CN1607258A - Gene chip linear amplification indirect marking technology - Google Patents

Abstract

A signal amplifying marking technology and kit thereof which uses enzymology or chemistry method and fluorescence and biochemistry luminescence etc variety of nano microsphere as high load signal carrier to greatly raise detection sensitivity. Said invention overcomes the shortage of low sensitivity and high cost in non -linear marking of direct marking method, solves the affection of cDNA hybridization efficiency to making process in gene chip direct making.

Description

Indirect method gene chip linear amplification labeling technique Indirect Linear Amplification Labeling for Microarray Assays[iLAL]

The technical literature that the present invention quoted

Su; People's U.S. Patent No.s such as Sheng-Hui 6,130,323.Non-nucleotide?linking?reagents。October 10,2000.

Nelsen, people such as p.S., Nuclear Acids Research.20:6253,1992.

Song Ke, Chinese patent application, gene chip enzyme process microballoon magnifying tags.Application number 031420818.On August 6th, 2003.People such as ROBIN L.STEARS, " A novel, sensitive detection system for high-density microarraysusing dendrimer technology ".Physiological Genomics3:93-99,2000; 1094-8341/00.DeRisi people such as JL, Exploring the metabolic and genetic control of gene expression on agenomic scale.Science 278:680-686,1997.

People such as Schena M, Quantitative monitoring of gene expression patterns with acomplementary DNA microarrays.Science 270,467-470.1995.

People such as Duggan D., Expression profiling using cDNA microarrays.Nature Genetic Supplement21:10-14,1999.

People such as Charlie C.Xiang.Amine-modified?random?primers?to?label?probes?for?DNAmicroarrays.Nature?Biotechnology。Vol20，738-742.2002

Affiliated field

The invention belongs to the gene chip applied technical field, particularly relate to the hybridization of gene chip, mark, wash-out, and signal detection technique.

The technology of the present invention background

Usually (be Cy with fluorescence dye during gene chip is analyzed at present ^TM3 or Cy ^TM5) thing that serves as a mark carries out mark.Marker can directly be introduced by reverse transcription [reverse transcription] technology when cDNA is synthetic, aRNA amplifies or back aRNA reverse transcription reaction [post-aRNA reverse transcription (RT) reaction], or connects by the chemical coupling reaction after any enzyme reaction.Directly mark needs enzyme reaction at cDNA, introduces the dNTP/NTP nucleotide structure analogue of Cy mark in aRNA or the RT product.

At present, on behalf of mRNA, gene chip transcribe product from laboratory sample with cDNA[with reference to sample in actually operating] with different fluorochrome labels, mixing and be hybridised on the chip.Measured fluorescence intensity can't be directly proportional with its actual abundance of transcribing product.

Though direct labelling method looks more convenient, there are some problems.At first, the quantity of the base U that is labeled of introducing will depend on the based composition of cDNA, i.e. the content of base A among the mRNA.Secondly, participate in the length that nucleosides quantity in the tagged molecule also depends on transcription product.Very long transcription product or/and contain a large amount of base A template can produce by the cDNA of stronger fluorescent signal mark.In the case, strong signal is not represented high expression abundance, and is therefore not proportional with the amount of the target sequence of actual hybridization to the mark of the target sequence in the hybridization, and this causes difficulty for the accurate quantification during gene chip is detected.The 3rd, Cy5 and Cy3 introducing level or efficient in reverse transcription reaction is not high, and efficient difference separately, can produce different quantum yield and various fluorescence intensity, therefore, will be very necessary to the normalization method of fluorescence intensity, this is the influence that downstream data is analyzed.The 4th, adopt the direct method mark to carry out signal and amplify the restriction that is subjected to magnification.Present most methods needs total RNA sample of minimum 20 micrograms, or 2 microgram poly (A) RNA samples people such as [, people such as Duggan D.] Schena M, often seems powerless for some rare tissues of research or the fewer sample of sample size.The 5th, the too much fluorescence molecule that participates in often causes hybridization efficiency or specificity to reduce.And have data to show, the multiple stability that can reduce their formed heteroduplexs that participates in that is labeled nucleosides in probe or the target gene.Data shows, have the synthetic oligonucleotide of the plain group of multi-fluorescence, the nucleic acid probe of biogenetic derivation that has the plain base of multi-biological and the stability of heteroduplex that has the synthetic oligonucleotide of vitamin H group and the fluorescein base group concurrently [Su that all descends, people such as Sheng-Hui, U.S. Patent No. 6,130,323].

Indirect labelling is introduced a base media usually on will be by the nucleic acid molecule of fluorochrome label.This base media can be the base of amido modified [amino allyl group-], then, and with Cy5 or the combination with it of Cy3 molecule.But in reverse transcription reaction, indirect method does not overcome the problem that fluorescence intensity in the direct mark depends on cDNA length and based composition.

Purpose of the present invention

The objective of the invention is for the gene chip product provide one the cover utilize zymetology or the chemistry method, utilize high lotus signal vehicle to promote detection sensitivity significantly, the signal magnifying tags technology and the test kit of economic and practical.Solve non-linear mark in the direct method mark simultaneously and occur can't accurate quantification, sensitivity deficiency, defective such as with high costs, the proportional relationship that makes the fluorescence signal intensity of generation and actual abundance in the genetic expression become to amplify.Technical scheme among the present invention has solved the influence problem of the hybridization efficiency of cDNA in the direct mark of gene chip to labeling process simultaneously.

The present invention compares the positively effect that is produced with background technology

The method that is provided among the present invention has not only improved the gene chip detection sensitivity, reduce cost, and overcome exist in the direct labeling technique can't accurately quantitative various technical factor, as the terminal enzyme (DNA) method and the represented method of acquisition sequence method that can be regarded as the tailing mark in the present invention.

Conclusion is got up, and the beneficial effect that the present invention brought has following several aspect:

1, the technical foundation of super sensitivity detection is provided for general biochip technology product, technology has more powerful signal amplification effect than existing in-vitro transcription [IVT] mark, Cy series fluorescence molecule labeling technique, can select the high lotus signal vehicle of different size that signal amplification factor is brought up to 10 as required ²-10 ⁷Not etc.

2, for cost and the running cost that reduces the gene chip treatment system provides technical support, help the popularization and application of gene chip;

3, overcome false positive problem in the gene amplification technology such as PCR;

4, overcome and utilize Cy3/Cy5 fluorescence molecule in the reverse transcription reaction to participate in the fluorescence signal intensity that the direct labeling technique of method produced also to depend on the uncertainty that cDNA length and based composition are caused, the fluorescence signal intensity that marking method produced among the present invention then only depends on the molecule number of cDNA, therefore can linear quantitative detection.Therefore, the actual abundance linear relationship in direct ratio of fluorescence signal intensity that technical solutions according to the invention produced and genetic expression.

5, the crossover process of the end-labelling in the indirect labelling is before labeled reactant, and the base of hybrid molecule was not modified before hybridization, overcome the problem of the low or mismatch hybridization of hybridization efficiency in the direct mark, improve the stability of hybrid molecule, also helped the rigorous hybridization of probe and target gene simultaneously.

Specific term is explained

Template [Template, T]

The nucleic acid molecule substrate of various enzymes [as DNA/RNA polysaccharase, reversed transcriptive enzyme etc.] is nucleic acid molecule to be detected in biological sample.Template is through processing

Primer [Primer]

Be positioned to be synthesized or the DNA that transcribes or segmental certain particular sequence of RNA and with one section nucleic acid fragment of this sequence nucleotide sequence complementary, live the synthetic of RNA or transcribe of startup DNA such as bootable corresponding D NA or RNA synthetic enzyme.

Probe [Probe]

What contain a certain specific nucleic acid or peptide nucleic acid(PNA) base sequence is fixed on the on-chip DNA of gene chip, RNA, oligonucleotide or peptide nucleic acid(PNA) [peptide nucleic acid, PNA] fragment.

Target sequence [Target Sequence, TS]

Be used for gene chip on the nucleic acid probe complementary nucleic acid or the peptide nucleic acid sequence of hybridizing.

Reporter group [Reporter Group, Y-]

In nucleic acid marking reaction, the present invention directly is connected to signal tracer [as the fluorescent signal molecule etc.] on the target sequence, but at first intermediary's group is connected on the target sequence, or produces a reporter group by the synthetic method on target sequence.Reporter group its objective is in next step labeled reactant as label, beacon or a joint, shows the generation of hybridisation events on certain probe molecule in certain site of gene chip, and preparation simultaneously is connected with tagged molecule.

Report primer [Reporter Primer]

By zymetology or chemical process synthetic, be connected with the primer sequence of reporter group.

Report target sequence [Report Target Sequence, RTS]

The target sequence that has one or several reporter groups at DNA, RNA, oligonucleotide or peptide propylhomoserin molecular end or other positions.Reporter oligonucleotide [Reporter Nucleotide]

Various [deoxidation or two deoxidation] Nucleotide or nucleotide analogs that have reporter group are as amino allyl group deoxidation Nucleotide [aminoallyl deoxynucleotides, aa-dUTP etc.], biotinyl dideoxy nucleotide [biotin-ddUTP] etc.Under enzyme or chemical action, these reporter oligonucleotides are impregnated in or are connected in report primer or the report target sequence.

The joint group [Y '-]

Can take place that specificity interacts with reporter group and bonded biological or chemical molecule or group with it by covalent linkage or non-covalent bonding force.

Report primer [Random Reporter Primer, RRP] at random

By the dna synthesizer synthetic have might base sequence the summation of report primer.The primer part of report primer is six poly-or eight polynucleotides normally.

Acquisition sequence

A nucleic acid or a peptide nucleic acid(PNA) fragment that is connected to a specific base sequence on the target sequence.Acquisition sequence plays a part the target sequence reporter group, for the labeled reactant of next step and high lotus signal vehicle [HSC] ready.It can be by forming being connected of a nucleic acid double chain with the complementary hybridization of the joint sequence that is fixed on the HSC surface.

Joint sequence

Fixing [connecting by covalent linkage or non covalent bond] is on the HSC surface, with one section nucleotide sequence of acquisition sequence base complementrity paired.

Know-why of the present invention

The present invention proposes a kind of indirect method gene chip linear amplification labeling technique, its principle is, to carry the reporter oligonucleotide [Reporter Nucleotides] of reporter group (Reporter Group) [Y-], with testing gene sequence in the sample is template, under enzyme or chemical action, be incorporated in the target sequence, synthetic report target sequence [Reporter target sequences], the latter and the probe array hybridization that is fixed on the gene chip, form the heterozygosis two strands of report target sequence and its complementary probe, the high lotus signal vehicle [HSC] that reporter group joint group [Y '-] arranged with finishing is with the double-stranded reaction of this heterozygosis and combine with it, thereby realizes the signal magnifying tags.

This gene chip is made up of with the probe that is no less than two arrays arrangements that is fixed on substrate [being abbreviated as S] or coating [being abbreviated as C] surface the coating or the coating on substrate and surface thereof, is used for the biological or chemical target material of test sample.The material of substrate and/or coating or coating can by but be not limited in the following material any one or more than one differing materials combination and make:

(1) inorganic sheet or tabular material type such as glass, quartz, mica, transparent conductive material such as transparent conductive oxide class [TCO] are as tin indium oxide [indium tin oxide, ITO, or be written as In ₂O ₃: Sn], InAs, SnO ₂: coating such as F, gallium arsenide [GaAs], magnesium oxide, stannic oxide [tin oxide, SnO], ZnO, CdO, CdIn ₂O ₄, Cd ₂SnO ₄, Zn ₂SnO ₄And In ₂O ₃-ZnO etc., and carbon/pottery composite conductive ceramic, various semiconductor materials etc., and porous plate pattern, as microwell plate, droplet plate,

(2) simple substance class: platinum, gold and silver, aluminium, chromium metals such as [Au, Ag, Pt, Cu, Rh, Pd, Al, Cr] and silicon etc. are nonmetal,

(3) organism class: each organic molecular species and the self-assembled monolayer of making thus thereof or self-assembly multimolecular film,

(4) high score subclass: nylon membrane, plastics, rubber, resin, nitrocellulose filter.

The structure of substrate can be but be not limited to following kind:

(1) thin slice/flat: as sheet glass, silicon chip, nylon membrane, plastic sheet,

(2) multiaperture-type: as microwell plate, droplet is board-like etc.,

(3) electric pole type: on the sheet type gene chip, arrange the various electrod-arrays form with certain rule, as the platinum electrode in substrates such as semi-conductor, isolator, made, gold electrode etc.,

(4) micro-tubular: by kapillary or other material of kapillary tubular intracavity constructional feature is arranged, arrange various matrix form kapillary gene chips [two dimensional surface arrangement], linear kapillary gene chip [one dimension linear array] that forms and the various matrix form parallel arranged of in substrate formula structure, producing [being microfluid electrophoresis chip etc.] that are no less than a microchannel formula cavity structure by the predefined position rule of correspondence

Probe, target sequence or template [T] that the said gene chip of the present invention is used for detecting can be, but be not limited to certain composition of one of following kind or different types of combination or following kind, extract:

(1) Yeast Nucleic Acid [RNA], various Yeast Nucleic Acid [" ribonucleic acid " and " RNA "], comprise messenger RNA(mRNA) [be called for short mRNA], change RNA[and be called for short tRNA], amplify Yeast Nucleic Acid [amplified RNA] or antisense rna [anti-sense RNA, be called for short aRNA], complementary RNA [cRNA] etc.

(2) thymus nucleic acid [DNA] is as DNA, complementary DNA [cDNA] etc.,

(3) oligonucleotide [oligonucleotide] comprises dna oligo [oligodeoxynucleotide is called for short ODNs], oligomerization Yeast Nucleic Acid [oligoribonucleotide, ORNs], and polynucleotide [polynucleotide],

(4) the nucleic acid construct resemblance [DNA-DNA, RNA-RNA or RNA-DNA mimic duplexes, triplexes or quadroplexes, etc.]] as peptide nucleic acid(PNA) [peptide nucleic acids, be called for short PNA],

(5) hybrid molecule of forms such as the complementary two strands of thymus nucleic acid, Yeast Nucleic Acid, peptide nucleic acid(PNA) and structural similitude thing thereof, three [weight] chain, four [weight] chain such as DNA-DNA, RNA-DNA, RNA-RNA, PNA-DNA, PNA-RNA, PNA-RNA-PNA, PNA-DNA-PNA] etc.

The present invention adopts high lotus signal vehicle (high molecular fluorescent microballoons or contain a large amount of fluorescence dyes in the gene chip treatment technology, the semiconductor nano quantum dot, the polymeric particles of electroluminescent molecule etc., a large amount of signaling molecules are polymerized to the various forms of huge signaling molecule of one by synthetic technology, silica optics magnetic bead [silica beads], Radioactive colloidal gold [or nm gold particles, colloidal gold nanoparticles], or pass through any other means with fluorescence dye, non-fluorescence dye, semiconductor-quantum-point, electroluminescent, chemoluminescence or noclilucence material signaling molecule etc. are fixed in the surface of particulate or are embedded in the high lotus signal vehicle of various ways such as its inside) thing that serves as a mark carries out molecular signal and amplifies.

The gene chip signal magnifying tags technology that the present invention will introduce is to be applied to the various forms of gene chip probes of mark, target or probe-target crossbred with various forms of high lotus signal vehicle or signal body.

Here said gene chip includes but not limited to following type: gene chip [comprises various DNA chips, as the glass gene chip, film chip, lab-on-a-chip, micro-fluid chip etc.], based on various biosensors of electrochemical principle or biological electronics principle etc.

The detection principle of gene chip used herein can be, but is not limited to the combination of one of following kind or different principle:

(1) carries out optical signalling [as fluorescence molecule, chemoluminescence etc.] mark by probe-target-specific affinity reaction bonded body or the crossbred that solid phase surface is positioned at different positions, then through exciting generation fluorescence, chemistry or noclilucence and detecting.With glass, silicon chip, nylon membrane etc. is that gene chip, protein chip, the immuno-chip of material taked this pattern more;

(2) by translational speed or the mobility in the liquid phase that has applied voltage detects to probe mark or unlabelled or target, be electrophoretic technique or combine with other technologies and the other technologies that produce by electrophoretic technique, as capillary electrophoresis [capillaryelectrophoresis, CE], capillary zone electrophoresis [capillary zone electrophoresis, CZE], capillary gel electrophoresis [CGE], HPCE [HPCE], capillary isoelectric focusing [capillary isoelectric focusing (CIEF)], isotachophoresis [isotachophoresis (ITP)], electrokinetic chromatography [electrokineticchromatography (EKC)], mcellar electrokinetic capillary chromatography [micellar electrokinetic capillarychromatography (MECC OR MEKC)], capillary electrochromatography method [capillaryelectrochromatography (CEC)], nonaqueous phase capillary electrophoresis [non-aqueous capillaryelectrophoresis (NACE)] etc.

(3) by probe, target or probe-target complex compound and the different avidity of their marker, carry out the chromatographic technique analysis, as affinity chromatography [affinity chromatography], SEC[size exclusion chromatography], GPC[gelpermeation chromatography], MCC[metal chelate chromatography] etc.

(4) utilization has the molecule of redox property or the part-structure of molecule [being chemical group or functional group], metal complex or (nucleic acid) intercalator be positioned and be fixed on probe on solid phase surface different positions or the electrode, target or probe-target complex compound combination is also adding under suitable mode or suitable big or small voltage (or potential difference) effect (volts DS or exchange current or pulsed electrical field or voltage), redox reaction takes place, cause from reductibility molecule or group (electron donor, electrondonor) transfer transport is to oxidisability molecule or group (electron acceptor(EA), electron acceptor), form stream of electrons, being sent to detection system by electrode measures, measured strength of current size is directly proportional with the amount of the double-strandednucleic acid of hybridizing and being labeled

(5) utilize the conductivity difference of double-strandednucleic acid and single-chain nucleic acid, or utilize by between the formed double chain acid molecule of the nucleic acid hybridization of the complete complementary pairing of sequence and double-strandednucleic acid (the nucleic acids duplexes) molecule that mispairing base pair (Mismatches) existence is arranged in conductivity difference, by various discriminatings to other aspects that nucleic acid molecule is reflected thus being carried out via the measurement of the stream of electrons size of nucleic acid molecule conduction, detect and investigate, as in transgenation, gene pleiomorphism, gene sequencing, gene expression profile research, the application of aspects such as pathogenic mechanism analysis and medical diagnosis on disease

(6) will occur on the nucleic acid molecule or and (include but not limited to by material with redox property, have the metal complex of redox property and be the polymkeric substance that monomer polymerization forms by the derivative of metal complex or metal complex, fluorescence molecule, nucleic acid intercalators etc.) electronics that is taken place or charge transfer reaction or stream of electrons convert other to and can detect the detection technique that maybe can investigate form, as by electroluminescent [Electroluminescence] principle, utilize the electroluminescent molecule that stream of electrons is converted to the detection that optical signal carries out

(7), thereby realize detection to the biological or chemical reaction that occurs in electrode surface by making marker electron-transfer reaction take place behind the mark near electrode surface directly or indirectly having oxidation-reduction quality material (as ferrocenyl) to probe or target molecule marker.ESensor Chips as Motorola Inc..

(8) pass through probe, target or probe-target complex compound and the different avidity of their marker, carry out the chromatographic technique analysis, as affinity chromatography [affinity chromatography], SEC[size exclusion chromatography], GPC[gelpermeation chromatography], MCC[metal chelate chromatography], electrokinetic chromatography [electrokinetic chromatography (EKC)], mcellar electrokinetic capillary chromatography [micellarelectrokinetic capillary chromatography (MECC OR MEKC)], capillary electrochromatography method [capillary electrochromatography (CEC)] etc.

Here the high lotus signal vehicle [HSC] of said gene chip be meant by any way with signaling molecule be concentrated in molecule inside, surface, carry from the teeth outwards or the three have both at the same time.These HSC include but not limited to following type; the high loading optics that exists with various molecule forms; magnetic; electronics or electrochemical signals carrier; it can be quantum dot microsphere [QD-tagged Microbeads]; superpolymer fluorescent microsphere or high molecular fluorescent microballoons [polymer microspheres] or colloid microballon [latex beads]; at particle surface; inner by fixing; embedding; modes such as chemistry covalent bonds are with signaling molecule such as luminescent dye molecule; chemiluminescent molecule; [magnetic] silica microballon [silica beads] that chromonic molecule etc. concentrates; the brilliant quantum dot [colloidal fluorescent semiconductornanocrystal quantum dots] of colloid luminescent semiconductor nanocrystal; dendroid high polymer nano ball [dendrimers]; star dendrimer or dendrimer [stardendrimers; dendrimeric stars or dendrimers]; the semiconductor nanoparticle of protein parcel or molecule or quantum dot [as wrapping up best results with Chaperonin GroEL albumen and T.th cpn albumen among the Chaperonin mediated semiconductor nanoparticles.Chaperonin]; micella [micelles]; molecular magnet [molecular magnets]; capsule-type microballoon [encapsulated spheres]; colloidal nano gold [colloidal gold nanoparticles]; electroluminescent molecule [electroluminescent molecules]; two Metallocenyl copolymer microspheres [polymetalcene block copolymers; as ferrocene-based polymer [polyferrocene block copolymers; PFC; or polyferrocenylsilanes; PFS] microballoon; poly zirconocene [polyzirconocene; PZC]; poly two Metallocenyls are modified or the grafted dendrimer [includes but not limited to; poly-ferrocene base grafted dendrimer (polyferrocenyl-brancheddendrimers)]; two Metallocenyls or poly two Metallocenyls are modified or grafted nanometer gold colloid [includes but not limited to; the nanometer gold colloid of amido ferrocenyl functionalization (Gold colloids functionalized with amidoferrocenyl structures) etc.]; copolymer microsphere or nanometer ball; contain fullerene structure or be the superpolymer or the multipolymer of one of monomer with the soccerballene; other contains fluorescence dye or chemoluminescence [chemiluminescence]; the carrier of noclilucence [bioluminescence] material; phycobiliprotein class [phycobiliproteins] and various derivative thereof or biological or chemical modifier; and in above-mentioned each carrier different parameters [as size; the optical emitting optical wavelength; electromagnetic property; physics or chemical property such as the modification of surface biological or chemical functional group] combination of carrier, or the mutual combination between the various different carriers.

One of signal amplification technique sight that the present invention will introduce, be a kind of terminal enzyme (DNA) [Terminal DeoxyribonucleotidylTransferase, be abbreviated as TdT] mediation gene chip signal magnifying tags technology, it is characterized in that, with the nucleosides [modifiednucleotides] modified and not modified nucleosides under the effect of this enzyme, utilize testing gene (DNA, RNA or oligonucleotide) in the sample for template, synthetic modification target sequence.Hybridize with this new synthetic target sequence and the gene probe that is fixed on the gene chip surface.Afterwards, through elution process, again with finishing have can with the high lotus signal vehicle of the biological or chemical group of reporter group generation specific reaction on this target sequence and gene chip on have a target sequence heteroduplex carry out chemical covalent linkage or non covalent bond coupling, realize specific signals magnifying tags to hybrid molecule.

TdT be an effect and template sequence irrelevant archaeal dna polymerase, can be 3 '-add a deoxynucleoside on the terminal hydroxyl, outstanding on two strands or the strand, retraction can become the substrate of TdT with 3 ' of tack-end.

Two of the signal amplification technique sight that the present invention will introduce is a kind of primer method [Random Reporter Primer is abbreviated as RRP] marks of reporting at random.This method is based on random priming.Promptly, gene chip random primer magnifying tags technology by the Klenow enzyme mediation that has 5 ' → 3 ' DNA composite reactive in the archaeal dna polymerase, it is characterized in that, with the nucleosides [modified nucleotides] modified and not modified nucleosides under the effect of this enzyme, with might sequence the mixture of oligonucleotide be primer, utilize testing gene (DNA or RNA) in the sample for template, synthetic modification the new nucleic acid sequence as target sequence [Target Sequence is abbreviated as TS].When this report primer was synthetic, we mixed amino allyl group deoxidation ribonucleoside [aminoallyl deoxynucleotide triphosphate is abbreviated as aa-dNTPs], generated to have one or several amido modified primers.We are referred to as to report primer [Reporter Primer].So,, have one or several amino at 5 '-end as primer synthetic TS.Be referred to as to report target sequence [Reporter Target Sequence is abbreviated as RTS].Number of amino groups in the report primer can be controlled when dna synthesizer is synthetic, and optimal number is one.Even be not one in fact, because the limited length of report primer, synthetic RTS can be because sterically hindered in follow-up mark, and reaching only has a HSC by covalent linkage coupling mark.

Hybridize with the gene probe that is fixed on the gene chip surface with this newly synthetic RTS.Afterwards, through elution process, again with finishing have can with the high lotus signal vehicle of the biological or chemical group of functional group generation specific reaction on this new template and gene chip on have a RTS heteroduplex carry out chemical covalent linkage or non covalent bond coupling, realize specific linear magnifying tags to hybrid molecule.

Synthetic might sequence the primer of report at random [RRP] mixture the template in the sample solution is carried out DNA or reactions such as RNA is synthetic, reverse transcription or in-vitro transcription, thereby the synthetic report target sequence [RTS] of hybridizing of can be used for accordingly., after complementation hybridizes on the gene chip probes, fixed and to have reacted with the HSC of reporter molecules reaction and generation covalent coupling at such RTS, realized the linear magnifying tags of signal with the surface.

Three of the technology sight that the present invention will introduce, be a kind of be that reverse transcription reaction carries out quantitatively by acquisition sequence.Utilize this method, unmodified or unlabelled dNTP are added to test cDNA and with reference among the cDNA respectively, and oligo (dT) primer is added in the RNA solution.Different is that the oligo here (dT) primer contains an acquisition sequence.TTTTT+++++++ represents to be added to the acquisition sequence with reference in the RNA solution such as, TTTTT------represents to be added in the test RNA solution.Two kinds of different acquisition sequences that are connected on the above-mentioned primer are used respectively------and ++ ++ ++ the expression.Then, the reverse transcription reaction synthetic is tested cDNA and is mixed and and gene chip hybridization with reference to cDNA.Bathe with high lotus signal vehicle [HSC] marker temperature after hybridization and the wash-out.The surface of each high lotus signal vehicle [HSC] is fixed with the nucleotide sequence with above-mentioned acquisition sequence complementary pairing respectively, is called joint sequence, is used for being hybridized by hybridization fixed acquisition sequence with the gene chip surface.

Like this, each cDNA molecule only can produce a strength of signal.And synthetic cDNA is not at the very start by the signal mark, but adds earlier an acquisition sequence on primer, just in incubation period just by the signal mark.

Four of the technology sight that the present invention will introduce, be the chemical coupling reaction taking place specifically and form the group that covalent linkage connects, as amino [NH by chemical method as DNA, RNA, PNA or the oligonucleotide of target gene or synthetic one of 5 '-end or 3 ' of oligomerization PNA-end ₂], sulfydryl [SH] etc.After the probe hybridization on this target gene and gene chip, this group as reporter molecules can be further with tagged molecule [HSC] on corresponding specific reaction group react, thereby form firm covalent linkage mark.Here synthetic can be the synthetic chemical functional group that carries out on DNA, RNA, PNA or oligonucleotide at biogenetic derivation or the oligomerization PNA.

Five of the technology sight that the present invention will introduce is directly when DNA, RNA or oligonucleotide acid molecule are synthetic, adds such specificity chemical functional group at its end, as the reporter molecules of follow-up labeling process.Be such nucleic acid or oligonucleotide acid sequence according to different molecular biology processes, different sequences and functional requirement can be arranged respectively.

If target gene is mRNA, so can be by synthetic 5 '-terminal poly (A) sequence complementary oligo (dT) with it.Simultaneously, 5 '-end at oligo (dT) adds groups such as amino or sulfydryl, formation 5 '-H ₂N-Oligo (dT)-3 ' or 5 '-HS-Oligo (dT)-3 ' etc.Afterwards, by reverse transcription reaction, on new synthetic primer, be template composition sequence complementary 5 '-H with target gene mRNA ₂N-Oligo (dT)-cDNA-3 ', or the target-gene sequence that 5 '-HS-Olig (dT)-cDNA-3 ' is new.This sequence will be used to gene chip on the gene probe direct cross.And be fixed with the high lotus signal vehicle of can be with above-mentioned specificity chemical functional group [amino, sulfydryl etc.] reaction and forming the group that covalent linkage be connected with follow-up surface and carry out the chemical coupling labeled reactant.

By Enzymology method [as terminal enzyme (DNA) [terminal transferase, be abbreviated as TT], reversed transcriptive enzyme, (Taq) archaeal dna polymerase, Klenow enzyme [be referred to as enzyme, and represent with letter e]] synthetic or chemical process synthetic labeled reactant can represent with following general formula

The formula of specimen preparation, hybridization and labeling process can be expressed as follows,

1. template (T can be DNA, RNA, oligonucleotide or the peptide nucleic acid(PNA) of various strands or two strands) is under enzyme (E) or chemical action, and formation target sequence ZNA (Y) n[ZNA can be DNA, RNA, oligonucleotide or nucleic acid analog sequence],

T→ZNA(Y)n??????????????????????????????[I]

2. the nucleic acid probe hybridization on target sequence ZNA (Y) n and the gene chip,

ZNA(Y)n+_XNA→_XNA:ZNA(Y)n????????????[II]

Finishing have can with the biological or chemical joint group Y ' of Y-reporter group specific reaction-high lotus signal vehicle and XNA:ZNA (Y) n on the gene chip carry out chemical covalent linkage or non covalent bond coupling,

_XNA:ZNA(Y)n+HSC(Y’)m→_XNA:ZNA(Y) _n-p[-Y-Y’-] _p-HSC(Y’) _m-p[III]

Said n, m, p are to infinitely-great arbitrary natural number numerical value 1,2,3... from 0.But to one of above-mentioned technology sight, three, be optimum value during p=1.

XNA is nucleic acid probe or the peptide nucleic acid probe sequence on the gene chip, and HSC is high lotus signal vehicle.

Y-can be but be not limited to: digoxin-, anti digoxin antibody-, amino [as amino allyl group-, aa-, N-hydroxy-succinamide base] etc. [seeing the following form].

The enzyme [E] and the zymetology effect that are prepared target sequence by template can be, but be not limited to the combination of one of following mode or different modes,

1. terminal enzyme (DNA), [Terminal Deoxyribonucleotidyl Transferase is called for short TdT] and add [dideoxyribonucleoside (a dideoxynucleotides at 3 ' of DNA-end, as 2 ', 3 '-two deoxyadenines-5 '-triphosphoric acid (2 ', 3 '-Dideoxy-adenosine 5 ' triphosphate, ddATP), ddUTP, ddTTP, ddCTP)] or a plurality of [deoxynucleoside (deoxynucleotides, as (dUTP, dTTP, dCTP etc.)] terminal transferase reaction of base

2. reversed transcriptive enzyme, include but not limited to Superscript II RT, avian leukosis poison (Avian MyoblastosisVirus, AMV) reversed transcriptive enzyme and mouse retrovirus (MMLV) reversed transcriptive enzyme, perhaps StrataScript reversed transcriptive enzyme, PowerScript reversed transcriptive enzyme, can be used for mixing in the reverse transcription process media nucleosides, as the Taq enzyme

3.DNA polysaccharase, as the Taq enzyme, escherichia coli or e. coli dna polymerase I,

4. the Klenow fragment that has the archaeal dna polymerase of 5 ' → 3 ' DNA composite reactive,

5.RNA polysaccharase, as SP6, T7 and T3 phage rna polymerase [phage RNA polymerases] can be used for in-vitro transcription [in vitro transcription] method mark, Superscript II RT,

6.DNA polysaccharase I[polymerase I].

Y-functional group modified nucleoside [modified nucleotides] can be, but be not limited to:

1. amino allyl group-nucleosides is as amino allyl group-dUTP[aa-dUTP],

2. biotinylated dideoxyribonucleoside, as biotinylated pair of Brdurd [biotin-16-ddUTP],

3. the dideoxyribonucleoside modified of digoxin, as DIG-11-ddUTP, two Brdurd triphosphoric acids [djdeoxyuridine-triphosphate], as (e.g-, IG-ddUTP),

Y-wherein and Y '-can be respectively, but be not limited to one of reaction pair as shown in the table or their combination;

Y-or Y '-

ZNA-Y、NTP-Y、

ZNA-、NTP、

	DNTP-Y and ddNTP example	DNTP, ddNTP example
			Phenyldiboronic acid, PDBA, the SHA-digoxin-, the DIG-digoxin-, anti digoxin antibody-amino, as amino allyl group-, aa-, the N-hydroxy-succinamide base-, amino-[aminolinker-], digoxin-, the DIG-NHS ester, N-hydroxy-succinamide base [NHS-]-, anti digoxin antibody-, biotinyl-, avidin-, Streptavidin-chloromethyl [chloromethyl], amino mercapto [sulfhydryl], halogen acetic acid base [Haloacetyls], alkylogen [Alkyl halide] derivative, maleimide [Maleimides], acridine [Aziridines]; Acryloyl [Acryloyl] derivative; Arylation [Arylating] reagent; Sulfydryl-acquisition sequences such as two sulphur compound displacer reagents, 5 '-ATTCGGAA-3 '; Joint sequence, 3 '-TAAGCCTT-5 '	PDBA-UTP DIG-11-UTP Digoxigenin-11-ddUTP aa-dUTP, aa-UTP[Ambion, (Cat.# 8437) DIG-X-NHS ester Biotin-UTP, biotin-dCTP, biotin-dUTP, aa-dUTP such as biotin-ddUTP, aa-UTP[Ambion, (Cat.# 8437) 5 '-HS-C6-Oligo (dT)-cDNA-OH-3 ', 5 '-HS-cDNA-OH-3 ' etc. carry the oligonucleotide acid primer [oligodeoxynucleotide primers containing capture secquences] of acquisition sequence, as 3 '-oligo (dT)-TAAGCCTT-5 ' or 3 '-oligo (dT)-GTAGCAAT-5 '	Ribonucleoside triphosphote-, UTP-, ATP-, CTP-, GTP-, TTP-bi-deoxyribose ribonucleoside triphosphote-[ddNTP-], as ddATP-, ddCTP-, ddUTP-, ddGTP-, ddTTP-. deoxyribonucleoside triphosphate [dNTP], as dATP, dCTP, dUTP, dGTP, dTTP. the same 5 '-cDNA-OH-3 ', 5 '-Oligo (dT)-3 ' oligonucleotide acid primer, as oligo (dT), oligo (dA) ...

The characteristics of these modified nucleosides are, can synthesize, transcribe at nucleic acid or the reverse transcription process in, can be under the effect of enzyme, be incorporated in the target-gene sequence after synthetic and go.

Indirect labelling of the present invention reaction can be, but be not limited to the combination of one of following process or various process,

(1) terminal deoxynucleotidyl transferase (Terminal Deoxyribonucleotidyl Transferase is abbreviated as TdT) effect down the single phosphoric acid nucleoside in the triphosphate deoxy-nucleotide is transferred to 3 of dna molecular '-reaction on the terminal hydroxy group.End deoxynucleotide [TdT] transfer process.

(2) with 5 ' → 3 ' DNA composite reactive of Klenow enzyme, be the DNA building-up process of primer with the mixture of the oligonucleotide acid of all possible base sequence, the random primer labelling reaction,

(3) under reversed transcriptive enzyme [Reverse transcriptase] effect to the reverse transcription reaction of mRNA,

(4) the hybridization labeled reactant that carries out of the oligonucleotide acid primer that connects with acquisition sequence.

HSC represents the high loading optics that exists with various molecule forms, magnetic, electronics or electrochemical signals carrier, it can be superpolymer or multipolymer fluorescent microsphere, surface or internal fixation have silica [perhaps silicon-dioxide] molecule [silicabeads] of fluorescence dye or other signaling molecules, the quantum dot of inorganicss such as semi-conductor [quantum dots], inorganicss such as semi-conductor nanocrystalline [nanocrystal particles], dendroid high polymer nano ball [dendrimers], the dendritic high polymer nano ball of star tree [dendrimeric stars], micella [micelles], molecular magnet [molecular magnets], capsule-type microballoon [encapsulated spheres], colloidal nano gold [colloidal gold nanoparticles], electroluminescent molecule [electroluminescent molecules], poly two metallocene copolymer microspheres [polymetalcene block copolymers, as poly ferrocene multipolymer [polyferrocene block copolymers, PFC] microballoon, poly zirconocene [polyzirconocene, PZC] copolymer microsphere or nanometer ball, contain fullerene structure or be the superpolymer or the multipolymer of one of monomer with the soccerballene, other contains fluorescence dye or chemoluminescence [chemiluminescence], the carrier of noclilucence [bioluminescence] material, and in above-mentioned each carrier different parameters [as size, the optical emitting optical wavelength, electromagnetic property, physics or chemical property such as the modification of surface biological or chemical functional group] combination of carrier, or the mutual combination between the various different carriers, its finishing have can with general formula (I), (II), (III) functional group of probe molecule joint the Y '-specific reaction in the compound or effect to Y-.

The end mark of the dideoxyribonucleoside that example one usefulness vitamin H or digoxin are modified

The characteristics of this kind method are only at each end that is labeled templet gene or target gene Y-of a specific function group of quantitative mark only, thereby can ensure that the signal wire sex ratio amplifies.When Y-is that biotinyl-or during the digoxin base, technical process is as follows:

One, specimen preparation

3 '-end mark that the two Brdurd triphosphoric acids [being abbreviated as DIG-ddUTP] modified with digoxin or biotinylated pair of Brdurd triphosphoric acid [being abbreviated as Biotin-ddUTP] nucleosides carry out oligonucleotide.

Terminal enzyme (DNA) [Terminal Transferase] can be used to add single modified two Brdurd triphosphoric acids [dideoxyuridine triphosphate] at 3 '-end of strand [ssDNA], double-stranded [dsDNA] and oligonucleotide [oligonucleotides], as (e.g, DIG-ddUTP).Process is as follows:

1. the test group oligonucleotide with purifying is dissolved in the aseptic distilled water,

2. in distilled water, prepare the X-ddUTP[X=DIG-of 1mM, Biotin-, amino allyl group (aminoallyl-) or amino hexyl (aminocaproyl-) etc.],

3. following compositions is joined micro-centrifuge tube in ice bath: 1) 5 of 4 μ l * reaction buffered soln [1M potassium, 0.125M Tris-HCl, 1.25mg/mlBSA; PH6.6,25 ℃] (test tube 1).2) the 25mM CoCl of 4 μ l ₂(test tube 2).3) 100pmole oligonucleotide [template or target gene, or cDNA].4) 1 μ l 1mM DIG-ddUTP (test tube 3), or 1 μ l 1mM Biotin-ddUTP.5) 1 μ l (50U) terminal enzyme (DNA) (200mM methyl-arsinic acid potassium [potassium cacodylate], 1mM EDTA, 200mM KCl, 0.2mg/ml, bovin serum albumin [BSA]), 50% glycerol [glycerol], pH6.5[25 ℃] (test tube 4) .6) add distilled water until middle capacity 20 μ l.

Above-mentioned reacted constituent thorough mixing is also simply centrifugal 4.,

5. 37 ℃ of following incubations 15 minutes, be placed on ice then.

6. with one of following method termination reaction:

1) the Glycofurol solution with 200 μ l 0.2M EDTA (pH8.0) and 1 μ l mixes [concentration is the distilled water of 20mg/ml].

2) add Glycofurol-EDTA mixed solution of 2 μ l at reaction mixture.

With following step precipitation mark oligonucleotide [optional step]: [1] adds the LiCl of 2.5 μ l 4M and 75 μ l in advance-15 ℃ to-25 ℃ iced absolute ethanol in reaction tube.Abundant mixing: [2] precipitate 30 minutes at-70 ℃; [3] under 13,000 * g and 2-8 ℃ of condition centrifugal 15 minutes; [4] abandon supernatant liquor; [5] with iced 70% (v/v) washing precipitation granule of 50 μ l; [6] under 2-8 ℃ of condition centrifugal 5 minutes; [7] abandon supernatant liquor; [8] drying precipitated granule under vacuum.

8. will precipitate granule and be dissolved in aseptic, the distilled water of trace, then in hybridization buffered soln the probe solution of dilution equivalent to be fit to directly use.

9. the cDNA or the oligonucleotide solution that prepare a reference group reporter molecules mark with above-mentioned and step 1-8 with quadrat method.

Two Brdurd triphosphoric acids [Digoxigenin-11-ddUTP] that biotinylated pair of Brdurd triphosphoric acid [Biotin-16-ddUTP], digoxin are modified, reorganization terminal enzyme (DNA) [from E.Coli] etc. are available from Roche Applied Science company.Other conventional reagent has commercially available.

Two, hybridization

Following process introduction hybridizes to an oligonucleotide microarray with the two kinds of samples reference group cDNA and the test group cDNA of the mark [] and gets on.

Material: cDNA that cubic capacity is 42ul test group and control group reporter molecules mark or oligonucleotide solution [each 21ul] 20 *, SSC (Ambion Cat#9765), 10%SDS, hybridization chamber, oligonucleotide microarray; 98 ℃ of heating block; 42 ℃ of water-baths; 2X is hybridized buffered soln: 50% formaldehyde, 10x SSC, 0.2%SDS

Techniqueflow:

1. add the competitive DNA mix of 12ul;

2. determine sample volume, and to add water be 54 microlitres up to cumulative volume,

3. sample was heated 3 minutes at 98 ℃,

4. 2x is hybridized buffered soln and above-mentioned cDNA or oligonucleotide reporter molecules label solution 42 ℃ of following incubations 20 minutes,

5. the slide microarray is heated 10min in 100 ℃ dH2O, uses light and slow airflow drying again,

6. cover glass is cleaned, dry and be placed on the slide microarray,

7. the 2X hybridization buffer with 60ul (1x volume) joins above-mentioned cDNA or oligonucleotide reporter molecules label solution and thorough mixing,

8. hybridization solution is loaded into below the cover glass; Allow hybridization solution slowly be attracted to the other end by capillary action, and cover the entire area of microarray from an end of microarray.

9. the 2X SSC with 2 each 5ul is filled into the unlapped position of hybridization solution.

10. sealing hybridization chamber and being immersed in 42 ℃ the water-bath is spent the night.

11. the slide microarray is removed from hybridization chamber.

12. it is immersed in the test tube that 45ml 2X SSC/0.1%SDS is housed of a 50ml, and cover glass is separated with the slide microarray by the action of gravity nature.

13. microarray is transferred in the test tube that 45ml 2X SSC/0.1%SDS newly is housed, and is at room temperature vibrated 5 minutes gently.

14. repeating step 13.

15. microarray is transferred in the test tube that 45ml 2X SSC newly is housed, and is at room temperature vibrated 5 minutes gently.

16. repeating step 15.

17. microarray is transferred in the test tube that 45ml 2X SSC newly is housed, and is at room temperature vibrated 5 minutes gently.

18. repeating step 17.

19. microarray is immersed in the solution of 0.06X SSC and transfers in the centrifuge tube.

20. that is walked from 0.06X SSC solution with the slide microarray, and puts into an empty test tube, and immediately at the centrifugal 2-3min of 800rpm.

Guarantee that microarray is in moisture state before centrifugal.

Three, mark

Labeling process promptly is to make the surperficial fixed of high lotus signal vehicle [HSC] can react and form covalent linkage link coupled process with it with the group of the reporter molecules specific reaction that is connected on the target gene or molecule.For this reason, need at first synthetic this HSC.With in the present embodiment as the vitamin H of reporter molecules, should synthetic surface be fixed with the HSC of avidin.[building-up process is referring to my Chinese invention patent application: gene chip enzyme process microballoon magnifying tags.Application number, 031420818].

Four, wash-out

Elution buffer solution A: SDS 2%, 2X SSC.Elution buffer solution B: SDS 1%, 0.06X SSC.

Elution buffer solution C: ddH ₂O.

1. cDNA or oligonucleotide microarray are taken out from the reaction chamber through the mark treating processes,

2. transfer to the Wash Station that 400ml 1X elution buffer solution A is housed.

3. at 20-25 ℃ of powerful down washing 5min.

4. transfer to the Wash Station. that 400ml 1X elution buffer solution B is housed

5. at 20-25 ℃ of powerful down washing 5min.

6. transfer to the Wash Station. that 400ml 1X elution buffer solution C is housed

7. at 20-25 ℃ of powerful down washing 2min.

8. the residual buffer solution on the microarray is drained with wiping paper with experiment.

9. centrifugal under 500 * g condition.

10. in scanning, microarray is scanned, obtain fluoroscopic image.

The end mark of two deoxyadenines that the example dual-purpose is amido modified

The principle of this kind method is identical with example one with process.Wherein, Y-is amino-[NH ₂].And Y '-be the NHS ester.

The dideoxyribonucleoside triphosphate that amido is modified be 3 '-amino-2 ', 3 '-two deoxyadenines-5 '-triphosphoric acid (3 '-amino-2 ', 3 '-dideoxyadenosine-5 '-triphosphate), be abbreviated as 3 '-amino-ddATP, or AddATP.(TriLinkBiotechnologies?Inc，San?Diego，CA)]。

The end mark of two deoxidation cytosine(Cyt)s that example three usefulness are amido modified

The principle of this kind method is identical with example one with process.Wherein, Y-is amino-[NH ₂].And Y '-be the NHS ester.

The dideoxyribonucleoside triphosphate that amido is modified is 3 '-amino-2 ', 3 '-two deoxidation cytosine(Cyt)s-5 '-triphosphoric acid (3 '-amino-2 ', 3 '-dideoxycytidine-5 '-triphosphate), be abbreviated as 3 '-amino-ddCTP, or AddCTP. (TriLinkBiotechnologies Inc, San Diego, CA)].

The end mark of the dideoxy guanine that the example four-function is amido modified

The principle of this kind method is identical with example one with process.Wherein, Y-is amino-[NH ₂].And Y '-be the NHS ester.

The dideoxyribonucleoside triphosphate that amido is modified is 3 '-amino-2 ', and 3 '-dideoxy guanine-5 '-triphosphoric acid (3 '-amino-2 ', 3 '-dideoxyguanosine-5 '-triphosphate), be abbreviated as 3 '-amino-ddGTP, or AddGTP.(TriLinkBiotechnologies?Inc，San?Diego，CA)]。

The end mark of the dideoxy guanine that example five usefulness are amido modified

The principle of this kind method is identical with example one with process.Wherein, Y-is amino-[NH ₂].And Y '-be the NHS ester.

The dideoxyribonucleoside triphosphate that amido is modified be 3 '-amino-2 ', 3 '-two Brdurds-5 '-triphosphoric acid (3 '-amino-2 ', 3 '-dideoxyuridine-5 '-triphosphate), be abbreviated as 3 '-amino-ddUTP, or AddUTP.(TriLinkBiotechnologies?Inc，San?Diego，CA)]。

Example six carries out quantitatively genetic expression by acquisition sequence and reverse transcription reaction

Stears, people such as Robin L. utilize dendrimer [Dendrimer] by acquisition sequence microarray to be carried out the signal amplification can reduce 16 times with RNA sample demand.Present embodiment uses fluorescent microsphere etc. to be HSC.Present method allows to carry out multi-channel detection on the individual gene chip.The same with dendrimer, because fluorescent microspheres etc. are better than the former on the signal amplifying power, thus can produce higher signal to noise ratio, and easier of the cDNA single counting of hybridization on gene chip, thereby comparing to dendrimer, establishment better counts standard.

In addition, the employed method of present embodiment is simpler, quick than DNA dendrimer method.

In reverse transcription reaction, the at present general Cy3/Cy5 method that the fluorescence molecule modified nucleotide is mixed into needs a large amount of initial sample, nearly the total RNA of 50 μ g or the mRNA of 1 μ g.

Present embodiment is HSC with high molecular fluorescent microballoons or silica fluorescent microballoon.

1.HSC synthetic modified reverse transcription (the Modified RT of bonding probes, be called for short mRT) synthetic following (by Integrated DNA Technologies company) (5 ' to 3 '): the cap3D1 of primer sequence, g gCg ggA CAg AAg ACg CgC AgT gAgTCg gCC, oligo d (T), cap3D2, A CCg gAg Cgg CAC ggg AAA ATg AgC AAC Agg, oligo dT.Total RNA that the mRT primer of 1 picomole can be used for 40 μ g analyzes.

Be used for the RT reaction 2.HSC contain following joint sequence: cpl3D1 (with the cap3D1 complementation), ggC CgA CTC ACT gCg CgTCTT CTg TCC CgC C; Cpl3D2 (with the cap3D2 complementation), CCT gTT gCT CTA TTT CCC gTg CCgCTC Cgg T (Genisphere).

3.RT reaction is according to the GIBCO of standard, SuperScript RT II techniqueflow carries out.Reaction adds the 0.5M NaOH/50mM EDTA of 3.5 μ l, and is heated to 65 ℃ of 10min so that the sex change of DNA/RNA heteroduplex is unwind after finishing.

4. with the 1M Tris of 5 μ l, this reaction that neutralizes of pH 7.5 solution.For the detection of double-tagging (double-colored mark), carry out with directly mixing the high-density DNA microarray direct labelling method of being introduced that method Cy3/Cy5RT reaction technology flow process sees people such as people such as Stears R.L. and DeRisi JL.

5. detect for HSC, the silica fluorescent microballoon of two kinds of different separately excitation/emission wavelength of 2.0 μ l [being respectively 543nm/570nm and 640nm/675nm] [the excitation/emission wavelength is near Cy3-and Cy5-Mk system] (Shanghai Mai Kewo biotechnology company) is suspended in 4 * SSC-2%SDS of 15 μ l again and is applied on the microarray sheet glass.Microarray is built with cover glass, incubation 6-8h in gene chip hybridization chamber (Telechem International) and under 65 ℃ of conditions.By people's such as people such as Stears R.L. and DeRisiJL the method hybridization of being introduced, wash-out and on ScanArray 3000 laser confocal scanning instrument (GSI Lumonics), detect.

6. signal and background noise carry out quantitative analysis with the quantitation software of Imagene.With the mean pixel light intensity signal in each circle in the microarray grid as the signal measuring value.Mean pixel light intensity with the blank spot that do not have cDNA in the microarray is average background noise [background] and the light intensity that is used as the outside mean pixel of grid circle, is used to calculate the specific signal value.

7.HSC it is HSC that reagent preparation choose finishing to have the silica fluorescent microballoon of above-mentioned two kinds of optical characteristics of primary amine groups [Shanghai Mai Kewo biotechnology company].By dna synthesizer synthetic 5 '-end be 5 of sulfydryl '-the SH-mRT primer sequence.When carrying out chemical coupling, HSC is precipitated as solid-state granule, is dissolved in the absolute ethanol by centrifugation.At ambient temperature, the absolute ethanol solution that adds polyvinyl alcohol [PEG] connecting arm [NHS-PEG-MAL] of above-mentioned HSC, 5 '-SH-mRT primer sequence and two exclusive-OR function bases in vitro is fixed with the HSC-(NH-CO-PEG-MAL-S-5 '-mRT-3 ') of joint sequence through chemical coupling reaction synthetic surface _n, be abbreviated as HSC-(mRT) _nReaction mixture again through centrifugation, abandoning supernatant, once more with resolution of precipitate in absolute ethanol, with absolute ethanol washing precipitation granule.In lucifuge ,-20 ℃ of storages.During use, 1%SDS solution dissolution precipitation granule to the solids concentration for preparing with aseptic double-distilled water is 1-5%.

8. semiconductor-quantum-point is more intense because of the fluorescence intensity consistence of each semiconductor-quantum-point as HSC, standard error is less, generally can be controlled at 4-5%, so the typical problem of the reagent that serves as a mark is determined referring to people's such as Stears Robin L. method.

9. to these two kinds of optics microballoons, though their standard error is big than dendrimer, generally along with volume increases, the diameter standard error diminishes as the stdn of HSC reagent for polymer or silica fluorescent microballoon.CV[coefficient of variation such as the high molecular fluorescent microballoons of Duke Scientific company] general diameter when 0.90um, CV＜3%; 0.03 during micron, the CV of diameter＜-20%.Because the labelling experiment of gene chip is the mark of a large amount of molecules normally, so more feasible method is that this mark is carried out the statistical data analysis.At first, the fluorescent microsphere in the localized area carries out artificial counting on the blank slide to being dispersed in body formula fluorescent microscope [Nikon or Olympus].Then, on gene chip laser confocal scanning instrument, the fluorescent signal total intensity that takes place on this localized area is carried out integration under excitation.So just can be in the hope of the strength of signal of average each HSC.Obtain after this mean value the number of the fixing hybrid gene at that point on its statistical significance that the fluorescence total intensity of certain the gene point that records in can the detection according to gene chip is asked.

Example seven carries out quantitatively genetic expression by reporter molecules and reverse transcription reaction

Present embodiment is close with example six.The reverse transcription reaction condition is identical or close with example six with process.It is directly when DNA, RNA or oligonucleotide acid molecule are synthetic, adds a specific biological or chemical functional group at its end, as the reporter molecules of follow-up labeling process.Among general formula I, II, the III, XNA is mRNA, and ZNA is 5 '-Oligo (dT)-primer sequence-cDNA-3 ', and ZNA (Y) n is 5 '-H ₂N-Oligo (dT)-primer sequence-cDNA-3 ' or 5 '-HS-Oligo (dT)-primer sequence-cDNA-3 ' etc., HSC-Y ' _nIn Y '-be succinimide ester or maleimide ester, E is reversed transcriptive enzyme [Reverse Transcriptase, RT], chemical coupling labeled reactant process is with example six.

Example eight is reported primer method (Random Reporter Primer Method is called for short the RRP method) at random

The 5 '-end that uses TriLink Biotechnologies company to produce has the amido modified oligonucleotide of C12 connecting arm.Synthetic have 5 '-C12-NH ₂Six polynucleotides [Hexamers] of various possibility sequences or eight polynucleotides [Octomers] [3 '-oligonucleotide-5 '-OPO ₃ ^--(CH2) _n-NH ₂, n is a natural number.Production number: 0-0003,0-0004,0-0005 etc.] as RT reaction primer, i.e. RRP.With people's such as Charlie C.Xiang RNA purifying, reverse transcription, hybridization, wash-out and testing conditions, test.

HSC can choose Duke Scientific, the fluorescence polystyrene microsphere of the finishing amino of companies such as Molecular Probes.Process-the chemical coupling of HSC labeled reactant is referring to example six.

The amino allyl group Nucleotide of example nine usefulness synthesizes RRP

When synthesizing cDNA, with amino allyl group Nucleotide, as amino allyl group dUTP[5-Aminoallyl-2 '-deoxyuridine-5 '-Triphosphate (TriLink Biotechnologies, Inc)] be RRP, by the synthetic cDNA that is used for gene chip hybridization of reverse transcription reaction.Other reaction process and condition can be referring to examples six.

The amino allyl group Nucleotide of example ten usefulness synthesizes RRP

When synthesizing cDNA, with amino allyl group Nucleotide, as amino allyl group dCTP[5-Aminoallyl-2 '-deoxycytidine-5 '-Triphosphate, TriLink Biotechnologies, Inc] be RRP, synthesize the cDNA that is used for gene chip hybridization by reverse transcription reaction.Other reaction process and condition can be referring to examples nine.

Example 11 '-amino-(5 '-amino-Oligo-dT Reporter Primer Method is called for short 5 '-H to Oligo-dT report primer method ₂N-OT-RP)

In cDNA is synthetic, with 5 '-NH ₂The Oligo-dT of modified synthesizes 5 '-NH ₂-Linker-Oligo (dT)-OH-3 ' is the report primer, by the synthetic 5 '-NH of reverse transcription reaction ₂-Linker-Oligo (dT)-cDNA-OH-3 '.Other reaction process and condition can be referring to examples nine.

Example 12 '-sulfydryl-Ollgo-dT-3 ' report primer method (abbreviation 5 '-HS-OT-RP)

In cDNA is synthetic, with the synthetic 5 '-HS-C6-Oligo (dT) of the Oligo-dT of 5 '-terminal C6 sulfydryl connecting arm modified-OH-3 ' [TriLink Biotechnologies, Inc]] be the report primer, by the synthetic 5 '-HS-C6-Oligo (dT) of reverse transcription reaction-cDNA-OH-3 '.Y ' among the HSC (Y ') is amino, and two exclusive-OR functions group connecting arm is still selected NHS-PEG-MAL or NHS-PEG-VS[succinimido-polyoxyethylene glycol (connecting arm)-vinyl sulphone for use].Coupling reaction process is seen example six.Other reaction process and condition are seen example nine.

Example 13 '-sulfydryl-RP-3 ' report primer method (being called for short 5 '-HS-RRP-3 ')

In cDNA is synthetic, six polynucleotides or eight the polynucleotides 5 '-HS-C6-RP-OH-3 ' of the synthetic various 5 '-end-C6-sulfydryl connecting arm modifieds that may sequences of the method for having made to order with the client, as 5 '-sulfydryl report at random primer (5 '-HS-RRP-3) [TriLinkBiotechnologies, Inc provide this business.Production code member: O-0016], by the synthetic 5 '-HS-cDNA-OH-3 ' of reverse transcription reaction.Y ' among the HSC (Y ') is amino, and two exclusive-OR functions group connecting arm is still selected NHS-PEG-MAL or NHS-PEG-VS[succinimido-polyoxyethylene glycol (connecting arm)-vinyl sulphone for use].Coupling reaction process is seen example six.Other reaction process and condition are seen example nine.

Example 14 '-carboxyl-RP-3 ' report primer method (being called for short 5 '-HOOC-RRP-3 ')

In cDNA is synthetic, six polynucleotides or eight the polynucleotides 5 '-HOOC-DADE-RP-OH-3 ' of the synthetic various 5 '-end-carboxyl-DADE connecting arm modifieds that may sequences of the method for having made to order with the client, as 5 '-sulfydryl report at random primer (5 '-HOOC-RRP-3) [TriLink Biotechnologies, Inc provide this business.Production code member: O-0015], by the synthetic 5 '-HOOC-cDNA-OH-3 ' of reverse transcription reaction.RP is six polynucleotides or eight polynucleotides.

Y ' among the HSC (Y ') is amino.Coupling reaction process need not use two exclusive-OR functions to roll into a ball connecting arm.Selecting water-soluble carbodiimide [1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide, EDAC] for use is coupling agent.Other reaction process and condition are seen example nine.The covalent coupling reaction is as follows:

Fluorescent microsphere, 10% solids concn, 250 μ l.EDAC，40mg。

Buffer A: NaH ₂PO ₄-10mM, pH 6.0.

Coupling method:

With might sequence 5 '-HOOC-RRP-3 ' mixture is dissolved among the buffered soln A.

2. in 5 milliliters glass test tube, the buffered soln A of 1000 μ l is mixed with the HSC of 250 microlitres 10%.

3. EDAC is dissolved among the buffered soln A, to ultimate density be 20mg/ml.

4. the dissolved EDAC with 125 μ l joins in the HSC suspension of dilution, mixes.

5. said mixture is at room temperature slightly vibrated incubation 2 hours, or under 4 ℃, spend the night.

6. also be suspended in again in the following hybridization buffered soln with buffered soln A washed twice.

The methylated HSC of example ten pentachloro-s and amino allyl group-RRP

When synthesizing cDNA, with amino allyl group Nucleotide, as basic dCTP[5-Aminoallyl-2 '-deoxycytidine-5 '-Triphosphate in the amino alkene, TriLink Biotechnologies, Inc] synthetic RRP, synthesize the cDNA that is used for gene chip hybridization by reverse transcription reaction again.

HSC (Y ') n select for use Merck Eurolab France company finishing the fluorescent microsphere of chloromethyl.Y '-be chloromethyl [CH ₂Clgroup].HSC-[CH ₂Cl] n is as follows with amino covalent coupling method:

1. with the ESTAPOR of 10% (100mg/ml) ^_Microballoon washing/coupling buffered soln (1ml/10ml) washed twice.Buffered soln is: PBS, pH7.5.

2. [ultrasonic wave or the method for waving are vibrated] again suspends in washing/coupling buffered soln of 5 milliliters.

3. RRP is dissolved in washing/coupling buffered soln of 5 milliliters.

4. in room temperature with under constantly vibrating, reacted 3-4 hour.Washing, and it is suspended in 10 milliliters the reacting terminating solution again, slight jolting mixing is 30 minutes under the room temperature.The primary amine groups source of reacting terminating solution: 30-40mM azanol [hydroxylamine], thanomin [ethanolamine] or glycine [glycine] etc., contain 0.05-1% (w/v) sealing molecule [Blocking molecules] (BSA, Casein, Pepticase, Tween 20, Triton X-100, PE G, Sera, perhaps IgG).

5. wash, and be suspended in storage buffered soln [storage buffered soln: pH 7-7.5 has 0.01-0.1% (w/v) sealing molecule and NaN3 (0.09%) .] again with 10mg/ml concentration.

6. store 4 ℃ into up to application.

Other reaction process and condition can be referring to examples nine.

Claims (10)

1. indirect method gene chip linear amplification labeling technique, it is characterized in that, with various nucleic acid molecule, certain fragment of structural nucleic acid molecule analogue or its molecule is a template, being prepared under by the biological or chemical effect can be directly and the target sequence of hybridizing of the probe molecule on the gene chip, end at this target sequence is connected with reporter group [Y-], this report group be used for hybridize the back be connected with it by the specific molecular recognition reaction, and the map group [Y '-] that is fixed on high lotus signal vehicle [HSC] surface carries out chemistry or biological coupling, thereby forms the specific signal magnifying tags at hybridisation events.

2. gene chip according to claim 1, coating or coating by substrate and surface thereof are formed with the capture probe that is no less than two arrays arrangements that is fixed on substrate [being abbreviated as S] or coating [being abbreviated as C] surface, the gene target gene that is used for test sample, the material of substrate and/or coating or coating can by but be not limited in the following material any one or more than one differing materials combination and make:

(1) inorganic sheet material type such as glass, quartz, mica, transparent conductive material such as transparent conductive oxide class [TCO] are as tin indium oxide [indium tin oxide, ITO, or be written as In ₂O ₃: Sn], SnO ₂: coating such as F, gallium arsenide [GaAs], magnesium oxide, stannic oxide [tin oxide, SnO], ZnO, CdO, CdIn ₂O ₄, Cd ₂SnO ₄, Zn ₂SnO ₄And In ₂O ₃-ZnO etc., and carbon/pottery composite conductive ceramic, various semiconductor materials etc.,

(2) simple substance class: platinum, gold and silver, aluminium, chromium metals such as [Au, Ag, Pt, Cu, Rh, Pd, Al, Cr] and silicon are nonmetal etc.,

(3) organism class: each organic molecular species and the self-assembled monolayer of making thus thereof or self-assembly multimolecular film,

(4) high score subclass: nylon membrane, cellulose acetate membrane, various plastics, rubber, resin, the structure of substrate can be but be not limited to following kind:

(1) sheet type: as sheet glass, silicon chip, nylon membrane, plastic sheet, microwell plate, droplet is board-like etc.,

(2) electric pole type: on the sheet type gene chip, arrange the various electrod-arrays form with certain rule, as the platinum electrode in substrates such as semi-conductor, isolator, made, gold electrode etc.,

(3) micro-tubular: by kapillary or other material of kapillary tubular intracavity constructional feature is arranged, arrange various matrix form kapillary gene chips [two dimensional surface arrangement], linear kapillary gene chip [one dimension linear array] that forms and the various matrix form parallel arranged of in substrate formula structure, producing [being microfluid electrophoresis chip etc.] that are no less than a microchannel formula cavity structure by the predefined position rule of correspondence

(4) particulate formula: with the molecule is solid phase carrier, biomolecules or chemical molecular are fixed on its surface, size by molecule, [luminous] thus character elements such as color, surface potential, surface charge density carry out the particulate that sort merge produces a large amount of various combinations, the molecule of combination [specific combination of a certain size and certain emission wavelength etc.---coding] of using a certain particular group parameter respectively is as the carrier of certain biological or chemical molecule, thereby can make gene chip with a large amount of differences and specific coding molecule.

3. template according to claim 1 [T], probe [P] and target sequence [TS] can be, but be not limited to one of following kind or different types of combination:

(1) Yeast Nucleic Acid [RNA], various Yeast Nucleic Acid [" ribonucleic acid " and " RNA], comprise messenger RNA(mRNA) [being called for short mRNA], transcribe rna [being called for short tRNA], antisense rna [aRNA], complementary RNA [cRNA] etc.,

(2) thymus nucleic acid [DNA] is as DNA, complementary DNA [cDNA] etc.,

(3) oligonucleotide [oligonucleotide] comprises dna oligo [oligodeoxynucleotide is called for short ODNs], oligomerization Yeast Nucleic Acid [oligoribonucleotide, ORNs], and polynucleotide [polynucleotide],

(4) the nucleic acid construct resemblance [DNA-DNA, RNA-RNA or RNA-DNA mimic duplexes, triplexes or quadroplexes, etc.]] as peptide nucleic acid(PNA) [pcptide nucleic acids, be called for short PNA],

(5) hybrid molecule of forms such as the complementary two strands of thymus nucleic acid, Yeast Nucleic Acid and structural similitude thing thereof, three [weight] chain, four [weight] chain such as DNA-DNA, RNA-DNA, RNA-RNA, PNA-DNA, PNA-RNA, PNA-RNA-PNA, PNA-DNA-PNA] etc., the detection principle of gene chip can be, but is not limited to the combination of one of following kind or different principle:

(1) probe by solid phase surface being positioned at different positions-target-specific hybrid reaction carrying out optical signalling [as fluorescence molecule, chemoluminescence etc.] mark produces fluorescence, chemistry or noclilucence and detects through exciting then,

(2) by translational speed or the mobility in the liquid phase that has applied voltage detects to probe mark or unlabelled or target, be electrophoretic technique or combine with other technologies and the other technologies that produce by electrophoretic technique, as capillary electrophoresis [capillary electrophoresis, CE], capillary zone electrophoresis [capillary zone electrophoresis, CZE], capillary gel electrophoresis [CGE], HPCE [HPCE], capillary isoelectric focusing [capillary isoelectric focusing (CIEF)], isotachophoresis [isotachophoresis (ITP)], electrokinetic chromatography [electrokinetic chromatography (EKC)], mcellar electrokinetic capillary chromatography [micellar electrokinetic capillary chromatography (MECC OR MEKC)], capillary electrochromatography method [capillary electrochromatography (CEC)], nonaqueous phase capillary electrophoresis [non-aqueous capillary electrophoresis (NACE)] etc.

(3) by probe, target or probe-target complex compound and the different avidity of their marker, carry out the chromatographic technique analysis, as affinity chromatography [affinity chromatography], SEC[size exclusion chromatography], GPC[gel permeationchromatography], MCC[metal chelate chromatography] etc.

(4) utilization has the molecule of redox property or the part-structure of molecule [being chemical group or functional group], metal complex or (nucleic acid) intercalator be positioned and be fixed on probe on solid phase surface different positions or the electrode, target or probe-target complex compound combination is also adding under suitable mode or suitable big or small voltage (or potential difference) effect (volts DS or exchange current or pulsed electrical field or voltage), redox reaction takes place, cause from reductibility molecule or group (electron donor, electron donor) transfer transport is to oxidisability molecule or group (electron acceptor(EA), electron acceptor), form stream of electrons, being sent to detection system by electrode measures, measured strength of current size is directly proportional with the amount of the double-strandednucleic acid of hybridizing and being labeled

(5) utilize the conductivity difference of double-strandednucleic acid and single-chain nucleic acid, or utilize by between the formed double chain acid molecule of the nucleic acid hybridization of the complete complementary pairing of sequence and double-strandednucleic acid (the nucleic acids duplexes) molecule that mispairing base pair (Mismatches) existence is arranged in conductivity difference, by various discriminatings to other aspects that nucleic acid molecule is reflected thus being carried out via the measurement of the stream of electrons size of nucleic acid molecule conduction, detect and investigate, as in transgenation, gene pleiomorphism, gene sequencing, gene expression profile research, the application of aspects such as pathogenic mechanism analysis and medical diagnosis on disease

(6) will occur on the nucleic acid molecule or and (include but not limited to by material with redox property, have the metal complex of redox property and be the polymkeric substance that monomer polymerization forms by the derivative of metal complex or metal complex, fluorescence molecule, nucleic acid intercalators etc.) electronics that is taken place or charge transfer reaction or stream of electrons convert other to and can detect the detection technique that maybe can investigate form, as by electroluminescent [Electroluminescence] principle, utilize the electroluminescent molecule that stream of electrons is converted to the detection that optical signal carries out

(7), thereby realize detection to the biological or chemical reaction that occurs in electrode surface by making marker electron-transfer reaction take place behind the mark near electrode surface directly or indirectly having oxidation-reduction quality material (as ferrocenyl) to probe or target molecule marker.

4. gene chip linear amplification labeling technique according to claim 1, its specimen preparation, hybridization, labeled reactant pattern can be, but be not limited to the combination of one of described sight of following general formula or different sight, the formula of specimen preparation, hybridization and labeling process can be expressed as follows

A) template (T, can be various strands or double-stranded DNA, RNA, oligonucleotide or peptide nucleic acid(PNA)) under enzyme (E) effect, chemical action or zymetology effect and chemical action acting in conjunction, forming target sequence ZNA (Y) n[ZNA can be DNA, RNA, oligonucleotide or nucleic acid analog sequence]

T→ZNA(Y)n???????????????????????????[I]

B) nucleic acid probe hybridization on target sequence ZNA (Y) n and the gene chip,

ZNA(Y)n+_XNA→_XNA:ZNA(Y)n?????????[II]

C) finishing have can with the biological or chemical map group Y ' of Y-reporter group specific reaction-high lotus signal vehicle and XNA:ZNA (Y) n on the gene chip carry out chemical covalent linkage or non covalent bond coupling,

_XNA:ZNA(Y)n+HSC(Y’)m→

_XNA:ZNA(Y) _n-p[-Y-Y’-] _p-HSC(Y’) _m-p????????[III]

Said n, m, p are from 0 to infinitely-great arbitrary natural number numerical value 1,2,3...,

XNA is nucleic acid probe or the peptide nucleic acid probe sequence on the gene chip,

HSC is high lotus signal vehicle.

5. enzyme and the zymetology effect for preparing target sequence by template according to claim 4 can be, but be not limited to the combination of one of following mode or different modes,

A) terminal enzyme (DNA) [Terminal Deoxyribonucleotidyl Transferase, be called for short TdT], and add [dideoxy nucleotide (a dideoxynucleotides at 3 ' of DNA-end, as 2 ', 3 '-two deoxyadenines-5 '-triphosphoric acid (2 ', 3 '-Dideoxy-adenosine 5 '-triphosphate, ddATP), ddUTP, ddTTP, or a plurality of [deoxynucleotide (deoxynucleotides ddCTP)], as (dUTP, dTTP, dCTP etc.)) have a terminal transferase reaction of the base of reporter group, thereby generate the report target sequence

B) has the Klenow fragment [enzyme] of the archaeal dna polymerase of 5 ' → 3 ' DNA composite reactive, by reporting primer method [Random Reporter Primer at random, be abbreviated as RRP] under the driving of the effect of this enzyme and the report primer [Reporter Primer] that is formed by connecting by covalent linkage by a reporter group and nucleic acid or peptide nucleic acid(PNA) primer two portions, synthetic report target sequence [Reporter Target Sequence, RTS]

C) reversed transcriptive enzyme, include but not limited to Superscript II RT, avian leukosis poison (AvianMyoblastosis Virus, AMV) reversed transcriptive enzyme and mouse retrovirus (MMLV) reversed transcriptive enzyme, perhaps StrataScript reversed transcriptive enzyme, PowerScript reversed transcriptive enzyme, can be used for mixing in the reverse transcription process media nucleosides, as the Taq enzyme

D) archaeal dna polymerase, as the Taq enzyme, escherichia coli or e. coli dna polymerase I,, dna polymerase i [polymerase I],

E) RNA polymerase, as SP6, T7 and T3 phage rna polymerase [phage RNA polymerases] can be used for in-vitro transcription [in vitro transcription] method mark, Superscript II RT.

6. the chemical action for preparing target sequence by template according to claim 4 can be, but is not limited to the combination of one of following mode or different modes,

A) report primer method [Random Reporter Primer is abbreviated as RRP] mark at random, report primer [Reporter Primer] that might sequence by synthetic institute be to synthesize from template to report that target sequence gets ready,

B) synthetic modification deoxynucleotide or dideoxy nucleotide, for the synthetic target sequence of terminal transfer method provides the nucleotide structure that has reporter group analogue,

C) oligonucleotide of anamorphic zone reporter group is as the report primer, as the synthetic oligomerization thymus pyrimidine [5 '-amino-Oligo-dT] that has 5 '-amino reporter group, for the synthetic cDNA target sequence that has 5 '-amino reporter group is got ready, with reporter group [Y-] can also be but to be not limited to sulfydryl, carboxyl, hydroxyl, chloromethyl, vitamin H and digoxin.

7. according to claim 1, claim 4 and the described Y-of claim 6 and Y '-can be respectively, but be not limited to one of reaction pair as shown in the table or their combination: Y-or Y '-[give an example, but be not limited to] ZNA-Y, NTP-Y, dNTP-Y and ddNTP example ZNA, NTP, dNTP, ddNTP example Phenyldiboronic acid, PDBA, the SHA-digoxin-, the DIG-digoxin-, anti digoxin antibody-amino, as amino allyl group, aa-, N-hydroxyl PDBA-UTP DIG-11-UTP Digoxigenin-11-ddUTP aa-dUTP, aa-UTP[Ambion, (Cat.# Ribonucleoside triphosphote, UTP, ATP, CTP, GTP, TTP bi-deoxyribose ribonucleoside triphosphote [ddNTP], as ddATP, ddCTP, ddUTP, ddGTP, ddTTP.

The base succinimido, amino [aminolinker-], digoxin-, the DIG-NHS ester, N-hydroxy-succinamide base [NHS-], anti digoxin antibody-, biotinyl, avidin-, Streptavidin-chloromethyl [chloromethyl], amino, carboxy thiol groups [sulfhydryl, HS-], halogen acetic acid base [Haloacetyls], alkylogen [Alkyl halide] derivative, maleimide [Maleimides], acridine [Aziridines]; Acryloyl [Acryloyl] derivative; Arylation [Arylating] reagent; Sulfydryl-acquisition sequences such as two sulphur compound displacer reagents, 5 '-ATTCGGAA-3 '; Joint sequence, 3 '-TAAGCCTT-5 ' 8437) DIG-X-NHS ester Biotin-UTP, biotin-dCTP, biotin-dUTP, aa-dUTP such as biotin-ddUTP, aa-UTP[Ambion, (Cat.# 8437) 5 '-HS-C6-Oligo (dT)-cDNA-OH-3 ', 5 '-HS-cDNA-OH-3 ' carries the oligonucleotide acid primer [oligodeoxynucleotide primers containing capture sequences] of acquisition sequence, as 3 '-oligo (dT)-TAAGCCTT-5 ' or 3 '-oligo (dT)-GTAGCAAT-5 ' Deoxyribonucleoside triphosphate [dNTP], as dATP, dCTP, dUTP, dGTP, dTTP.The same 5 '-cDNA-OH-3 ', 5 '-Oligo (dT)-3 ' oligonucleotide acid primer, as oligo (dT), oligo (dA) ...

8. according to the high lotus signal vehicle [HSC] described in the claim 1 can be; but be not limited to; the high loading optics that exists with various molecule forms; magnetic; electronics or electrochemical signals carrier; it can be quantum dot microsphere [QD-tagged Microbeads]; superpolymer fluorescent microsphere or high molecular fluorescent microballoons [polymer microspheres] or colloid microballon [latex beads]; at particle surface; inner by fixing; embedding; modes such as chemistry covalent bonds are with signaling molecule such as luminescent dye molecule; chemiluminescent molecule; [magnetic] silica microballon [silica beads] that chromonic molecule etc. concentrates; the brilliant quantum dot [colloidal fluorescent semiconductor nanocrystal quantumdots] of colloid luminescent semiconductor nanocrystal; dendroid high polymer nano ball [dendrimers]; star dendrimer or dendrimer [stardendrimers; dendrimeric starsor dendrimers]; the semiconductor nanoparticle of protein parcel or molecule or quantum dot [as wrapping up best results with Chaperonin GroEL albumen and T.th cpn albumen among the Chaperonin mediatedsemiconductor nanoparticles.Chaperonin]; micella [micelles]; molecular magnet [molecular magnets]; capsule-type microballoon [encapsulated spheres]; colloidal nano gold [colloidal goldnanoparticles]; electroluminescent molecule [electroluminescent molecules]; two Metallocenyl copolymer microsphere [polymetalcene blockcopolymers; as ferrocene-based polymer [polyferrocene block copolymers; PFC; or polyferrocenylsilanes; PFS] microballoon; poly zirconocene [polyzirconocene; PZC]; poly two Metallocenyls are modified or the grafted dendrimer [includes but not limited to; poly-ferrocene base grafted dendrimer (polyferrocenyl-branched dendrimers)]; two Metallocenyls or poly two Metallocenyls are modified or grafted nanometer gold colloid [includes but not limited to; the nanometer gold colloid of amido ferrocenyl functionalization (Gold colloidsfunctionalized with amidoferrocenyl structures) etc.]; copolymer microsphere or nanometer ball; contain fullerene structure or be the superpolymer or the multipolymer of one of monomer with the soccerballene; other contains fluorescence dye or chemoluminescence [chemiluminescence]; the carrier of noclilucence [bioluminescence] material; phycobiliprotein class [phycobiliproteins] and various derivative thereof or biological or chemical modifier; and in above-mentioned each carrier different parameters [as size; the optical emitting optical wavelength; electromagnetic property; physics or chemical property such as the modification of surface biological or chemical functional group] combination of carrier, or the mutual combination between the various different carriers.

9. enzyme process labeled reactant according to claim 4 can be, but be not limited to the combination of one of following process or various process,

(2) reverse transcription or reverse transcription process [Reverse Transcription],

(3) in-vitro transcription process, [in vitro transcription]

(4) nick translation process or breach transfer process or nick translation process [Nick Translation],

(5) end deoxynucleotide transfer process,

(6) terminal nucleotide transfer process,

(7) synthesis reaction of DNA process or RNA synthetic reaction process,

(8) DNA ligation,

(9) under the effect of DNA[restriction enzyme] endonuclease reaction,

(10) under the effect of phosphorus enzyme enzyme 5 ' in the DNA end-end phosphate is removed, is made its 5 '-end become the reaction of OH base,

(11) T4 polynueleotide kinase (T4 polynucleotide kinase) effect down the phosphate on the γ position among the ATP is transferred to have 5 '-DNA of terminal hydroxy group or the reaction on the RNA molecule,

(12) terminal deoxynucleotidyl transferase (Terminal Deoxyribonucleotidyl Transferase is abbreviated as TdT) effect down the single phosphoric acid nucleoside in the triphosphate deoxy-nucleotide is transferred to 3 of dna molecular '-reaction on the terminal hydroxy group,

(13) ox pancreas deoxyribonuclease I (DNaseI) effect down the degraded double-stranded DNA be 5 '-reaction of phosphoric acid oligonucleotide,

(14) nuclease RNaseA effect acts on the reaction of pyrimidine nucleotide down specifically,

(15) under the nuclease RNaseH effect, the RNA molecule in the specificity ground decomposing D NA/RNA hybrid molecule, thereby the reaction of in the cDNA building-up process, removing the RNA chain among the eDNA-RNA.

10. indirect method gene chip linear amplification mark [iLAL] test kit of making according to claim 1, it is the iLAL test kit, comprise gene chip and handled the needed whole biochemical reagents of each process, it is characterized in that, contain but be not limited to core component related in the following general formula of claim 4: general formula (I), general formula (II), general formula (III).