transgenic product of the present invention detects oligonucleotide chip, and the present invention also can be individually fixed in " general sieve ", " kind detection ", " strain evaluation " three class probes on the different slides, makes independently chip of three classes;

Fig. 3 is that the detected result synoptic diagram A of it " general sieve " probe of transgenic product detection oligonucleotide chip of the present invention is the rape detected result, CaMV35S promotor, Nos terminator, FMV35 promotor, Bar, PAT, the NptII positive;

B is the corn detected result, CaMV35S promotor, Nos terminator, 35S terminator, the Bar positive;

C is the soybean detected result, CaMV35S promotor, the Nos terminator positive;

D is the cotton detected result, CaMV35S promotor, Nos terminator, the NptII positive;

Fig. 4 is that the detected result synoptic diagram A of it " plant variety " detection probes of transgenic product detection oligonucleotide chip of the present invention is the detected result of genetically engineered soybean;

B is the detected result of transgenic corns;

C is the detected result of transgene cotton;

Fig. 5 is that transgenic product of the present invention detects the detected result synoptic diagram that it " plant lines " of oligonucleotide chip is identified probe

A is the detected result of RRS;

B is the detected result of T25;

C is the detected result of GA21;

D is the detected result of Mon810;

Embodiment

The modification of embodiment 1 slide glass

Clean slide is dipped in 2min in 95% acetone soln that contains 1%3-aminopropyl trimethyl silane, acetone embathes 5min, repeat 6 times, 110 ℃ of baking 45min, slide is dipped in 2h in the 10% pyridine/DMF solution that contains 0.2% 1,4-phenylene diisocyanate, acetone soln washes once again, room temperature is dried, be put in 4 ℃ standby.

The design of embodiment 2 probes

The poly-oligonucleotide probe of design 15-35 at difference target gene to be checked, and will adjust to basic identical at the Tm value (melting temperature(Tm)) of different probe as far as possible.

Allos is inserted gene	Probe	Length	???Tm
				The CaMV35S promotor	???????TGCTCCACCATGTTGACGAAG<210>1	???21	??61.6
The FMV35S promotor	???????AAAACCAAGAAGGAACTCCCATC<210>4	???23	??60.7
				The CAMV35 terminator	?????GAAACCCTTAGTATGTATTTGTATTTGTAA<210>7	???30	??60.0
The Nos promotor	?????????GACCTTAGGCGACTTTTGAACG<210>10	???22	??60.9
				The Nos terminator	??????????AATCCTGTTGCCGGTCTTGC<210>13	???22	??62.3
????NptII	??????????GCTCCTGCCGAGAAAGTATCC<210>16	???21	??60.6
				????Bar	???????????CTGTGCCTCCAGGGACTTCA<210>19	???20	??60.5
????PAT	???????????GCCACAACACCCTCAACCTCA<210>22	???21	??62.8
				????Barnase	???????????GTCTGAAGATAATCCGCAACCC<210>25	???22	??60.4
????Barstar	???????????ACCTGGACGCTTTATGGGATT<210>28	???21	??60.3
				EPSPS (soybean)	????????????GGAAAGGCCAGAGGATTTGC<210>31	???20	??61.1
EPSPS (rape)	???????????GGGTCTTGTTGGTGTTTACGATT<210>34	???23	??60.1
				???Cry?I?A(b)	????????????CAGCACGGGGTTGGTGTAGAT<210>37	???21	??62.1
???Cry?I?A(c)	???????????TCTGGTAGATGTGGATGGGAAGT<210>40	???23	??60.1
				????CryIIIA	?????????????AAGCTGCCAACACCCACTTG<210>43	???20	??60.8
?????Cry9C	?????????CAAGTACACCAACTACTGCGAGACC<210>46	???25	??62.3
				??????GOX	??????????GTTCTTCAGAACTGGCAGGAGC<210>49	???22	??60.7
????CaMV-CP	??????????TATGACAACTACCGACGACTCGA<210>52	???23	??60.0

The plant species internal standard gene	Probe	Length	???Tm
				????Lectin	????TCTATCAGATCCATCAAAACGACG<210>55	???24	??61.1
????Zein	?????TTCTGGCTCTAACTTGGTTCCG<210>58	???22	??61.3
				????Fbp	??????TGCTACTCGTTTCCCGCTCA<210>61	???20	??61.7
Rbcl (cotton)	?????GGTGGGCTTGATTTTACCAAAG<210>64	???22	??60.7
				Rbcl (potato)	?????TGTCTTCGAGGTGGACTTGATTT<210>67	???23	??60.5
Plant distinguished boundary sequence	Probe	Length	???Tm
				????Bt9c	??????AGAGAAATGACCCCGTGTTCTC<210>70	???22	??60.0
????Bt176	??????GGGGTTGTTGTCCATTGTTGG<210>73	???21	??62.2
				????GA21	???????GATCCGGTGCATGGCCG<210>76	???17	??63.2
????Mon810	??????GGGCAATGGCAAAGGATGTT<210>79	???20	??62.1
				????BT11	?????GACGCTCAGTGGAACGAAAACT<210>82	???22	??61.0
????BT25	?????CTCCGGAGACATGTCGACTCTAG<210>85	???23	??60.6
				????RRS	????AAAATAAACATAGGGAACCCAAATG<210>88	???25	??60.5
Luxuriant No. one of China	??????GATTACGAATTCCCATGGAGTC<210>91	???22	??60.0

In addition, choose one section people HLA sequence, and design corresponding probe (CTGGAACAGCCAGAAGGAC＜210〉105, length 19, Tm 61) and, be used for monitoring the non-specific hybridization of hybridization as negative control;

Choose one section with 18S rRNA PCR product paired oligonucleotide fragment (CGCGCAAATTACCCAATCCTGA＜210〉102, length 22, Tm 60.4) and design corresponding primer mutually:

Target gene	Primer sequence (5 '-3 ')	???Tm	Product length (bp)
				?18s?RNA	????GAGAAACGGCTACCACATCCA<210>103	??60.0	????254
????CGTGCCATCCCAAAGTCCAA<210>104	??60.8

As positive control, be used for monitoring the false-negative appearance of hybridization; Simultaneously, also as the locating point of probe array, be used for determining the position of probe array hybridization region;

In order to reduce the sterically hindered of when hybridization, when synthetic, oligonucleotide fragment 5 ' end adds the preceding paragraph connecting arm (polyT), and the length that experiment showed, connecting arm is 5-20 when poly-, and crossbreeding effect is good.In addition, can be fixed on the slide, need also that 5 ' end carries out amido modified at it in order to make oligonucleotide probe.

Fixing of embodiment 3 probes

Synthetic above-mentioned probe (synthetic) by TaKaRa company, press institute to the nmol number, add the stock solution that TE (PH7.5) is made into 100uM, get 10ul with 5 times of TE (PH7.5) dilutions, be made into 20uM concentration 50ul (after the most handy GeneQuant is quantitative concentration being adjusted unanimity), therefrom get 20ul and 20ul 0.1M PH 9.0 Na ₂CO ₃/ NaHCO ₃It is the sampling liquid 40ul of 10uM that the damping fluid two-fold dilution becomes final concentration.Setting up the sampling liquid that does not contain oligonucleotide fragment simultaneously is blank, is used to monitor the background after the hybridization.

Get each 8ul of above-mentioned sampling liquid in 384 orifice plates, become desired matrix with the point sample instrument point.Each bar probe repeats point sample 5 times, dot spacing 500um.Place 1-2h for 37 ℃, 1% ammoniacal liquor washes once, dries standby.

The application of embodiment 4 oligonucleotide chips of the present invention

A. design corresponding pcr amplification primer according to target gene to be checked, be respectively:

The target gene title	Primer sequence (5 '-3 ')	???Tm	Product length (bp)
				CaMV 35S promotor	?????AGACTGGCGAACAGTTCATACAGA<210>2	??60.8	???188
???????GCAATGGAATCCGAGGAGGT<210>3	??61.1
		The FMV35S promotor	???????CAGCATTCCAGATTGGGTTCA<210>5		??60.7	???172
??????CTTTTGGCTAATGGTTTGGAGAC<210>6	??60.1
			The CaMV35s terminator	??????CCCAGATAAGGGAATTAGGGTTC<210>8	??60.5		???134
??????CCCTGGATTTTGGTTTTAGGAAT<210>9	??60.9
		The Nos terminator		???????TGAATCCTGTTGCCGGTCTT<210>13	??60.2	???138
?????AAATGTATAATTGCGGGACTCTAATC<210>14	??60.1
			????NptII	???????GGAAGGGACTGGCTGCTATTG<210>17	??61.3		???157
???????GATGTTTCGCTTGGTGGTCG<210>18	??61.1
		The Nos promotor		???????GAACCGCAACGATTGAAGGA<210>11	??60.8	???177
??????TGGATACCGAGGGGAATTTATG<210>12	??60.7
			??Cry?I?A(b)	???????TGTTCCCCAACTACGACTCCA<210>38	??60.2		???151
????????AGGTGCGGGCTCCTGATGCT<210>39	??66.5
		??Cry?I?A(c)		??????AGACTCAACAGCAGTGGAAATAACA<210>41	??60.2	???123
???????GTGAATAGGGGTCACAGAAGCATA<210>42	??60.3
			????Cry9C	?????????CTGCTGCTGCTGAAGGATGC<210>47	??61.7		???199

	???????CCCTGCGGAACTGGTGGTAG<210>48	??62.6
				??CryIIIA	????GGTTCTTTTCCTCACTACCTATGCTC<210>44	??61.6	????101
?????CCTCTTTCTCGTATCCCCACTCTT<210>45	??62.3
		????GOX	???????AGTTCGCTGGTCTCACTGCTG<210>50		??60.3	????133
???????GGACGGAAACCCATCCACTT<210>51	??60.9
			EPSPS (soybean)	?????AGAGCCGTGGATAGATTAAGGAAG<210>32	??60.6		????149
??????AGACCGCCGAACATGAAGGA<210>33	??62.6
		EPSPS (rape)		????GGGTCTTGTTGGTGTTTACGATT<210>35	??60.1	????182
?????TGTAGGTGATTGGCGTTGGAG<210>36	??60.9
			??Barnase	?????TCCGTTTGTTTACTGGTGCTTG<210>26	??60.5		????192
?????GAGGGCTTGTGCTTCTGATTTT<210>27	??60.4
		??Barstar		????AACAAATCAGAAGTATCAGCGACCT<210>29	??60.9	????145
?????AACTGCCTCCATTCCAAAACG<210>30	??61.6
			????Bar	?????GCTCCACGCTCTACACCCAC<210>20	??60.2		????181
?????AAACCCACGTCATGCCAGTT<210>21	??60.0
		????PAT		?????GACAGAGCCACAAACACCACAA<210>23	??60.7	????144
?????CAATCGTAAGCGTTCCTAGCCT<210>24	??60.8
			???CaMV-CP	???GGGACCAAATTATTGATCTAACCTCT<210>53	??61.0		????148
????TCTCCTGAATCGCTTTCGTCTT<210>54	??60.9
		???Lectin		?????CAAGTCGTCGCTGTTGAGTTTG<210>56	??61.0	????165
?????GCTGGTGGAGGCATCATAGGT<210>57	??61.3
			????Fbp	?????CCTCACCTCAGCCACGAATC<210>62	??60.1		????169
????GTCAGTGTTTGAAGCTCATACCCA<210>63	??61.0
		????Zein		????CCAAGAACTGGATGAACGGTTAG<210>59	??60.7	????184
????GAGAAGCCGGTCGAGGTAGATAG<210>60	??61.5
			Rbcl (cotton)	???GATTATCCGCTAAGAATTACGGTAGA<210>65	??60.4		????134
????TTCGGCACAAAATAGGAAACG<210>66	??60.6
		Rbcl (potato)		??GAGCGGGATAAATTGAACAAGTATG<210>68	??61.7	????198
????TTCGGCACAAAACAAGAAACG<210>69	??61.4
			???18s?RNA	????GAGAAACGGCTACCACATCCA<210>103	??60.0		????254
????CGTGCCATCCCAAAGTCCAA<210>104	??60.8
		????Bt9c		????CCTTCGCAAGACCCTTCCTCTATA<210>71	??63.3	????190
????GTAGCTGTCGGTGTAGTCCTCGT<210>72	??60.3
			????Bt176	?????CTCTCGCCGTTCATGTCCGT<210>74	??60.0		????213
????GGTCAGGCTCAGGCTGATGT<210>75	??60.0
		????GA21		????ACGGTGGAAGAGTTCAATGTATG<210>77	??60.0	????270
??????TCTCCTTGATGGGCTGCA<210>78	??59.6
			???Mon810	????TCGAAGGACGAAGGACTCTAACG<210>80	??62.7		????170
????TCCATCTTTGGGACCACTGTC<210>81	??61.4

????Bt11	??????GCTGAAGCCAGTTACCTTCGG<210>83	??61.6	????212
				??????TATCATCGACTTCCATGACCA<210>84	??60.0
	????T25	?????CCTTCGCAAGACCCTTCCTCTATA<210>86				??62.1	????231
?????AGATCATCAATCCACTCTTGTGGTG<210>87			??60.6
		????RRS		???????TGTAGGAGCCACCTTCCTTTTC<210>89	??61.2	????177
???????AACCCTTCAATTTAACCGATGC<210>90	??60.2
			Luxuriant No. one of China	???????TACGGCGAGTTCTGTTAGGTCC<210>92	??62.0		????146
??????CCAGGCTTTACACTTTATGCTTC<210>93	??62.3

B. genome DNA extraction:

Adopt the Promega paramagnetic particle method, use Wizard Magnetic DNA purification system for food (PromegaFF3750) test kit, working method is seen specification sheets.

The c.PCR reaction marking

Reaction system adopts the 25ul reaction system.Each composition dosage is 10 * PCR damping fluid (containing Mg2+) 2.5ul, d (ATG) TP (10mM) 0.5ul, dCTP (1mM) 0.5ul, Cy5-dCTP (0.1mM) 0.5ul, each primer final concentration 0.3uM, template DNA 100ng, Taq enzyme 0.5ul, ddH2O supplies volume to 25ul.

PCR response procedures: 94 ℃ of 3min sex change; 94 ℃ of 15S, 55 ℃ of 15S, 72 ℃ of 15S, 35 circulations; 72 ℃ of 10min.Experimentation is noted lucifuge.

D. hybridize and detect

Get 6ul PCR reaction solution, add the hybridization solution 6 μ l through 55 ℃ of preheatings, 95 ℃ 3 minutes, 0 ℃ point sample zone of all transferring to chip after 5 minutes behind the mixing adds cover glass (noting not having bubble); Hybridization adds several dripping in the cabin, to keep humidity; Chip is put into the hybridization cabin, and sealing hybridization cabin is put into then in 55 ℃ of water-baths and is incubated 1 hour.

Open the hybridization cabin, take out chip, wash out cover glass, then chip is put into the staining jar that fills 0.2%SDS, placed 5 minutes, use ddH with 0.2%SDS ₂O flushing two times.Room temperature lucifuge drying.

Slide is put into laser confocal scanning instrument (GMS418 scanner) scanning, and by software kit analyzing and testing result.

Sequence table

＜110〉Boxing Gene Chip Co., Ltd., Shanghai

Star gene chip research institute is won in Shanghai

Animal and Plant Quarantine Institute, AQSIQ

＜120〉transgenic product detects the making method and the application thereof of oligonucleotide chip

<130>??1

<160>??105

<170>??PatentIn?version?3.1

<210>??1

<211>??21

<212>??DNA

<213>??Artificial

<400>??1

tgctccacca?tgttgacgaag??????????????????????????????????????????????????21

<210>??2

<211>??24

<212>??DNA

<213>??Artificial

<400>??2

agactggcga?acagttcata?caga??????????????????????????????????????????????24

<210>??3

<211>??20

<212>??DNA

<213>??Artificial

<400>??3

gcaatggaat?ccgaggaggt???????????????????????????????????????????????????20

<210>??4

<211>??23

<212>??DNA

<213>??Artificial

<400>??4

aaaaccaaga?aggaactccc?atc???????????????????????????????????????????????23

<210>??5

<211>??21

<212>??DNA

<213>??Artificial

<400>??5

cagcattcca?gattgggttca??????????????????????????????????????????????????21

<210>??6

<211>??23

<212>??DNA

<213>??Artificial

<400>??6

cttttggcta?atggtttgga?gac???????????????????????????????????????????????23

<210>??7

<211>??30

<212>??DNA

<213>??Artificial

<400>??7

gaaaccctta?gtatgtattt?gtatttgtaa????????????????????????????????????????30

<210>??8

<211>??23

<212>??DNA

<213>??Artificial

<400>??8

cccagataag?ggaattaggg?ttc???????????????????????????????????????????????23

<210>??9

<211>??23

<212>??DNA

<213>??Artificial

<400>??9

ccctggattt?tggttttagg?aat???????????????????????????????????????????????23

<210>??10

<211>??22

<212>??DNA

<213>??Artificial

<400>??10

gaccttaggc?gacttttgaa?cg????????????????????????????????????????????????22

<210>??11

<211>??20

<212>??DNA

<213>??Artificial

<400>??11

gaaccgcaac?gattgaagga???????????????????????????????????????????????????20

<210>??12

<211>??22

<212>??DNA

<213>??Artificial

<400>??12

tggataccga?ggggaattta?tg????????????????????????????????????????????????22

<210>??13

<211>??20

<212>??DNA

<213>??Artificial

<400>??13

aatcctgttg?ccggtcttgc???????????????????????????????????????????????????20

<210>??14

<211>??20

<212>??DNA

<213>??Artificial

<400>??14

tgaatcctgt?tgccggtctt???????????????????????????????????????????????????20

<210>??15

<211>??26

<212>??DNA

<213>??Artificial

<400>??15

aaatgtataa?ttgcgggact?ctaatc????????????????????????????????????????????26

<210>??16

<211>??21

<212>??DNA

<213>??Artificial

<400>??16

gctcctgccg?agaaagtatc?c?????????????????????????????????????????????????21

<210>??17

<211>??21

<212>??DNA

<213>??Artificial

<400>??17

ggaagggact?ggctgctatt?g?????????????????????????????????????????????????21

<210>??18

<211>??20

<212>??DNA

<213>??Artificial

<400>??18

gatgtttcgc?ttggtggtcg???????????????????????????????????????????????????20

<210>??19

<211>??20

<212>??DNA

<213>??Artificial

<400>??19

ctgtgcctcc?agggacttca???????????????????????????????????????????????????20

<210>??20

<211>??20

<212>??DNA

<213>??Artificial

<400>??20

gctccacgct?ctacacccac???????????????????????????????????????????????????20

<210>??21

<211>??20

<212>??DNA

<213>??Artificial

<400>??21

aaacccacgt?catgccagtt???????????????????????????????????????????????????20

<210>??22

<211>??21

<212>??DNA

<213>??Artificial

<400>??22

gccacaacac?cctcaacctc?a????????????????????????????????????????????????21

<210>??23

<211>??22

<212>??DNA

<213>??Artificial

<400>??23

gacagagcca?caaacaccac?aa???????????????????????????????????????????????22

<210>??24

<211>??22

<212>??DNA

<213>??Artificial

<400>??24

caatcgtaag?cgttcctagc?ct???????????????????????????????????????????????22

<210>??25

<211>??22

<212>??DNA

<213>??Artificial

<400>??25

gtctgaagat?aatccgcaac?cc??????????????????????????????????????22

<210>??26

<211>??22

<212>??DNA

<213>??Artificial

<400>??26

tccgtttgtt?tactggtgct?tg??????????????????????????????????????22

<210>??27

<211>??22

<212>??DNA

<213>??Artificial

<400>??27

gagggcttgt?gcttctgatt?tt??????????????????????????????????????22

<210>??28

<211>??21

<212>??DNA

<213>??Artificial

<400>??28

acctggacgc?tttatgggat?t???????????????????????????????????????21

<210>??29

<211>??25

<212>??DNA

<213>??Artificial

<400>??29

aacaaatcag?aagtatcagc?gacct???????????????????????????????25

<210>??30

<211>??21

<212>??DNA

<213>??Artificial

<400>??30

aactgcctcc?attccaaaac?g???????????????????????????????????21

<210>??31

<211>??20

<212>??DNA

<213>??Artificial

<400>??31

ggaaaggcca?gaggatttgc?????????????????????????????????????20

<210>??32

<211>??24

<212>??DNA

<213>??Artificial

<400>??32

agagccgtgg?atagattaag?gaag????????????????????????????????24

<210>??33

<211>??20

<212>??DNA

<213>??Artificial

<400>??33

agaccgccga?acatgaagga??????????????????????????????????????????????20

<210>??34

<211>??23

<212>??DNA

<213>??Artificial

<400>??34

gggtcttgtt?ggtgtttacg?att??????????????????????????????????????????23

<210>??35

<211>??23

<212>??DNA

<213>??Artificial

<400>??35

gggtcttgtt?ggtgtttacg?att??????????????????????????????????????????23

<210>??36

<211>??21

<212>??DNA

<213>??Artificial

<400>??36

tgtaggtgat?tggcgttgga?g????????????????????????????????????????????21

<210>??37

<211>??21

<212>??DNA

<213>??Artificial

<400>??37

cagcacgggg?ttggtgtaga?t????????????????????????????????????????????????21

<210>??38

<211>??21

<212>??DNA

<213>??Artificial

<400>??38

tgttccccaa?ctacgactcc?a????????????????????????????????????????????????21

<210>??39

<211>??20

<212>??DNA

<213>??Artificial

<400>??39

aggtgcgggc?tcctgatgct??????????????????????????????????????????????????20

<210>??40

<211>??23

<212>??DNA

<213>??Artificial

<400>??40

tctggtagat?gtggatggga?agt??????????????????????????????????????????????23

<210>??41

<211>??25

<212>??DNA

<213>??Artificial

<400>??41

agactcaaca?gcagtggaaa?taaca???????????????????????????25

<210>??42

<211>??24

<212>??DNA

<213>??Artificial

<400>??42

gtgaataggg?gtcacagaag?cata????????????????????????????24

<210>??43

<211>??20

<212>??DNA

<213>??Artificial

<400>??43

aagctgccaa?cacccacttg?????????????????????????????????20

<210>??44

<211>??26

<212>??DNA

<213>??Artificial

<400>??44

ggttcttttc?ctcactacct?atgctc??????????????????????????26

<210>??45

<211>??24

<212>??DNA

<213>??Artificial

<400>??45

cctctttctc?gtatccccac?tctt???????????????????????????????24

<210>??46

<211>??25

<212>??DNA

<213>??Artificial

<400>??46

caaggtacacc?aactactgcg?agacc?????????????????????????????25

<210>??47

<211>??20

<212>??DNA

<213>??Artificial

<400>??47

ctgctgctgc?tgaaggatgc????????????????????????????????????20

<210>??48

<211>??20

<212>??DNA

<213>??Artificial

<400>??48

ccctgcggaa?ctggtggtag??????????????????????????????????????????20

<210>??49

<211>??22

<212>??DNA

<213>??Artificial

<400>??49

gttcttcaga?actggcagga?gc???????????????????????????????????????22

<210>??50

<211>??21

<212>??DNA

<213>??Artificial

<400>??50

agttcgctgg?tctcactgct?g????????????????????????????????????????21

<210>??51

<211>??20

<212>??DNA

<213>??Artificial

<400>??51

ggacggaaac?ccatccactt??????????????????????????????????????????20

<210>??52

<211>??23

<212>??DNA

<213>??Artificial

<400>??52

tatgacaact?accgacgact?cga?????????????????????????23

<210>??53

<211>??26

<212>??DNA

<213>??Artificial

<400>??53

gggaccaaat?tattgatcta?acctct??????????????????????26

<210>??54

<211>??22

<212>??DNA

<213>??Artificial

<400>??54

tctcctgaat?cgctttcgtc?tt??????????????????????????22

<210>??55

<211>??24

<212>??DNA

<213>??Artificial

<400>??55

tctatcagat?ccatcaaaac?gacg????????????????????????24

<210>??56

<211>??22

<212>??DNA

<213>??Artificial

<400>??56

caagtcgtcg?ctgttgagtt?tg???????????????????????????22

<210>??57

<211>??21

<212>??DNA

<213>??Artificial

<400>??57

gctggtggag?gcatcatagg?t????????????????????????????21

<210>??58

<211>??22

<212>??DNA

<213>??Artificial

<400>??58

ttctggctct?aacttggttc?cg???????????????????????????22

<210>??59

<211>??23

<212>??DNA

<213>??Artificial

<400>??59

ccaagaactg?gatgaacggttag???????????????????????????23

<210>??60

<211>??23

<212>??DNA

<213>??Artificial

<400>??60

gagaagccgg?tcgaggtaga?tag????????????????????????????????????????????23

<210>??61

<211>??20

<212>??DNA

<213>??Artificial

<400>??61

tgctactcgt?ttcccgctca????????????????????????????????????????????????20

<210>??62

<211>??20

<212>??DNA

<213>??Artificial

<400>??62

cctcacctca?gccacgaatc????????????????????????????????????????????????20

<210>??63

<211>??24

<212>??DNA

<213>??Artificial

<400>??63

gtcagtgttt?gaagctcata?ccca???????????????????????????????????????????24

<210>??64

<211>??22

<212>??DNA

<213>??Artificial

<400>??64

ggtgggcttg?attttaccaa?ag?????????????????????????????????????????????22

<210>??65

<211>??26

<212>??DNA

<213>??Artificial

<400>??65

gattatccgc?taagaattac?ggtaga?????????????????????????????????????????26

<210>??66

<211>??21

<212>??DNA

<213>??Artificial

<400>??66

ttcggcacaa?aataggaaac?g??????????????????????????????????????????????21

<210>??67

<211>??23

<212>??DNA

<213>??Artificial

<400>??67

tgtcttcgag?gtggacttga?ttt???????????????????????????????????????23

<210>??68

<211>??25

<212>??DNA

<213>??Artificial

<400>??68

gagcgggata?aattgaacaa?gtatg?????????????????????????????????????25

<210>??69

<211>??21

<212>??DNA

<213>??Artificial

<400>??69

ttcggcacaa?aacaagaaac?g?????????????????????????????????????????21

<210>??70

<211>??22

<212>??DNA

<213>??Artificial

<400>??70

agagaaatga?ccccgtgttc?tc????????????????????????????????????????22

<210>??71

<211>??24

<212>??DNA

<213>??Artificial

<400>??71

ccttcgcaag?acccttcctc?tata???????????????????????????24

<210>??72

<211>??23

<212>??DNA

<213>??Artificial

<400>??72

gtagctgtcg?gtgtagtcct?cgt????????????????????????????23

<210>??73

<211>??21

<212>??DNA

<213>??Artificial

<400>??73

ggggttgttg?tccattgttgg????????????????????????????????21

<210>??74

<211>??20

<212>??DNA

<213>??Artificial

<400>??74

ctctcgccgt?tcatgtccgt????????????????????????????????20

<210>??75

<211>??20

<212>??DNA

<213>??Artificial

<400>??75

ggtcaggctc?aggctgatgt??????????????????????????????????????20

<210>??76

<211>??17

<212>??DNA

<213>??Artificial

<400>??76

gatccggtgc?atggccg?????????????????????????????????????????17

<210>??77

<211>??23

<212>??DNA

<213>??Artificial

<400>??77

acggtggaag?agttcaatgt?atg??????????????????????????????????23

<210>??78

<211>??18

<212>??DNA

<213>??Artificial

<400>??78

tctccttgat?gggctgca????????????????????????????????????????18

<210>??79

<211>??20

<212>??DNA

<213>??Artificial

<400>??79

gggcaatggc?aaaggatgtt??????????????????????????????20

<210>??80

<211>??23

<212>??DNA

<213>??Artificial

<400>??80

tcgaaggacg?aaggactcta?acg??????????????????????????23

<210>??81

<211>??21

<212>??DNA

<213>??Artificial

<400>??81

tccatctttg?ggaccactgtc?????????????????????????????21

<210>??82

<211>??22

<212>??DNA

<213>??Artificial

<400>??82

gacgctcagt?ggaacgaaaa?ct???????????????????????????22

<210>??83

<211>??21

<212>??DNA

<213>??Artificial

<400>??83

gctgaagcca?gttaccttcg?g?????????????????????????????21

<210>??84

<211>??21

<212>??DNA

<213>??Artificial

<400>??84

tatcatcgac?ttccatgacc?a?????????????????????????????21

<210>??85

<211>??23

<212>??DNA

<213>??Artificial

<400>??85

ctccggagac?atgtcgactc?tag???????????????????????????23

<210>??86

<211>??24

<212>??DNA

<213>??Artificial

<400>??86

ccttcgcaag?acccttcctc?tata??????????????????????24

<210>??87

<211>??25

<212>??DNA

<213>??Artificial

<400>??87

agatcatcaa?tccactcttg?tggtg?????????????????????25

<210>??88

<211>??25

<212>??DNA

<213>??Artificial

<400>??88

aaaataaaca?tagggaaccc?aaatg?????????????????????25

<210>??89

<211>??22

<212>??DNA

<213>??Artificial

<400>??89

tgtaggagcc?accttccttt?tc????????????????????????22

<210>??90

<211>??22

<212>??DNA

<213>??Artificial

<400>??90

aacccttcaa?tttaaccgat?gc?????????????????????????????22

<210>??91

<211>??22

<212>??DNA

<213>??Artificial

<400>??91

gattacgaat?tcccatggag?tc?????????????????????????????22

<210>??92

<211>??22

<212>??DNA

<213>??Artificial

<400>??92

tacggcgagt?tctgttaggt?cc?????????????????????????????22

<210>??93

<211>??23

<212>??DNA

<213>??Artificial

<400>??93

ccaggcttta?cactttatgc?ttc????????????????????????????23

<210>??94

<211>??225

<212>??DNA

<213>??Artificial

<400>??94

gtggattgat?gtgatatctc?cactgacgta?agggatgacg?cacaatccca?ctatccttcg????60

caagaccctt?cctctatata?aggaagttca?tttcatttgg?agagaacacg?gggtcatttc????120

tctattactt?cagccataac?aaaagaactc?ttttctcttc?ttattaaacc?aaaaaccatg????180

gctgactacc?tgcagatgac?cgacgaggac?tacaccgaca?gctac????????????????????225

<210>??95

<211>??380

<212>??DNA

<213>??Artificial

<400>??95

tttcctcccg?atacgcccct?ccatctctcg?ccgttcatgt?ccgtggctgg?ctgccctccg????60

tgggagcagg?ctggccgcac?tcgttccccg?ccgcagccgg?atccaacaat?ggacaacaac????120

cccaacatca?acgagtgcat?cccctacaac?tgcctgagca?atcccgaggt?ggaggtgctg????180

ggcggcgagc?gcatcgagac?cggctacacc?cccatcgaca?tcagcctgag?cctgacccag????240

ttcctgctga?gcgagttcgt?gcccggcgcc?ggcttcgtgc?tgggcctggt?ggacatcatc????300

tggggcatct?tcggccccag?ccagtgggac?gccttcctgg?tgcagatcga?gcagctgatc????360

aaccagcgca?tcgaggagtt????????????????????????????????????????????????380

<210>??96

<211>??459

<212>??DNA

<213>??Artificial

<400>??96

atggtggctc?cgttcaccgg?ccttaagtcc?aacgccgcct?tccccaccac?caagaaggct????60

aacgacttct?ccacccttcc?cagcaacggt?ggaagagttc?aatgtatgca?ggtgtggccg????120

gcctacggca?acaagaagtt?cgagacgctg?tcgtacctgc?cgccgctgtc?tatggcgccc????180

accgtgatga?tggcctcgtc?ggccaccgcc?gtcgctccgt?tccaggggct?caagtccacc????240

gccagcctcc?ccgtcgcccg?ccgctcctcc?agaagcctcg?gcaacgtcag?caacggcgga????300

aggatccggt?gcatggccgg?cgccgaggag?atcgtgctgc?agcccatcaa?ggagatctcc????360

ggcaccgtca?agctgccggg?gtccaagtcg?ctttccaacc?ggatcctcct?actcgccgcc????420

ctgtccgagg?ggacaacagt?ggttgataac?ctgctgaac???????????????????????????459

<210>??97

<211>??196

<212>??DNA

<213>??Artificial

<400>??97

tcgaaggacg?aaggactcta?acgtttaaca?tcctttgcca?ttgcccagct?atctgtcact????60

ttattgtgaa?gatagtggaa?aaggaaggtg?gctcctacaa?atgccatcat?tgcgataaag????120

gaaaggccat?cgttgaagat?gcctctgccg?acagtggtcc?caaagatgga?cccccaccca????180

cgaggagcat?cgtgga????????????????????????????????????????????????????196

<210>??98

<211>??332

<212>??DNA

<213>??Artificial

<400>??98

ggcagcagcc?actggtaaca?ggattagcag?agcgaggtat?gtaggcggtg?ctacagagtt????60

cttgaagtgg?tggcctaact?acggctacac?tagaagaaca?gtatttggta?tctgcgctct????120

gctgaagcca?gttaccttcg?gaaaaagagt?tggtagctct?tgatccggca?aacaaaccac????180

cgctggtagc?ggtggttttt?ttgtttgcaa?gcagcagatt?acgcgcagaa?aaaaaggatc????240

tcaagaagat?cctttgatct?tttctacggg?gcctgacgct?cagtggaacg?aaaactcacg????300

ttaagggatt?ttggtcatgg?aagtcgatga?ta??????????????????????????????????332

<210>??99

<211>??432

<212>??DNA

<213>??Artificial

<400>??99

aaagatggac?ccccacccac?gaggagcatc?gtggaaaaag?aagacgttcc?aaccacgtct????60

tcaaagcaag?tggattgatg?tgatatctcc?actgacgtaa?gggatgacgc?acaatcccac????120

tatccttcgc?aagacccttc?ctctatataa?ggaagttcat?ttcatttgga?gaggacaggg????180

tacccgggga?tcctctagag?tcgacatgtc?tccggagagg?agaccagttg?agattaggcc????240

agctacagca?gctgatatgg?ccgcggtttg?tgatatcgtt?aaccattaca?ttgagacgtc????300

tacagtgaac?tttaggacag?agccacaaac?accacaagag?tggattgatg?atctagagag????360

gttgcaagat?agataccctt?ggttggttgc?tgaggtggag?ggtgttgtgg?gctggtattg????420

cttacgctgg?gc????????????????????????????????????????????????????????432

<210>??100

<211>??390

<212>??DNA

<213>??Artificial

<400>??100

gatagtggga?ttgtgcgtca?tcccttacgt?cagtggagat?atcacatcaa?tccacttgct????60

ttgaagacgt?ggttggaacg?tcttcttttt?ccacgtgotc?ctcgtgggtg?ggggtccatc????120

tttgggacca?ctgtcggcag?aggcatcttc?aacgatggcc?tttcctttat?cgcaatgatg????180

gcatttgtag?gagccacctt?ccttttccat?ttgggttccc?tatgtttatt?ttaacctgta????240

tgtatgatct?tattttgaat?gaaatgcaat?aagttatttc?tagtaaaaaa?aaataaacat????300

ttgatagaaa?caaattaaag?catgcaaaaa?taactcatta?gcatcggtta?aattgaaggg????360

tttgaataat?ttgcacaagg?ttctgaattc?????????????????????????????????????390

<210>??101

<211>??526

<212>??DNA

<213>??Artificial

<400>??101

cagagactaa?aaggaagacc?attgatattc?ttgcaaactg?catttaagga?ggcaataaag??60

tagggacttt?acacttgcat?attctttaaa?aacagacaaa?aataggcggg?aaacgacaat??120

ctgatcatga?gcggagaatt?aagggagtca?cgttatgacc?cccgccgatg?acgcgggaca??180

agccgtttta?cgtttggaac?tgacagaacc?gcaacgttga?aggagccact?cagccgcggg??240

tttctggagt?ttaatgagct?aagcacatac?gtcagaaacc?attattgcgc?gttcaaaagt??300

cgcctaaggt?cactatcagc?tagcaaatat?ttcttgtcaa?aaatgctcca?ctgacgttcc??360

ataaattccc?ctcggtatcc?aattagagtc?tcatattcac?tctcaatcca?aataatctgc??420

accggatctg?gatcgtttcg?catgattgaa?caagatggat?tgcacgcagg?ttctccggcc??480

gcttgggtgg?agaggctatt?cggctatgac?tgggcacaac?agacaa?????????????????526

<210>??102

<211>??22

<212>??DNA

<213>??Artificial

<400>??102

cgcgcaaatt?acccaatcct?ga??????????????????????????????????????????22

<210>??103

<211>??21

<212>??DNA

<213>??Artificial

<400>??103

gagaaacggc?taccacatcc?a???????????????????????????????????????????21

<210>??104

<211>??20

<212>??DNA

<213>??Artificial

<400>??104

cgtgccatcc?caaagtccaa?????????????????????????????????20

<210>??105

<211>??19

<212>??DNA

<213>??Artificial

<400>??105

ctggaacagc?cagaaggac??????????????????????????????????19

Claims (11)

1. a transgenic product detects oligonucleotide probe array, it is characterized in that, and the poly-probe of design 15-35, the Tm value (melting temperature(Tm)) of probe is basic identical.

Allos is inserted gene probe length T m

CaMV35S promotor TGCTCCACCATGTTGACGAAG＜210〉1 21 61.6

FMV35S promotor AAAACCAAGAAGGAACTCCCATC＜210〉4 23 60.7

CAMV35 terminator GAAACCCTTAGTATGTATTTGTATTTGTAA＜210〉7 30 60.0

Nos promotor GACCTTAGGCGACTTTTGAACG＜210〉10 22 60.9

Nos terminator AATCCTGTTGCCGGTCTTGC＜210〉13 22 62.3

Npt?II???????????GCTCCTGCCGAGAAAGTATCC<210>16????????????21????60.6

Bar??????????????CTGTGCCTCCAGGGACTTCA<210>19?????????????20????60.5

PAT??????????????GCCACAACACCCTCAACCTCA<210>22????????????21????62.8

Barnase??????????GTCTGAAGATAATCCGCAACCC<210>25???????????22????60.4

Barstar??????????ACCTGGACGCTTTATGGGATT<210>28????????????21????60.3

EPSPS (soybean) GGAAAGGCCAGAGGATTTGC＜210〉31 20 61.1

EPSPS (rape) GGGTCTTGTTGGTGTTTACGATT＜210〉34 23 60.1

Cry?I?A(b)???????CAGCACGGGGTTGGTGTAGAT<210>37????????????21????62.1

Cry?I?A(c)???????TCTGGTAGATGTGGATGGGAAGT<210>40??????????23????60.1

CryIIIA??????????AAGCTGCCAACACCCACTTG<210>43?????????????20????60.8

Cry9C????????????CAAGTACACCAACTACTGCGAGACC<210>46????????25????62.3

GOX??????????????GTTCTTCAGAACTGGCAGGAGC<210>49???????????22????60.7

CaMV-CP??????????TATGACAACTACCGACGACTCGA<210>52??????????23????60.0

Plant species

Probe length Tm

Internal standard gene

Lectin???????????TCTATCAGATCCATCAAAACGACG<210>55?????????24????61.1

Zein?????????????TTCTGGCTCTAACTTGGTTCCG<210>58???????????22????61.3

Fbp??????????????TGCTACTCGTTTCCCGCTCA<210>61?????????????20????61.7

Rbc1 (spending mutually) GGTGGGCTTGATTTTACCAAAG＜210〉64 22 60.7

Rbc1 (potato) TGTCTTCGAGGTGGACTTGATTT＜210〉67 23 60.5

Plant is special

Probe length Tm

Border sequence

Bt9c????????????AGAGAAATGACCCCGTGTTCTC<210>70???????22????60.0

Bt176???????????GGGGTTGTTGTCCATTGTTGG<210>73????????21????62.2

GA21????????????GATCCGGTGCATGGCCG<210>76????????????17????63.2

Mon810??????????GGGCAATGGCAAAGGATGTT<210>79?????????20????62.1

BT11????????????GACGCTCAGTGGAACGAAAACT<210>82???????22????61.0

BT25????????????CTCCGGAGACATGTCGACTCTAG<210>85??????23????60.6

RRS?????????????AAAATAAACATAGGGAACCCAAATG<210>88????25????60.5

Luxuriant GATTACGAATTCCCATGGAGTC＜210 of China〉91 22 60.0

Transfer on the slide that chemical group is modified by point sample instrument, handle and make through SDS, pure water.

2. oligonucleotide probe array as claimed in claim 1 is characterized in that the modification group of described slide is selected from one of aldehyde radical, isothiocyano, sulfydryl.

3. the making method of a transgenic product detection oligonucleotide probe array is characterized in that, comprises the steps:

(1) modification of slide glass;

(2) probe design and synthetic;

(3) probe is fixing;

4. the making method of oligonucleotide probe array as claimed in claim 3 is characterized in that, the modification of described slide glass is selected from down one of group of methods:

(1) clean slide is dipped in 2min in 95% acetone soln that contains 1%3-aminopropyl trimethyl silane, acetone embathes 5min, repeat 6 times, 110 ℃ of baking 45min, again slide is dipped in and contains 0.2%1,2h in 10% pyridine of 4-phenylene diisocyanate/DMF solution, acetone soln washes once, room temperature is dried, be put in 4 ℃ standby;

(2) clean slide is dipped in boil in the 1N NaOH solution and carried out the hydroxylation pre-treatment in 10 minutes.Pretreated slide is dipped in 2min in 95% acetone soln that contains 1%3-aminopropyl trimethyl silane, and acetone embathes 5min, repeats 6 times, and 110 ℃ of baking 45min are dipped in slide in 5% glutaraldehyde water solution and spend the night under 50 ℃ of conditions.Take out slide, with 0.2%SDS washing 5 minutes, repeat 3 times, room temperature is dried standby; Or

(3) with clean slide with 25% ammoniacal liquor soaked overnight, the MilliQ water logging is given a baby a bath on the third day after its birth minute, dehydrated alcohol washes once, under the room temperature slide is immersed in 1%MPTS, 95% ethanol, the 16mM acetic acid solution 30 minutes, 95% ethanol, 16mM acetic acid solution wash once N ₂Jar in 6～8 hours.

5. the making method of oligonucleotide probe array as claimed in claim 3 is characterized in that, 5 ' end of oligonucleotide probe adds the preceding paragraph connecting arm (polyT), and the length of connecting arm is 5-20 when poly-, and 5 ' end carries out chemical group and modifies.

6. the making method of oligonucleotide probe array as claimed in claim 5 is characterized in that, the modification group of described oligonucleotide probe 5 ' end is selected from amino or sulfydryl.

7. the making method of oligonucleotide probe array as claimed in claim 3 is characterized in that: the Na that probe is dissolved in 0.1MpH9.0 ₂CO ₃/ NaHCO ₃Make the sampling liquid that final concentration is 10 μ M in the solution, sampling liquid is added in the point template, become desired matrix with the point sample instrument point, place 1-2h for 37 ℃, 1% ammoniacal liquor washes once, dries standby.

8. the application of oligonucleotide probe array as claimed in claim 1 or 2 in transgenic product detects, it is characterized in that, choose one section oligonucleotide fragment that has nothing to do with target gene to be checked,, be used for monitoring the non-specific hybridization of hybridization as negative control;

Choose one section with 18S rRNA PCR product paired oligonucleotide fragment mutually, as positive control, be used for monitoring the false-negative appearance of hybridization; Simultaneously, also as the locating point of probe array, be used for determining the position of probe array hybridization region;

Choose the sampling liquid that does not contain oligonucleotide fragment,, be used to monitor the background after the hybridization as blank.

9. the application of oligonucleotide probe array as claimed in claim 1 or 2 in transgenic product detects is characterized in that, according to target gene design to be checked corresponding pcr amplification primer arranged, and is respectively:

Product length

Target gene title primer sequence (5 '-3 ') Tm

(bp)

CaMV?35S????AGACTGGCGAACAGTTCATACAGA<210>2??????????????60.8

188

Promotor GCAATGGAATCCGAGGAGGT＜210〉3 61.1

FMV35S??????CAGCATTCCAGATTGGGTTCA<210>5?????????????????60.7

172

Promotor CTTTTGGCTAATGGTTTGGAGAC＜210〉6 60.1

CaMV35s?????CCCAGATAAGGGAATTAGGGTTC<210>8???????????????60.5

134

Terminator CCCTGGATTTTGGTTTTAGGAAT＜210〉9 60.9

TGAATCCTGTTGCCGGTCTT<210>13?????????????????60.2

The Nos terminator

138

AAATGTATAATTGCGGGACTCTAATC<210>14???????????60.1

GGAAGGGACTGGCTGCTATTG<210>17????????????????61.3

NptII

157

GATGTTTCGCTTGGTGGTCG<210>18?????????????????61.1

GAACCGCAACGATTGAAGGA<210>11?????????????????60.8

The Nos promotor

177

TGGATACCGAGGGGAATTTATG<210>12???????????????60.7

TGTTCCCCAACTACGACTCCA<210>38????????????????60.2

Cry?I?A(b)

151

AGGTGCGGGCTCCTGATGCT<210>39?????????????????66.5

AGACTCAACAGCAGTGGAAATAACA<210>41?????60.2

CryIA(c)???????????????????????????????????????????????????123

GTGAATAGGGGTCACAGAAGCATA<210>42??????60.3

CTGCTGCTGCTGAAGGATGC<210>47??????????61.7

Cry9C??????????????????????????????????????????????????????199

CCCTGCGGAACTGGTGGTAG<210>48??????????62.6

GGTTCTTTTCCTCACTACCTATGCTC<210>44????61.6

CryIIIA????????????????????????????????????????????????????101

CCTCTTTCTCGTATCCCCACTCTT<210>45??????62.3

AGTTCGCTGGTCTCACTGCTG<210>50?????????60.3

GOX????????????????????????????????????????????????????????133

GGACGGAAACCCATCCACTT<210>51??????????60.9

EPSPS???????AGAGCCGTGGATAGATTAAGGAAG<210>32??????60.6

149

(soybean) AGACCGCCGAACATGAAGGA＜210〉33 62.6

EPSPS???????GGGTCTTGTTGGTGTTTACGATT<210>35???????60.1

182

(rape) TGTAGGTGATTGGCGTTGGAG＜210〉36 60.9

TCCGTTTGTTTACTGGTGCTTG<210>26????????60.5

Barnase????????????????????????????????????????????????????192

GAGGGCTTGTGCTTCTGATTTT<210>27????????60.4

AACAAATCAGAAGTATCAGCGACCT<210>29?????60.9

Barstar????????????????????????????????????????????????????145

AACTGCCTCCATTCCAAAACG<210>30?????????61.6

GCTCCACGCTCTACACCCAC<210>20??????????60.2

Bar????????????????????????????????????????????????????????181

AAACCCACGTCATGCCAGTT<210>21??????????60.0

GACAGAGCCACAAACACCACAA<210>23????????60.7

PAT????????????????????????????????????????????????????????144

CAATCGTAAGCGTTCCTAGCCT<210>24????????60.8

GGGACCAAATTATTGATCTAACCTCT<210>53????61.0

CaMV-CP????????????????????????????????????????????????????148

TCTCCTGAATCGCTTTCGTCTT<210>54????????60.9

CAAGTCGTCGCTGTTGAGTTTG<210>56????????61.0

Lectin?????????????????????????????????????????????????????165

GCTGGTGGAGGCATCATAGGT<210>57?????????61.3

CCTCACCTCAGCCACGAATC<210>62??????????60.1

Fbp????????????????????????????????????????????????????????169

GTCAGTGTTTGAAGCTCATACCCA<210>63??????61.0

CCAAGAACTGGATGAACGGTTAG<210>59???????60.7

Zein???????????????????????????????????????????????????????184

GAGAAGCCGGTCGAGGTAGATAG<210>60???????61.5

Rbc1????????GATTATCCGCTAAGAATTACGGTAGA<210>65????60.4

134

(cotton) TTCGGCACAAAATAGGAAACG＜210〉66 60.6

Rbc1????????GAGCGGGATAAATTGAACAAGTATG<210>68?????61.7

198

(potato) TTCGGCACAAAACAAGAAACG＜210〉69 61.4

GAGAAACGGCTACCACATCCA<210>103????????60.0

18s?RNA????????????????????????????????????????????????????254

CGTGCCATCCCAAAGTCCAA<210>104?????????60.8

CCTTCGCAAGACCCTTCCTCTATA<210>71??????63.3

Bt9c???????????????????????????????????????????????????????190

GTAGCTGTCGGTGTAGTCCTCGT<210>72???????60.3

CTCTCGCCGTTCATGTCCGT<210>74??????????60.0

Bt176??????????????????????????????????????????????????????213

GGTCAGGCTCAGGCTGATGT<210>75??????????60.0

ACGGTGGAAGAGTTCAATGTATG<210>77???????60.0

GA21???????????????????????????????????????????????????????270

TCTCCTTGATGGGCTGCA<210>78????????????59.6

TCGAAGGACGAAGGACTCTAACG<210>80??????62.7

Mon810????????????????????????????????????????????????????170

TCCATCTTTGGGACCACTGTC<210>81????????61.4

GCTGAAGCCAGTTACCTTCGG<210>83????????61.6

Bt11??????????????????????????????????????????????????????212

TATCATCGACTTCCATGACCA<210>84????????60.0

CCTTCGCAAGACCCTTCCTCTATA<210>86?????62.1

T25???????????????????????????????????????????????????????231

AGATCATCAATCCACTCTTGTGGTG<210>87????60.6

TGTAGGAGCCACCTTCCTTTTC<210>89???????61.2

RRS???????????????????????????????????????????????????????177

AACCCTTCAATTTAACCGATGC<210>90???????60.2

TACGGCGAGTTCTGTTAGGTCC<210>92???????62.0

Luxuriant No. one 146 of China

CCAGGCTTTACACTTTATGCTTC<210>93??????62.3

10. the application of oligonucleotide probe array as claimed in claim 9 in transgenic product detects, it is characterized in that, extracting DNA of plants to be checked, with primer target gene to be checked is carried out pcr amplification, and carry out hybridizing with oligonucleotide probe array again behind the fluorescent mark, obtain the relevant information of plant to be checked by scanner scanning.

11. the application of oligonucleotide probe array as claimed in claim 10 in transgenic product detects is characterized in that, describedly target gene to be checked is carried out fluorescent mark can adopt labeled primer method, fluorescence to mix method or TDT enzyme addition.

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