CN101100764A - Molecule substitution label sequencing parallel detection method-oligomictic nucleic acid coding label molecule library micro-sphere array analysis - Google Patents

Abstract

Molecular displacement label sequence parallel inspection - oligonucleo coded label molecular pool micro-ball array analysis includes: using artificial sequential oligo nucleo-labeled DNA coded molecular pool, coating micro-balls with ligands in correspondent pools to form catching micro-balls, caught ligands combined with labeled ciding molecular pools to form labeled micro-balls array pools. Sample molecules competitively combined with micro-balls catching ligands when sample contains molecules same as those in labeled micro-balls array pools, calculating amount of displaced coded molecular sequence computing kinds and mounts of molecules to be tested. It verifies high flux of inspection with good repeatability, high reliability and sensitivity, so that it is suitable to precisely parallel quantitative analysis concerned with cancer-inhibiting genes and cancer genes pools, cell factor pools, etc.

Description

The molecule substitution label sequencing parallel detection method-oligomictic nucleic acid coding label molecule library micro-sphere array analysis

Technical field

The technical field that the present invention relates to is the microballoon suspension array system of high-flux parallel Molecular Detection, claims liquid biochip again.Especially at the competitive metathetical parallel detecting method of microsphere surface bonded nucleic acid oligomer code tag molecular library.

Background technology

Along with finishing of gene order-checkings such as the mankind, and enter the functional protein group epoch, demand detecting simultaneously sample in batches urgently in the face of the biomolecules of enormous quantity extremely, the high-flux parallel detection technique of many indexs replaces traditional molecular detecting method one by one.This notion from computer chip of biochip (claiming microarray again) is just arisen at the historic moment.Biochip essence be on the little substrate surface of area in an orderly manner dot matrix arranged a series of addressable identification molecules that are fixed in certain position, and through fluorescent mark or chemoluminescence demonstration, laser scanning/CCD record of making a video recording, the result passes through computer software analysis.Biochip just becomes the focus of biological study maximum once being born, and advances just with surprising rapidity.Each university research mechanism, company enter this field one after another, have created many biochip notions and device (biochip, second edition, Ma Liren chief editor,, Chemical Industry Press in 2002).

Biochip mainly is divided into gene chip and protein chip two big classes.The kind of gene chip exploitation and use many, most of company use automatic system BioRobot with the DNA spot printing be stamped in wave plate or filter membrane on.Relatively more accurate chip is based on the Genechip of Affymetrix company, be the segmental detection of nucleic acids microarray of utilization semi-conductor photograph plate technique original position synthetic oligonucleotide on the two dimensional surface slide (1998, U.S.Pat.No.5,837,832 and 2001, U.S.Pat.No.6,309,831).Protein chip is by Ciphergen company the biochip (2001, U.S.Pat.No.6,225,047) that laser desorption ionisation flight time mass spectrum technology is applied to protein detection to be strengthened on the surface after gene chip.The domestic Wu Jian of Southeast China University has successfully developed in male laboratory the synthetic high-density gene chip of molecular seal original position; Beijing Bo Ao company has developed the laboratory-micro-fluid chip on the chip (2003, Chinese patent 01145118.1 and 2004, Chinese patent 031051080.1).These chip technologies have improved the degree of integration of Molecular Detection greatly.But still have some planar chip institute inherent defectives: the chip manufacture on (1) physical significance is loaded down with trivial details, and a large amount of industrial production efficient are not high, costliness.(2) have certain difference because of physical method processing between the chip array site, cause difference between the different loci, the difference between the different chips causes nearly 10% background deviation, poor repeatability.(3) molecule and the dynamic (dynamical) deficiency of chip array surface bonding are slow as molecular reaction liquid mixing, because of the chip surface size causes the slow skewness of reaction solution molecular diffusion etc.(4) the easy distortion of physical simulation conversion of signals.These are just not high in developing chip technology practicality.

In order to overcome the intrinsic deficiency of two dimensional surface biochip, Beckton-Dickson company and Luminex company etc. has created based on the liquid suspension array analysis of fluorescence-encoded micro-beads.At first infiltrate microballoon coding (addressable) mark in different ratios, can form 140 kinds of different codings at most by two kinds of different fluorescent substances.The third fluorescent substance marker detection probe, the coding microball bag is hunted down and combines with testing molecule behind the molecule, again with fluorescent probe in conjunction with after flow cytometer to transmit into one-dimensional linear single-row, one by one by two bundle laser detection, its a branch of micrometer ball color is determined the coding kind, another bundle detection probes fluorescence intensity quantitative analysis.(1999, U.S.Pat.No.5,981,180 and 2001, U.S.Pat.No.6,268,222).The biochip of this liquid notion has promoted its practicality greatly.

Its major advantage:

(1), high-speed: the fastest reach 10000 tests/hour;

(2), highly sensitive: detect lower bound and can reach 10pg/ml;

(3), low cost: the easy batch machining of microballoon, cost is low, reagent dosage is few, can effectively reduce the reagent cost of clinical application;

(4), detect in real time: show during fruitage, but both parallel detection genes, but parallel detection protein again, liquid phase reaction is more suitable for biomolecules, only needs micro-sample (10ul), can reach 140 indexs at most;

(5), good reproducibility: a collection of production packing of microballoon is used, and difference is little between the microballoon unit, and each index has 5000 reaction members, analyzes 100 times and averages;

(6), linearity range is wide: sensing range reaches 6 orders of magnitude.

Microballoon microarray analysis advantage based on fluorescence is a lot, but it is limited to be limited to fluorescent substance physical markings group/cording quantity, detect flux even a lot of less, the easy distortion of simulating signal of fluorescent signal conversion simultaneously, the real time automatic detection of relatively more suitable more clinical indexs than the two dimensional surface chip.Micro-sphere array analysis approach based on label sequencing of the invention adopts the nucleic acid oligomer label coding molecular library of artificial sequence to comprise DNA library or protein library, rather than the direct coding microballoon.Microballoon is used corresponding library of molecules part (DNA complementary strand, or protein ligands such as the corresponding antibody of antigen) bag quilt again, forms and catches microballoon.The capture ligands of microballoon bag quilt combines the back and forms the micro-sphere array storehouse with the molecular library of label coding.When the molecular library that has contained with label when sample to be measured had same molecular, testing molecule combined with the microsphere surface capture molecules competitively, and the coding molecule that competition replace is by the series connection of nucleic acid oligomer label, the order-checking evaluation of clone back.This shows, except the limitation that can not detect in real time, kept advantage, and expanded some new intrinsic superiority based on the micro-sphere array analysis of fluorescence based on the micro-sphere array analysis that checks order.

Nucleic acid oligomer code tag molecular library micro-sphere array analysis of the present invention is a kind of existing ripe biological cloning, sequencing technologies of making full use of, and avoids the new ideas approach of physical chemistry technology.Based on the connection of nucleic acid oligomer dna fragmentation, clone technology and dna sequencing technology.Go through more than 20 year development of molecular biology, these technology are full-fledged to a very high realistic scale.

Since nineteen fifty-three, Watson﹠amp; Crick proposes dna double spiral theory and has established the molecular biology basis, thereby has been born vitro recombination, numerous molecule clone technologies such as gene amplification (molecular cloning, 2001, J.Sambrook, et, al.3^RdEdit).1980 ' s restriction enzyme after the age particularly, ligase enzyme, the practicality of PCR and heat-resisting polymerase and plasmid vector etc., DNA vitro recombination clone technology has become the most basic sophisticated routine techniques in the biology.

The dna sequencing technology is also gone through many different trials, as enzyme digestion, and chemical method.Even the initial main purpose of gene chip also is the mensuration of dna sequence dna.Concentrate on the dideoxy chain termination (SangerF, 1975, J.Mol.Biol.94:441-448 and Sanger F, 1977, PNAS 74:5463-5467) of Sanger at present.And go through isotopic labeling, and fluorescein-labelled, the improvement of Sequenase, speed has increased tens of times.Particularly the enforcement of human genome order-checking plan has promoted the development of sequencing technologies greatly, from the slab gel to the capillary electrophoresis, and has produced high-throughout automatization sequenator and order-checking robot.Greatly improved efficient once more.

Summary of the invention

The present invention is directed to the deficiency of existing biochip, a kind of new liquid biochip based on sequencing technologies-molecule substitution label sequencing parallel detection method is provided, can guarantees high-throughout detection, simultaneously this method good reproducibility, the reliability height, and have very high sensitivity.

A kind of molecule substitution label sequencing parallel detection method is characterized in that comprising the steps: (1) crosslinked microballoon that capture ligands is arranged, and the molecule in the molecular library of absorption label coding forms tag molecule micro-sphere array analysis storehouse; Described label is one section separate nucleic acid oligomer double-stranded DNA that has nothing to do with the library molecular sequences, sense strand 5 ' the end of described nucleic acid oligomer is connected with biotin labeling and extension increasing sequence, 3 ' end of nucleic acid oligomer is preset with and terminal complementary sequence of respective labels and restriction enzyme E1 recognition site, described label and library are molecule crosslinked, and described capture ligands is the specific binding ligand of library molecule; (2) mix with sample to be tested in the micro-sphere array storehouse, the same molecular in the competition displacement tape label molecular library; (3) being displaced the tag molecule of coming detects by label series connection, order-checking.

Described tag molecule length is 12-100bp.

The preferred 16-30bp of tag molecule length.

Described molecular library is DNA library or protein library.

Described label and dna molecular are crosslinked to be crosslinked by the design primer amplification.

Described label and cross linking of protein molecule are crosslinked by sulfhydrylation DNA and activation of protein.

Described microballoon size arrives micron order (1nm-1000um) for nano level.

Described microballoon material is plastics (polyethylene, polyvinyl chloride, polystyrene etc.); Or resin (resol, vibrin, polyamide resin etc.); Or gel (agarose, dextran, polyacrylamide etc.); Or other macromolecule polymeric material.

Described microballoon can also be particulate in irregular shape or the planar medium with active surface.

Label series connection in the describedstep 3, order-checking detect, and it is characterized in that comprising the steps: the nucleic acid oligomer double-stranded DNA of (a) code tag for the artificial distinguished sequence be made up of two complementary 16-30 base oligonucleotide; (b) a pair of not homotactic label of each molecular designing in the library of molecules, 5 ' end of each label sense strand is marked with vitamin H, 3 ' terminal portions complementation between every pair, and also 3 ' terminal and library of molecules junction is provided with the restriction enzyme site of restriction enzyme E1; (c) every pair of label of competitive metathetical by the little magnetic beads for purifying of streptavidin, forms the bigeminy label through its 3 ' terminal complementary polymerase extension after the E1 enzyme is cut; (d) the library of molecules molecule is divided into two groups of odd number (Odd), even numbers (Even), every pair of bigeminy label of odd molecule, and a side adds special sequence F in advance, opposite side adds Odd sequence and E2 restriction enzyme site; Every pair of bigeminy label of even number molecule, a side adds different R sequences in advance, opposite side adds and Odd complementary Even sequence and E2 restriction enzyme site; (e) every pair of bigeminy label is cut at random through the E2 enzyme and is connected, or the complementation of Odd-Even sequence is extended and generation tetrad label after the E2 enzyme is cut.Use F/R primer amplification 4 labels again, the PCR product is cut further series connection insertion cloning vector of back, clone, positive plasmid order-checking with the default E3 enzyme in the F/R outside.Identify the kind and the quantity of each code tag.

The present invention is a kind of microballoon based on short nucleic acid sequences code tag sequencing analysis, microparticle, or the suspension array analytical procedure of microballon.Ultimate principle is a molecule that the unknown is to be measured and the known molecular library of tape label relatively (competitiveness displaces the identical indication molecule of tape label), derives the kind of testing molecule and what according to the label coding of same molecular again.The connecting of the structure (synoptic diagram such as Fig. 1) that operation is divided into label molecule library and label coding, detection (synoptic diagram such as Fig. 2) two big steps.

The first step is according to known molecular library sequential structure information, each molecule is chosen the nucleic acid oligomer label that representational part or all mark adds the weak point of particular sequence coding in the library, makes up the molecular library that each molecule of a synthetical all has nucleic acid oligomer double-stranded DNA code tag.Get business-like homogeneous polystyrene plastic microballoon or activatory sepharose particle again, number grouping according to molecule in the library of molecules, the part of corresponding molecule is (as complementary DNA in every group of microsphere surface covalent cross-linking library of molecules, Ag-Ab etc.), the molecule indication storehouse of the microballoon adsorption zone label of mixing, the unconjugated molecule of flush away generates tag molecule microballoon storehouse.With the reaction of sample to be measured and microballoon storehouse, in the sample to be tested with the library in identical molecule just can displace the same molecular of the tape label of corresponding microsphere surface competitively.Separate at this point in the tape label molecule and the microballoon library of dissociating out.Preset restriction enzyme E1 in the nearly molecule of label junction in the molecule of tape label, behind endonuclease reaction, label and molecular separation, label again through one side 5 ' end with biotin labeling, be bonded to the crosslinked little magnetic ball of streptavidin, be further purified.

The library sequence label be one section about 20bp (16-30bp) long with the irrelevant artificial nucleic acid oligomer sequence of library molecular sequences.Pre-set limit restriction endonuclease E1 recognition site between library molecule and label, E1 can be any restriction enzyme in theory, but need from label series connection aspect, only discern 4bp site and enzyme and cut back sense strand 3 ' end and only remain the enzyme of a base of 4bp recognition site such as BafI etc., make the easier design operation of label 3 ' end sequence.

The second step code tag connects, increases, connects, clones, and order-checking at last detects.The connection of 2 and 4 aggressiveness of short label, if be based on 3 ' terminal portions complementation of two its sense strands of DNA chain, just each other primer by pcr amplification and continuous principle design.For being linked to each other, code tag tail-tail forms two aggressiveness labels, the default a pair of two different strip labels of each molecule in the molecular library, wherein a strip label sense strand 3 ' end and another strip label sense strand 3 ' terminal portions complementation are extended and tail-tail formation 2 labels that link are right through PCR.The right end of each molecule two label adds the preceding paragraph sequence more in advance,molecule 1 wherein, 3,5 ... use same sequence F on the odd molecule label,molecule 2,4,6 ... use another identical sequence R conduct forward and reverse amplimer of 4 labels subsequently Deng the even number molecular label.The other end pre-set limit restriction endonuclease E2 restriction enzyme site that each label is right, the selection principle of E2 is with restriction enzyme E1.

Two aggressiveness, 2 labels make between every pair of label of library of molecules molecule through the T4 ligase enzyme to connect at random after cutting purifying with default restriction enzyme E2 enzyme again.Perhaps every pair oflabel E 2 restriction enzyme site ends add that in advance complementary sequence makesodd molecule 1,3,5 ... label one end is same sequence, evennumber molecule 2,4,6 ... label one end is a complementary sequence.Through PCR odd molecule label pair and even number molecular label are extended to an end is complementary, and head-head join.If the library molecule number is less, do not wait for preventing the odevity molecule, remaining every pair of label of free connects with the T4 ligase enzyme again, forms 4 labels.

The 4 tag amplified default sequence F at 4 label two ends and the 5 ' ends of R of being to use that connection is good add that the E3 restriction enzyme site is as 4 label forward primers and reverse primer amplification, purifying, enzyme are cut, the repurity connection is connected into the continuous chain series connection label of individual 4 labels of 2-6, is cloned into the pUC19 carrier, choose tens of and even hundreds of clones of positive colony and extract plasmid, sequencing analysis.E3 can be any enzyme of cloning vector multiple clone site.

This shows, the present invention combines the micro-sphere array analysis technology with the biological cloning sequencing technologies, based on the micro-sphere array analysis of label sequencing except the limitation that can not detect in real time, kept advantage based on the micro-sphere array analysis of fluorescence, and expanded some inherent superiority: (1) high-throughput, nucleic acid oligomer label coding number substantially without limits.(2) highly sensitive, label series connection be after pcr amplification cloning and sequencing again, detection level only can reach in theory several, the dozens of molecular level, far above the pg level.(3) good reproducibility, the biotinylated nucleic acid sequence is accurate far beyond physical signalling, and reliability is high especially.(4) micro-sphere array analysis based on order-checking does not need to arrange just energy original position competitive assay through one-dimensional linear, and the reaction in of this point more approaches biological natural response, is more suitable for the detection of biomolecules than physics, chemical reaction.Especially be fit to cancer suppressor gene, oncogene library, tumor-marker gene pool, proprietary genotype such as cytokine storehouse or specific function phenotype library parallel accurately qualitative, quantitative analysis.

Be divided into gene library micro-sphere array and protein library micro-sphere array analysis again according to library molecule difference.

One, gene library micro-sphere array principle and operation:

Determine the gene library of a limited range according to Bioinformatic Data Base biomolecule information database, as the cytokine gene storehouse, lymphocytic cell surface flag library etc.Each dna molecular in the library, the machine aided design selects the sequence of guarding, do not have space structure of one section about 100-500bp length as the indication molecule library as calculated.Select the long sequence of each about 20-30bp of its both-side ends of each molecule as amplimer.The dna molecular marker label be utilize the PCR primer only 3 ' end mate dependency fully, and 5 ' terminal sequence mispairing does not influence the amplimer combination, the characteristic that prolongs, sequence label is added in library molecular cloning forward primer 5 ' end front and reverse primer 5 ' end front, after treating pcr amplification, the primer of sequence label by tape label is with regard to the part that becomes the amplifier molecule end and mark.And the primer 5 ' end of tape label is by vitamin H on the synthetic tense marker of Oligo.Default pair of sequences two label oligonucleotides inequality of each dna molecular in the library, they have the complementation of 6-10bp sequence by 3 ' end afterbody, so that primer and extend to the two aggressiveness labels that the tail tail links each other.Thelibrary DNA molecule 1, default label tagla; 1b,molecule 2, default tag2a, 2b; Molecule 3, default tag3a, 3b ... Deng by that analogy.One end of every pair of label adds the preceding paragraph sequence in advance,molecule 1 wherein, 3,5 ... all use same sequence F etc. the odd number group molecular label,molecule 2,4,6 ... all use another identical sequence R as the forward and reverse amplimer of 4 labels etc. the even number set molecular label, the other end pre-set limit restriction endonuclease E2 restriction enzyme site of every pair of label.Every pair oflabel E 2 restriction enzyme site ends design 8-12bp complementary common sequences more in advance makesodd molecule 1,3,5 ... label one end and evennumber molecule 2,4,6 ... label one end complementation.Form and catch microballoon with the crosslinked microsphere surface that is fixed in of one section sequence in the middle of the reverse amplimer in library or the molecule.

The molecular dna storehouse of the tape label of pcr amplification is adsorbed to the mixing microballoon of band capture ligands, and the unconjugated molecule of flush away forms molecule microballoon storehouse again.During detection sample to be tested is mixed with the microballoon storehouse, if have in the sample to be tested with the library in identical molecule, then the same molecular of label coding is just cemented out competitively, vitamin H by the label end, come out by the streptavidin magnesphere purifying, cut through the E1 enzyme, 90 ℃ of heat denatured of the oligonucleotide tag library of streptavidin magnetic microsphere purifying 5 minutes, 4 to 5 thermal cyclings, 90 ℃ 30 seconds, 20 ℃ 10 minutes, 65 ℃ of 1 minute pfu enzymatic amplifications make complementary extension of pair of tag 3 ' end of each molecule and tail-tail links to each other.After pair of tag tail-tail links to each other in each molecule of molecular library, cut purifying with restriction enzyme E2 enzyme again, make between every pair of label of library of molecules molecule with the T4 ligase enzyme to connect at random.After perhaps the E2 enzyme is cut purifying, 92 ℃ of heat denatured 5 minutes, 4 to 5 thermal cyclings, 92 ℃ 30 seconds, 26 ℃ 10 minutes, 68 ℃ of 1 minute Tag enzymatic amplifications make the pair of tag of any odd molecule complementary end identical with the pair of tag of even number molecule arbitrarily primer extension and connecting each other.For preventing that molecule number does not wait between every pair of label, do not connect every pair of label of free and spend the night with the connection of T4 ligase enzyme again, form 4 labels.

For make the series connection label have some amount and length be convenient to clone and the order-checking, 4 labels must through pcr amplification again enzyme cut further series connection.With the F primer of restriction enzyme site of band E3 and R primer through 20-25 cyclic amplification 4 labels, the overwhelming majority is the product at two ends for F/R, few part is the product at F/F, R/R two ends, cut purifying through the E3 enzyme and be connected into 2-6 4 labels that link to each other through the T4 ligase enzyme again, adding the pUC19 that the E3 enzyme cuts again further connects, transform DH5 α bacterium, PCR selects tens of to hundreds of positive colonies, extract plasmid respectively, last plasmid order-checking series connection label, kind and the quantity of deriving testing molecule by the kind and the quantity of computer software analysis label.

Operating process:

(1) the right design of DNA library primer and label, synthetic:

Choose the long sequence front of about 20-30base, each molecule two ends, DNA library and add that respectively sequence label and restriction enzyme site and F/R sequence constitute the amplified library primer.

Scheme one:

Forward primer 1a formula is (sense strand): 5 '-(F)--(1a------)--and (E1)--(1/5 '------)-3 '

(F) represent 4 label forward primer sequences, (1a------) sequence of every pair of label of representative (E1) is default restriction enzyme site, (1/5 '------) represent 5 ' ofmolecule 1 to hold preceding 20base sequence.

Forward primer 1b formula is (sense strand): 5 '-(E2)--(1b------)--(E1)--(1/5 '-----)-3 '

Reverse primer 1 formula is (antisense strand): 3 '-(------1/3 ')--5 ' synthetic its complementary sequence is as reverse primer.

(------1/3 ') represent 3 ' ofmolecule 1 to hold last 20base sequence, (E1) be default restriction enzyme site, (1b------) every pair of another different sequence of label of representative, 3 ' the terminal 6-10base complementation of its 3 ' end and 1a.(--E2) represent second kind of default restriction enzyme site and default every pair oflabel E 2 restriction enzyme site end part complementary sequences.

Molecule 3,5,7,---all by that analogy;Molecule 2,4,6,---F changes R into, and all the other are analogized equally.

Scheme two:

The forward primer formula is (sense strand): 5 '-(F/R)--(1a------)--and (E1)--(1/5 '------)-3 '; The odd number group molecule is F, and the even number set molecule is R.

(F/R) represent 4 label primer sequences, (1a------) sequence of every pair of label of representative (E1) is default restriction enzyme site, (1/5 '------) represent 5 ' ofmolecule 1 to hold preceding 20base sequence.

The reverse primer formula is (antisense strand): 3 '-(------1/3 ')--(E1)--(1b------)--(--E2)-5 ' synthetic its complementary sequence is as reverse primer.

(------1/3 ') represent 3 ' ofmolecule 1 to hold last 20base sequence, (E1) be default restriction enzyme site, (1b------) every pair of another different sequence of label of representative, 3 ' the terminal 6-10base complementation of its 3 ' end and 1a.(--E2) represent second kind of default restriction enzyme site and default every pair oflabel E 2 restriction enzyme site end part complementary sequences.

One section sequence was as capture ligands in the middle of this kind scheme also must be got each molecule.

The synthetic ABI company dna synthesizer that adopts the solid phase phosphoramidite triester method of primer.Or commerce is ordered synthetic.

Synthetic primer is to carry out 5 ' end biotin labeling by infiltrating biotinylated dNTP.

(2) DNA reverse primer or capture ligands cross-linked agarose gel particle:

Cross-linking method draws from fine works molecular biology experiment guide (F.M.Ausubel, et.ul., 1999, " Short Protocols inMolecular Biology " 4^ThEdit.p535).

Cyanogen bromide (CNBr) activation Sepharose CL-4B is available from Pharmacia company.

Whole cross-linking process is divided into parallel two groups, the sense strand Oligo of one group of crosslinked DNA reverse primer, and another organizes the antisense strand Oligo of crosslinked DNA reverse primer.Below with poly-representative of step of one group:

1) claims working of 1g Sepharose 4B agar in a 15ml conical centrifuge tube, add the 1M HCl aquation agar of 10ml, put upside downlight mixing 1 minute, move in the B of a 60ml, add the 1M HCl washing of 500ml again, about 15 minutes of swelling agar.

2) use 100ml dH₂O washs agar, uses 100ml 10mM potassiumphosphate (PH8.0) washing again.

3) in one 15 blind nut centrifuge tube of agar immigration, adding about 4ml 10mM potassiumphosphate (PH8.0) becomes uniform thick slurry until medium.

4) add 50ul Oligo aqueous dna immediately, place under the room temperature that mixing spends the night on the runner.

5) in stink cupboard, agar is moved in the B 100ml dH₂O washes 2 times, and 100ml 1M diethanolamine hydrochloride (PH8.0) is washed once.

6) agar moves in the 15ml centrifuge tube, adds 1M diethanolamine hydrochloride (PH8.0) to pasty state, room temperature runner mixing 2-4 hour.

7) move to B, use 100ml 10mM potassiumphosphate (PH8.0), 100ml 1M potassiumphosphate (PH8.0), 100ml1M Repone K, 100ml dH successively₂O washs agar.

8) the crosslinked agar of Oligo places storage buffer 10mM Tris-Cl (PH7.8) to add 1mM EDTA, 0.3M NaCl.Two groups of cross-linking agents are mixed in 4 ℃ and can stablizestorage 1 year.

(3) mark in DNA library increases and is adsorbed to and catches microballoon:

Amplification library molecule 1,molecule 2,molecule 3,

Template: dna library 0.5ul

5uM library forwardprimer 1 0.5ulforward primer 2,forward primer 3

5uMlibrary reverse primer 1 0.5ul reverse primer 2,reverse primer 3

10×PCR buffer 2ul

10mM dNTP 0.5ul

pfu 0.5ul

dH₂O 15ul

20ul

Pcr amplification in 96 hole micro-reaction plates, 94 ℃ 4 minutes, then 25 the circulation 94 ℃ 30 seconds .52 ℃ 30 seconds, 72 ℃ 40 seconds.Then 72 ℃ 5 minutes.

The PCR product is through Qiagen 96 hole purifying plate purifying.

Catch the DNA library of microballoon absorption mark: the PCR dna library of purifying mixes, and 100 ℃ of heating place cooled on ice after 5 minutes fast.The crosslinked microballoon of catching spends the night in 50-70 ℃ of dna library combination that adds purifying, and washs the unconjugated molecule of flush away 3-5 time.

(4) the competitive displacement in sample to be measured and microballoon storehouse DNA:

Sample to be measured at first adopts the DNA purification column test kit purifying of Qiagen to extract sample DNA.Sample DNA places dry ice then fast 100 ℃ of heat denatured 10 minutes.

The microballoon storehouse is at first washed the unconjugated library of flush away molecule three times before the displacement test again in 50-70 ℃ temperature is filled.Be warming up to 90 ℃ of sex change again moved in the 50-70 ℃ of incubation in 2 minutes immediately.The sample DNA of sex change is added in the microballoon storehouse 50-70 ℃ continued the incubation replacement(metathesis)reaction 1-2 hour.Centrifugal, careful sucking-off supernatant (keep off and contact agarose microbeads).Move in the fresh centrifuge tube Qiagen purification column purifying.Heavily be dissolved in 50ul dH₂O.Restriction enzyme E1 enzyme was cut 5-6 hour, added crosslinked little magnetic ball (Dynal Biotech) washing of streptavidin, magnet combination, purifying bonded tag library.

(5) 2 of label connect:

90 ℃ of heat denatured of the oligonucleotide tag library of streptavidin magnetic microsphere purifying 5 minutes, 4 to 5 thermal cyclings, 90 ℃ 30 seconds, 20 ℃ 10 minutes, 65 ℃ of 1 minute pfu enzymatic amplifications make complementary extension of pair of tag 3 ' end of each molecule and tail-tail links to each other.

The amplification of (6) 4 labels and series connection:

After pair of tag tail-tail links to each other in each molecule of molecular library, cut purifying with restriction enzyme E2 enzyme again, make between every pair of label of library of molecules molecule with the T4 ligase enzyme to connect at random.After perhaps the E2 enzyme is cut purifying, 92 ℃ of heat denatured 5 minutes, 4 to 5 thermal cyclings, 92 ℃ 30 seconds, 26 ℃ 10 minutes, 68 ℃ of 1 minute Tag enzymatic amplifications make the pair of tag ofmolecule 1 be connected with the pair of tag ofmolecule 2,molecule 3 and molecule 4 ... by that analogy.For preventing that molecule number does not wait between every pair of label, do not connect every pair of label of free and spend the night with 15 ℃ of connections of T4 ligase enzyme again, form 4 labels.

(7) clone:

4 labels of cutting through the E3 enzyme connect into 2-6 placed in-line long label again and are inserted into the pUC19 carrier (15:2ul) that the E3 enzyme cuts and add 2ul 10 * connection damping fluid again, and 1ul T4 dna ligase spends the night for 16 ℃.

Ligation transforms DH5 α: get 50ul competence DH5 α, add 10ul ligation liquid in 4 ℃ ofice baths 1 hour, 42 ℃ of heat-shockeds 45 seconds add the fresh LB substratum of 500ul, and 37 ℃ were shaken 1 hour, coated the Peri LB culture plate of diameter 20cm.

In 96 well culture plates that contain the LB substratum, each clone's toothpick is washed in the corresponding Confucius of corresponding 96 hole PCR Sptting plates (using the F/R primer) again with the hundreds of mono-clonal bacterium colonies of sterilization toothpick picking.The PCR screening positive clone.

(8) high-throughput extracts plasmid:

Adopt 96 hole micro plate high-flux parallel operations, Qiagen plasmid micro plate extracts test kit.

1) gets each positive colony of 200ul and spend the night the nutrient solution of jolting in 96 each hole of hole micro plate, centrifugal 1000rpm * 2 minute

2) bacterial precipitation adds Solution I (G/TE+RNase) 50ul, mixing, and room temperature 5-10 minute,

3) add Solution II (0-2NaOH+1%SDS) 50ul, light mixing, room temperature is no more than 2-3 minute.

4) add Solution III (5M KoAc, PH4.8) 70ul, light mixing, centrifugal 2500rpm * 10 minute immediately

5) supernatant is transferred to the centrifugal 1000rpm of Qiagen 96 hole plasmids trace purifying plates * 2 minutes, adds Wash buffer 200ul, and centrifugal washing once adds that 200ul Wash buffer is centrifugal to be washed for the second time again.

6) residual washing lotion is removed in centrifugal 2500rpm * 1 minute.

7) add every hole and add 10ul dH₂O wash-out plasmid.

(9) high-flux sequence

Adopt PE company's test kit and 96 holes heating microtiter plate, fluorescence stops thing (BigDye stops thing) cycle sequencing.

1) mix by the following amount of reagent of every reaction, the preparation feedback mixture is as follows:

4ul terminating nucleotide mixture (dNTP, ddNTP)

2ul 5 * order-checking damping fluid (0.4M Tris-Cl PH9.0 plus 10mM MgCl)

0.2ul Ampitaq FS

0.2ul pmol primer (T7/T3)

About 200ngBAC DNA

Add water to the 20ul volume

2) following thermal cycle reaction is set:

1 circulation: 5min, 95 ℃

20 circulations: 30s, 94 ℃

20s，54℃

4min，60℃

Cooling also keeps 4 ℃.

3) remove excessive termination thing with the centrifugal plate of Centri-Sep.The vacuum-drying sample.

4) add 2ul methane amide sample loading buffer termination reaction, last machine.

Automatic application of sample 96 passages of ABI3700 kapillary sequenator check order simultaneously.

(10) computer software analysis:

Adopt the DNA-Star software analysis or write a basic sequence statistics program

Two, protein library micro-sphere array principle and operation:

According to the similarity of proteinic function or phenotype, dependency is selected the protein library of one group of association, as the cytokine protein pool, and tumor cell surface flag library etc.Surveying protein molecular as required still detects its antibody molecule and determines label antibody molecule or proteantigen.If the detection protein molecule is then selected nucleic acid oligomer code tag labelled protein molecular antibody, antibody is monoclonal antibody preferably, also can be the genetically engineered small molecular antibody.If detected object is proteinic antibody, then select the label proteantigen, antigen can be the whole protein molecule, or the gene engineering expression protein fragments.

Antigen, antibody and nucleic acid oligomer DNA crosslinked labeling can adopt uviolizing protein nucleic acid mixture to cause covalent cross-linking, but efficient at most only 10% needs further enrichment.Main path be to use terminal methylthio groupization (nucleic acid oligomer modified of sulfenyl-SH) terminal with protein or polypeptide maleimide activatory aminothiopropionic acid residue covalent cross-linking (Ghosh S S, et.al. " Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzymeconjugate hybridization probes, " Bioconjug Chem 1990 January-February; 1 (1): 71-6).

Recently developed efficient cross-linking method between a kind of modifying DNA and the expressing protein (I.Burbulis, et.al., 2005, NatureMethods Vol.2 No.1, p31-37 and M.Lovrinovic, et.al., 2005 Mol.Biosyst., 1, p64-69).Ultimate principle is that the target protein plasmagene inserts PTYB1 or PTW1I1 carrier (NEB company) is expressed target protein and Intein intein-CBD (chitin binding domain) fusion rotein; Intein (intein) is that albumen from yeast etc. is from cutting element; sulfhydryl compound such as DTT can induce its halfcystine place from shearing, and produce the thioester bond that target protein C-terminal activatory is protected by thiophenol.The sulfydryl that nucleic acid one side 5 ' end is infiltrated the double-stranded DNA of halfcystine class residue is replaced shear protein protection thiophenol and crosslinked with the expressing protein end.Still mark vitamin H of the opposite side 5 of double-stranded DNA label ' hold.

Two irrelevant nucleic acid oligomer DNA labels of the default pair of sequences of each protein molecule in the library, 3 ' right end afterbody of label is established E1 restriction enzyme site and the complementation of 6-10bp sequence, so that primer and complementaryly extend to the dimer that the tail tail links to each other each other.Be protein molecular 1, bidding is signed tag1a, 1b, andmolecule 2 is established tag2a, 2b,molecule 3 is established tag3a, 3b ... Deng by that analogy, odd number odd molecular label is to the default same sequence F of a side, and the b side is established same sequence odd+E2 restriction enzyme site; Even number even molecular label is to the default same sequence R of a side, and the b side is established same and odd complementary sequence even+E2 restriction enzyme site.Make the terminal energy complementation of odd molecule bigeminy label b end and even number bigeminy label b end odd-even after the E2 enzyme is cut and combine extension, form 4 aggressiveness that head-head links to each other.Antibody or antigenic part covalent cross-linking cyanogen bromide (CNBr) activatory Sepharose CL-4B sepharose particle.

The protein library of nucleic acid oligomer label is adsorbed to the mixing microballoon of band capture ligands, and the unconjugated molecule of flush away forms molecule microballoon storehouse again.During detection sample to be tested is mixed with the microballoon storehouse, if have in the sample to be tested with the library in identical molecule, then the same molecular of label coding is just cemented out competitively, vitamin H by the label side end, come out by the streptavidin magnesphere purifying, cut through the E1 enzyme, 90 ℃ of heat denatured of the nucleic acid oligomer tag library of streptavidin magnetic microsphere purifying 5 minutes, through 4 to 5 thermal cyclings, 90 ℃ 30 seconds, 20 ℃ 10 minutes, 65 ℃ of 1 minute pfu enzymatic amplifications make complementary extension of pair of tag 3 ' end of each molecule and tail-tail links to each other.After pair of tag tail-tail links to each other in each molecule of molecular library, cut purifying with restriction enzyme E2 enzyme again, make between every pair of label of library of molecules molecule with the T4 ligase enzyme to connect at random.After perhaps the E2 enzyme is cut purifying, 92 ℃ of heat denatured 5 minutes, 4 to 5 thermal cyclings, 92 ℃ 30 seconds, 26 ℃ 10 minutes, 68 ℃ of 1 minute Tag enzymatic amplifications make that the pair of tag of any odd molecule and the pair of tag of even number molecule arbitrarily are complementary extends and be connected.For preventing that molecule number does not wait between every pair of label, do not connect every pair of label of free and spend the night with the connection of T4 ligase enzyme again, form 4 labels.

Nucleic acid oligomer 4 labels through pcr amplification again enzyme cut further series connection.With the F primer of restriction enzyme site of band E3 and R primer through 20-25 cyclic amplification 4 labels, the overwhelming majority is the product at two ends for F/R, few part is the product at F/F, R/R two ends, cut purifying through the E3 enzyme and be connected into 2-6 4 labels that link to each other through the T4 ligase enzyme again, adding the pUC19 that the E3 enzyme cuts again further connects, transform DH5 α bacterium, PCR selects tens of to hundreds of positive colonies, extract plasmid respectively, last plasmid order-checking series connection label, kind and the quantity of deriving testing molecule by the kind and the quantity of computer software analysis label.

Operating process:

(1) design of the protein library label of nucleic acid oligomer label, synthetic:

It is right to design a series of 20bp (16-30bp) nucleic acid oligomer, protein molecular 1 corresponding 1a, 1b label tag,molecule 2 corresponding 2a, 2b tag,molecule 3 corresponding 3a, 3b tag. ... .. by that analogy, the Tag sequence is separate irrelevant, synthetic respectively sense strand of each tag two strands and antisense strand oligonucleotide.Wherein a chain 5 ' end infiltrates halfcystine class residue, another chain 5 ' mark vitamin H.The reheat sex change mixes after the room temperature renaturation.

The synthetic ABI company dna synthesizer that adopts the solid phase phosphoramidite triester method of primer.Or commerce is ordered synthetic.

(2) protein-dna of Intein intein mediation is crosslinked:

1) EcoR I and HindIII enzyme behind the target protein gene amplification purifying are cut.Be cloned into PTYB1 or PTW1I1 carrier, transformed into escherichia coli is chosen positive colony.

2) the positive expression engineering bacteria is expressed target protein-Intein-CBD fusion rotein under aerobic conditions, with the crosslinked Sepharose 4B affinitive layer purification of chitin.

3) fusion rotein that contains target protein adds 0.2% thiophenol and 1mM TCEP (similar DTT on chitinous purification column, more stable), the nucleic acid oligomer DNA label that application of sample halfcystine class residue is modified, incubated at room 16 to 20 hours, the PBS wash-out, the crosslinked back of modifying DNA displacement thiophenol and target protein is eluted and is able to open and purifying with the Intein-CBD branch.

(3) antibody ligand or antigen part covalent cross-linking cyanogen bromide (CNBr) activatory Sepharole 4B sepharose particle.

Get and add on isopyknic albumen (1-10uM pr/1ml agar) carbonate buffer solution (0.2-0.25M carbonate contains 0.5M NaCl pH9) runner the mixing room temperature after the activatory Sepharole CL-4B swelling washing that 2g does and spent the night in 2 hours or 4 ℃.

Adding in advance with the HCl adjust pH is that 9 ethanolamine solutions stirs reaction termination in 15 minutes crosslinking reaction down.

At room temperature wash sorbent material, place the cloth funnel, successively use 100ml 50mMNaHCO3 (containing 0.5M NaCl), 0.1M Na to remove superfluous albumen and ethanolamine solutions₂B₄O₇(containing 0.5M NaCl), pH8.5 solution was washed, and washed with 0.5M NaCl solution finally, drained.

4 ℃ of storages in the 0.5M NaCl liquid that contains 0.02%NaN3.

Catch the protein of microballoon absorption label: the microballoon of catching of part covalent cross-linking mixes with the label protein library of purifying, and room temperature is in conjunction with a few hours, and washs the unconjugated molecule of flush away 3-5 time.

(4) protein of the competitive displacement in sample to be measured and microballoon storehouse tape label:

The protein antibody of purifying or antigen specimen add the protein microsphere array storehouse of thorough washing, and room temperature is in conjunction with a few hours, and is centrifugal, careful sucking-off supernatant (keep off and contact agarose microbeads).Move in the fresh centrifuge tube Qiagen purification column purifying.Heavily be dissolved in 50ul dH₂O.Restriction enzyme E1 enzyme was cut 5-6 hour, added crosslinked little magnetic ball (DynalBiotech) washing of streptavidin, magnet combination, purifying bonded DNA tag library.

(5) 2 of label connect:

90 ℃ of heat denatured of the nucleic acid oligomer tag library of streptavidin magnetic microsphere purifying 5 minutes, 4 to 5 thermal cyclings, 90 ℃ 30 seconds, 20 ℃ 10 minutes, 65 ℃ of 1 minute pfu enzymatic amplifications make complementary extension of pair of tag 3 ' end of each molecule and tail-tail links to each other.

The amplification of (6) 4 labels and series connection:

After pair of tag tail-tail links to each other in each molecule of molecular library, cut purifying with restriction enzyme E2 enzyme again, make between every pair of label of library of molecules molecule with the T4 ligase enzyme to connect at random.After perhaps the E2 enzyme is cut purifying, 92 ℃ of heat denatured 5 minutes, 4 to 5 thermal cyclings, 92 ℃ 30 seconds, 26 ℃ 10 minutes, 68 ℃ of 1 minute Tag enzymatic amplifications make the pair of tag ofmolecule 1 be connected with the pair of tag ofmolecule 2,molecule 3 and molecule 4 ... by that analogy.For preventing that molecule number does not wait between every pair of label, do not connect every pair of label of free and spend the night with 15 ℃ of connections of T4 ligase enzyme again, form 4 labels.

(7) clone:

The 2-6 that enzyme is cut placed in-line 4 labels are connected to the pUC19 carrier (15:2ul) that the E3 enzyme cuts and add 2ul 10 * connection damping fluid again, and 1ul T4 dna ligase spends the night for 16 ℃.

Ligation transforms DH5 α: get 50ul competence DH5 α, add 10ul ligation liquid in 4 ℃ ofice baths 1 hour, 42 ℃ of heat-shockeds 45 seconds add the fresh LB substratum of 500ul, and 37 ℃ were shaken 1 hour, coated the Petri LB culture plate of diameter 20cm.

In 96 well culture plates that contain the LB substratum, each clone's toothpick is washed in the corresponding Confucius of corresponding 96 hole PCR Sptting plates (using the F/R primer) again with the hundreds of mono-clonal bacterium colonies of sterilization toothpick picking.The PCR screening positive clone.

(8) high-throughput extracts plasmid:

Adopt 96 hole micro plate high-flux parallel operations, Qiagen plasmid micro plate extracts test kit.

1) gets each positive colony of 200ul and spend the night the nutrient solution of jolting in 96 each hole of hole micro plate, centrifugal 1000rpm * 2 minute

2) bacterial precipitation adds Solution I 50ul, mixing, and room temperature 5-10 minute,

3) add Solution II 50ul, light mixing, room temperature is no more than 2-3 minute.

4) add Solution III 70ul immediately, light mixing, centrifugal 2500rpm * 10 minute

5) supernatant is transferred to the centrifugal 1000rpm of Qiagen 96 hole plasmids trace purifying plates * 2 minutes, adds Wash buffer 200ul, and centrifugal washing once adds that 200ul Wash buffer is centrifugal to be washed for the second time again.

6) residual washing lotion is removed in centrifugal 2500rpm * 1 minute.

7) add every hole and add 10ul dH₂O wash-out plasmid.

(9) high-flux sequence:

Adopt 96 holes heating microtiter plate, fluorescence stops thing (BigDye stops thing) cycle sequencing.

1) mix by the following amount of reagent of every reaction, the preparation feedback mixture is as follows:

4ul terminating nucleotide mixture

2ul 5 * order-checking damping fluid

0.2ul Ampitaq FS

0.2ul pmol primer

About 200ng BAC DNA

Add water to the 20ul volume

2) following thermal cycle reaction is set:

1 circulation: 5min, 95 ℃

20 circulations: 30s, 94 ℃

20s，54℃

4min，60℃

Cooling also keeps 4 ℃.

3) remove excessive termination thing with the centrifugal plate of Centri-Sep.The vacuum-drying sample.

4) add 2ul methane amide sample loading buffer termination reaction, last machine.

Automatic application of sample 96 passages of ABI3700 kapillary sequenator check order simultaneously.

(10) computer software analysis

Adopt the DNA-Star software analysis or write a basic sequence statistics program.

Description of drawings

Fig. 1. represent molecule substitution label micro-sphere array synoptic diagram.

First row, circle is represented the part of the crosslinked differing molecular of microsphere surface.The molecular library differing molecular is with the rectangular expression of different shapes end, and represents code tag to link to each other by E1 restriction enzyme enzyme recognition site with the section of DNA sequence.Sequence one end stain is represented the mark vitamin H.Microballoon forms the micro-sphere array storehouse by the library of molecules that capture ligands absorption label connects.

Second row, the testing molecule rectangular representative of band suspension points arrow, the structure that rectangular representative is identical with molecule in the molecular library.Sample to be tested and micro-sphere array storehouse just displace the identical indication molecule of tape label in conjunction with the back testing molecule, see the molecule of the 3rd row's signal free tape label.

The 4th row represents isolating label, and after the molecule E1 enzyme of the 3rd row's tape label was cut, the little magnetic ball crosslinked through streptavidin adsorbed the vitamin H of an end mark and separate purifying with molecule.

Fig. 2. represent connection, the series connection synoptic diagram of nucleic acid oligomer label.

Numeral 1a, 1b; 2a, 2b ... represent differing molecular 1,2 in the molecular library ... code tag; The sequence of band suspension points is represented the sense strand of nucleic acid oligomer label double-stranded DNA; Stain is represented the vitamin H of 5 ' end mark.Suspension points is represented the sequence of the different labels of each molecule, tail-tail complementation between its 3 ' terminal every pair of label, and 5 ' end adds F or R respectively according to the label difference, or the E2 sequence.

At first 3 ' terminal sex change between the every couple of label a and the b, renaturation complementation through polymerase extension and tail-tail links to each other,forms 2 labels; Label forms 4 labels to cutting through the E2 enzyme, connecting at random through the T4 ligase enzyme behind the purifying again.End after perhaps the E2 enzyme is cut is preset as similar complementary sequence between a and the b, also can complementary the extension and head-head joins, and 4 labels that are connected between formation odd number group molecule and the even number set molecule.

Through F/R primer PCR amplification, cut, be connected to form bigger series connection and be cloned into the carrier through E3 enzyme cut again by the E3 enzyme for nucleic acid oligomer 4 labels, and transform bacteria is selected positive colony.

Embodiment

Adopt embodiment that the present invention is described in further detail below.

Embodiment: cancer suppressor gene pRB, p16, p53 and the parallel detection of contrast Tubulin gene:

Oncogene and cancer suppressor gene play a part most critical in the cell growth cycle regulation and control.Especially cancer suppressor gene pRB, p16, the metabolism of p53 and inactivation are being brought into play most important effect in tumour cell loses the mechanism of growth control.Nearly all tumour all relates to they or one of them certain change that is associated.Yet because traditional research means is single, the method that research object is analyzed is one by one taken a part for the whole unavoidably, does not reduce whole real metabolic images.

Present embodiment makes up a human tumor suppressor gene pRB, p16, the indication molecule storehouse of p53, and add that the proteic Tubulin of people's cellularstructure (tubulin) gene is as the build together pRB of a tape label of constant contrast indication molecule, p16, p53 and Tubulin indication DNA are adsorbed to the micro-sphere array storehouse of Seplarose CL-4B.The whole metabotic change of the cancer suppressor gene before and after the parallel parsing U2OS cell transformation p16 gene.

Cancer suppressor gene pRB get its C-terminal (792aa-928aa) E2F in conjunction with about 400bp sequence in territory as the pRB indication molecule, its both-side ends sequence is:

5’-cac att cct cga agc cct tac aag ttt cct ag------c agc aga aac tgg caa gaa atg acg gaa gag-3’

P16 gets the conserved sequence of one section nearly 300bp of its gene Exon2 as the p16 indication molecule, and its both-side ends sequence is:

5’-ccg atc cag gtc atg atg atg ggc agc gcc------cgg tac ctg cgc gcg gct gcg ggg ggc acc-3’

P53 gets the conservative nearly 300bp sequence of its gene N latter end as the p53 indication molecule, and its both-side ends sequence is:

5’-atg gag gag ccg cag tca gat cct agc------cct aca ccg gcg gcc cct gca cca gcc-3’

The N latter end 300bp sequence that structural protein Tubulin gene is got gammal contrasts indication molecule as confidential reference items, and its both-side ends sequence is:

5’-atg ccg agg gaa atc atc acc------ctg tcg gaa cat gga gga gga gct ggc-3’

At first define following sequence:

F primer sequence: 5 '-cca cag gaa aca gta ttc-3 '

R primer sequence: 5 '-gac gcg cga ggc aga tcg-3 '

The 1a label+E1: 5 '-atg tcc cct ata cta gca tgC tag-3 ', E1 is Bfa I.

The 1b label+E1: 5 '-cct taa ttt tcc aat gca tgC tag-3 '

2a label+E1:5 '-ctt gtg caa ccc act gtc gaC tag-3 '

2b label+E1:5 '-aag ata ttc caa aag gtc gaC tag-3 '

3a label+E1:5 '-gaa aag tat gaa gag gag ctC tag-3 '

3b label+E1:5 '-acc ttc atc gcg tca gag ctC tag-3 '

4a label+E1:5 '-ggc gaa aca aag agt gcc ggC tag-3 '

4b label+E1:5 '-gaa act cca aac cca gcc ggC tag-3 '

(1) design of indication molecule storehouse primer and nucleic acid oligomer label, synthetic:

1) molecule 1-pRB primer

The 1a forward primer: the F+1a label+E1+ pRB 5 ' terminal sense strand sequence, promptly

5’-cca cag gaa aca gta ttc+atg tcc cct ata cta gca tgc tag+cac att cct cga agc cct tac-3’

The 1b forward primer:E2+ 1b label+E1+ pRB 5 ' terminal sense strand sequence, promptly

5’atg cgc gat atc ggc+cct taa ttt tcc aat gca tgc tag+cac att cct cga agc cct tac-3”，

E2 is HinP1I

Molecule 1 reverse primer: pRB 3 ' terminal antisense strand sequence, promptly

5’-ctc ttc cgt cat ttc ttg cca gtt tct gct g-3’

2) molecule 2-p16 primer

The 2a forward primer: the R+2a label+E15 ' the terminal sense strand sequence of+p16, promptly

5’-gac gcg cga ggc aga tcg+ctt gtg caa ccc act gtc gac tag+ccg atc cag gtc atg atg-3’

The 2b forward primer:E2+ 2b label+E15 ' the terminal sense strand sequence of+p16, promptly

5’atg cgcgat atc gtg+aag ata ttc caa aag gtc gac tag+ccg atc cag gtc atg atg-3”

3 ' the terminal antisense strand sequence ofmolecule 2 reverse primers: p16, promptly

5’-ggt gcc ccc cgc agc cgc gcg cag gta ccg-3’

3) molecule 3-p53 primer

The 3a forward primer: the F+3a label+E15 ' the terminal sense strand sequence of+p53, promptly

5’-cca cag gaa aca gta ttc+gaa aag tat gaa gag gagctc tag+atg gag gag ccg cag tc-3’

The 3b forward primer:E2+ 3b label+E15 ' the terminal sense strand sequence of+p53, promptly

5’-gag cgcgta tac gca+acc ttc atc gcg tca gag ctc tag+atg gag gag ccg cag tc-3”

Molecule 3 reverse primers: pRB 3 ' terminal antisense strand sequence, promptly

5’-ggt tgg tgc agg ggc cgc cgg tgt agg-3’

4) molecule 4-Tubulin primer

The 4a forward primer: the R+4a label+E1+ Tubulin 5 ' terminal sense strand sequence, promptly

5’-gac gcg cga ggc aga tcg+ggc gaa aca aag agt gcc ggc tag+atg ccg agg gaa atc atc-3’

The 4b forward primer:E2+ 4b label+E1+ Tubulin 5 ' terminal sense strand sequence, promptly

5’gag cgc gta tac gtc+gaa act cca aac cca gcc ggc tag+atg ccg agg gaa atc atc-3”

Molecule 4 reverse primers: Tubulin 3 ' terminal antisense strand sequence, promptly

5’-gcc agc tcc tcc tcc atg ttc cga cag-3’

The synthetic ABI company dna synthesizer that adopts the solid phase phosphoramidite triester method of primer.Or commerce is ordered synthetic.Synthetic primer is to carry out 5 ' end biotin labeling by infiltrating biotinylated dNTP.

(2) DNA reverse primer cross-linked agarose gel particle:

Cyanogen bromide (CNBr) activation Sepharose CL-4B is available from Pharmacia company.

Whole cross-linking process is divided into parallel two groups, the sense strand Oligo of one group of crosslinked DNA reverse primer, and another organizes the antisense strand Oligo of crosslinked DNA reverse primer.Below with poly-representative of step of one group:

1) claims working of 1g Sepharose4B agar in a 15ml conical centrifuge tube, add the 1M HCl aquation agar of 10ml, put upside downlight mixing 1 minute, move in the B of a 60ml, add 500ml 1M HCl washing again, about 15 minutes of swelling agar.

2) use 100ml dH₂O washs agar, uses 100ml 10mM potassiumphosphate (PH8.0) washing again.

3) in one 15 blind nut centrifuge tube of agar immigration, adding about 4ml 10mM potassiumphosphate (PH8.0) becomes uniform thick slurry until medium.

4) add 50ul Oligo aqueous dna immediately, place under the room temperature that mixing spends the night on the runner.

5) in stink cupboard, agar is moved in the B 100ml dH₂O washes 2 times, and 100ml 1M diethanolamine hydrochloride (PH8.0) is washed once.

6) agar moves in the 15ml centrifuge tube, adds 1M diethanolamine hydrochloride (PH8.0) to pasty state, room temperature runner mixing 2-4 hour.

7) move to B, use 100ml 10mM potassiumphosphate (PH8.0), 100ml 1M potassiumphosphate (PH8.0), 100ml1M Repone K, 100ml dH successively₂O washs agar.

8) the crosslinked agar of Oligo places storage buffer 10mM Tris-Cl (PH7.8) to add 1mM EDTA, 0.3M NaCl.Two groups of cross-linking agents are mixed in 4 ℃ and can stablizestorage 1 year.

(3) mark in DNA library increases and is adsorbed to and catches microballoon

Amplification library molecule 1 a,molecule 2a,molecule 3a,

Template: pUHD-pRB 0.5ul pUHD-p16 pUHD-p53 pUC19-tubulin

Molecule 1 a forward primer 0.5ulforward primer 2a,forward primer 3a

Molecule 1 reverse primer 0.5ul reverse primer 2,reverse primer 3

10×PCR buffer 2ul

10mM dNTP 0.5ul

pfu 0.5ul

dH2O 15ul

20ul

Amplification library molecule 1 b,molecule 2b,molecule 3b,

Template: pUHD-pRB 0.5ul pUHD-p16 pUHD-p53 pUC19-tubulin

Molecule 1b forward primer 1 0.5ulforward primer 2b,forward primer 3b

Molecule 1reverse primer 1 0.5ul reverse primer 2,reverse primer 3

10×PCRbuffer 2ul

10mM dNTP 0.5ul

pfu 0.5ul

dH2O 15ul

20ul

Pcr amplification in 96 hole micro-reaction plates, 94 ℃ 4 minutes, then 25 the circulation 94 ℃ 30 seconds .52 ℃ 30 seconds, 72 ℃ 40 seconds.Then 72 ℃ 5 minutes.

The PCR product is through Qiagen 96 hole purifying plate purifying.

Catch the DNA library of microballoon absorption mark: the PCR dna library of purifying mixes, and 100 ℃ of heating place cooled on ice after 5 minutes fast.The crosslinked microballoon of catching spends the night in 66 ℃ of dna library combinations that add purifying, and washs the unconjugated molecule of flush away 3-5 time.

(4) the competitive displacement in sample to be measured and microballoon storehouse DNA:

Sample U2OS cell to be measured and pUHD-p16 transform the RNA purifying of U2OS cell: adopt the guanidinium isothiocyanate single stage method to extract total RNA (Chomczynski, P.et, al.1987 Anal.Biochem.Vol 162,156).

Sample adds Trizole (the 1ml 4M guanidinium isothiocyanate of 1ml in the EP pipe, 1ml phenol, 0.1ml sex change liquid cracking 2M sodium-acetate PH4.0), strong vortex oscillation or aspirate repeatedly with syringe needle, add 200ul chloroform vibration again, centrifugal 5 minutes, get and reset and add equivalent 0.5ml Virahol and put-20 ℃ of centrifugations again in 2 hours, 70% washing with alcohol once adds the dH that 50ul DEPC handles₂The O dissolving.

Oligo (dT) paramagnetic beads purified mRNA is undertaken by the Promega specification sheets.The cDNA reverse transcription adopts the Invitrogen test kit.

CDNA first chain is synthetic:

mRNA(1mg/ml) 10ul

Oligo(dT)12-18(1mg/ml) 10ul

1M Tris-Cl(pH7.6) 2.5ul

1MKCl 3.5ul

205mM MgCl₂ 2.0ul

DNTP (every kind of 10mM) 5ul

0.1M DTT 2.0ul

Rnase inhibitor 25umits

dH₂O 23ul

Add 2ul Mo-MLV ThermoScript II again, 37 ℃ are incubated one hour.

Second chain is synthetic: directly add in the first chain reaction thing

10mM MgCl₂ 70ul

2M Tris-Cl(pH7.4) 5ul

1M(NH₄)₂SO₄ 1.5ul

Rnase H(1Umits/ul) 1.0ul

E. coli dna polymerase I (10umits/ul) 1.0ul

Be incubated 4 hours down in 16 ℃,

Add 50mNAD 1.0ul again

E. coli dna ligase (100umit/ul) 1.0ul

T4 polynueleotide kinase (3umit/ul) 1.0ul

Insulation added 0.5M EDTA (pH8.0) 5ul termination reaction after 15 minutes under the room temperature.

Sample cDNA places dry ice then fast 100 ℃ of heat denatured 10 minutes then.

The microballoon storehouse is at first washed the unconjugated library of flush away molecule three times before the displacement test again in 66 ℃ temperature is filled.Be warming up to 90 ℃ of sex change again moved in 66 ℃ of incubations in 2 minutes immediately.The sample DNA of sex change is added in the microballoon storehouse 66 ℃ continued the incubation replacement(metathesis)reaction 1-2 hour.Centrifugal, careful sucking-off supernatant (keep off and contact agarose microbeads).Move in the fresh centrifuge tube Qiagen purification column purifying.Heavily be dissolved in 50ul dH₂O.Restriction enzyme E1-BfaI enzyme was cut 5-6 hour, added crosslinked little magnetic ball (Dynal Biotech) washing of streptavidin, magnet combination, purifying bonded tag library.

(5) 2 of label connect:

90 ℃ of heat denatured of the oligonucleotide tag library of streptavidin magnetic microsphere purifying 5 minutes, 4 to 5 thermal cyclings, 90 ℃ 30 seconds, 20 ℃ 10 minutes, 65 ℃ of 1 minute pfu enzymatic amplifications make complementary extension of pair of tag 3 ' end of each molecule and tail-tail links to each other.

The amplification of (6) 4 labels and series connection:

After pair of tag tail-tail links to each other in each molecule of molecular library, cut purifying with restriction enzyme E2-Hinp1I enzyme again, make between every pair of label of library of molecules molecule with the T4 ligase enzyme to connect at random.After perhaps the E2 enzyme is cut purifying, 92 ℃ of heat denatured 5 minutes, 4 to 5 thermal cyclings, 92 ℃ 30 seconds, 26 ℃ 10 minutes, 68 ℃ of 1 minute Tag enzymatic amplifications make the pair of tag ofmolecule 1 be connected with the pair of tag ofmolecule 2,molecule 3 and molecule 4 ... by that analogy.For preventing that molecule number does not wait between every pair of label, do not connect every pair of label of free and spend the night with 15 ℃ of connections of T4 ligase enzyme again, form 4 labels.

(7) clone:

4 labels of cutting through the E3-BamHI enzyme connect into 2-6 placed in-line long label again and are inserted into the pUC19 carrier (15:2ul) that the E3 enzyme cuts and add 2ul 10 * connection damping fluid again, and 1ul T4 dna ligase spends the night for 16 ℃.

Ligation transforms DH5 α: get 50ul competence DH5 α, add 10ul ligation liquid in 4 ℃ ofice baths 1 hour, 42 ℃ of heat-shockeds 45 seconds add the fresh LB substratum of 500ul, and 37 ℃ were shaken 1 hour, coated the Petri LB culture plate of diameter 20cm.

In 96 well culture plates that contain the LB substratum, each clone's toothpick is washed in the corresponding Confucius of corresponding 96 hole PCR Sptting plates (using the F/R primer) again with the hundreds of mono-clonal bacterium colonies of sterilization toothpick picking.PCR selects 50 positive colonies of every group of cell.

(8) high-throughput extracts plasmid:

Adopt 96 hole micro plate high-flux parallel operations, Qiagen plasmid micro plate extracts test kit.

1) gets each positive colony of 200ul and spend the night the nutrient solution of jolting in 96 each hole of hole micro plate, centrifugal 1000rpm * 2 minute

2) bacterial precipitation adds Solution I 50ul, mixing, and room temperature 5-10 minute,

3) add Solution II 50ul, light mixing, room temperature is no more than 2-3 minute.

4) add Solution III 70ul immediately, light mixing, centrifugal 2500rpm * 10 minute

5) supernatant is transferred to the centrifugal 1000rpm of Qiagen 96 hole plasmids trace purifying plates * 2 minutes, adds Wash buffer 200ul, and centrifugal washing once adds that 200ul Wash buffer is centrifugal to be washed for the second time again.

6) residual washing lotion is removed in centrifugal 2500rpm * 1 minute.

7) add every hole and add 10ul dH2O wash-out plasmid.

(9) high-flux sequence: adopt PE company test kit.

Selected 20 positive colony plasmids of every group of cell, fluorescence stops thing (BigDye stops thing) cycle sequencing.

1) mix by the following amount of reagent of every reaction, the preparation feedback mixture is as follows:

4ul terminating nucleotide mixture

2ul 5 * order-checking damping fluid

0.2ul Ampitaq FS

0.2 ul pmol primer

About 200ng BAC DNA

Add water to the 20ul volume

2) following thermal cycle reaction is set:

1 circulation: 5min, 95 ℃

20 circulations: 30s, 94 ℃

20s，54℃

4min，60℃

Cooling also keeps 4 ℃.

3) remove excessive termination thing with the centrifugal plate of Centri-Sep.The vacuum-drying sample.

4) add 2ul methane amide sample loading buffer termination reaction, last machine.

Automatic application of sample 96 passages of ABI3700 kapillary sequenator check order simultaneously.

(10) sequencing analysis result:

Adopt the DNA-Star software analysis

The result:

Number oftags	1a	1b	2a	2b	3a	3b		4a		4b
											U2OS	26	25	0	0	37	36	89	90
PUHD-p16 transforms U2OS	11	10	106	99	9	10	32	31

Detect 2a and 2b label before the U2OS cell transformation, detect 2a and the 2b label of a large amount of p16 behind the pUHD-p16 conversion U2OS cell less than p16.Label indication result is consistent with p16 gene variation before and after the U2OS conversion, conforms to.

Claims (11)

1. a molecule substitution label sequencing parallel detection method is characterized in that comprising the steps: 1) the crosslinked microballoon that capture ligands is arranged, the molecule in the molecular library of absorption label coding forms tag molecule micro-sphere array analysis storehouse; Described label is one section separate nucleic acid oligomer double-stranded DNA that has nothing to do with the library molecular sequences, the sense strand 5 of described nucleic acid oligomer, end is connected with biotin labeling and extension increasing sequence, 3 ' end of nucleic acid oligomer is preset with and terminal complementary sequence of respective labels and restriction enzyme E1 recognition site, described label and library are molecule crosslinked, and described capture ligands is the specific binding ligand of library molecule; (2) mix with sample to be tested in the micro-sphere array storehouse, the same molecular in the competition displacement tape label molecular library; (3) being displaced the tag molecule of coming detects by label series connection, order-checking.

2. detection method according to claim 1, described tag molecule length is 12-100bp.

3. detection method according to claim 2, described tag molecule length is 16-30bp.

4. detection method according to claim 1, described molecular library are DNA library or protein library.

5. detection method according to claim 1, described label and dna molecular are crosslinked to be crosslinked by the design primer amplification.

6. detection method according to claim 1, described label and cross linking of protein molecule are crosslinked by sulfydryl modification DNA and maleimide activation of protein.

7. detection method according to claim 1, described microballoon size arrives micron order for nano level.

8. detection method according to claim 1, described microballoon material is plastics, resin, gel or other macromolecule polymeric material, described plastics are polyethylene, polyvinyl chloride or polystyrene, described resin is resol, vibrin or polyamide resin, and described gel is agarose, dextran or polyacrylamide.

9. detection method according to claim 1, described microballoon can also be particulate in irregular shape or the planar medium with active surface.

10. detection method according to claim 1, the series connection of described label, order-checking detect and comprise the steps: the nucleic acid oligomer double-stranded DNA of (a) code tag for the artificial distinguished sequence be made up of two complementary Oligo (oligonucleotide); (b) each molecular designing has a pair of not homotactic label in the library of molecules, and 5 ' end of each label sense strand is marked with vitamin H, 3 ' terminal portions complementation between every pair, and also 3 ' terminal and library of molecules junction is provided with the restriction enzyme site of restriction enzyme E1; (c) the every pair of label that displaces by the little magnetic beads for purifying of streptavidin, forms the bigeminy label through its 3 ' terminal complementary polymerase extension after the E1 enzyme is cut; (d) the library of molecules molecule is divided into two groups of odd number (Odd), even numbers (Even), every pair of bigeminy label of odd molecule, and a side adds special sequence F in advance, opposite side adds Odd sequence and E2 restriction enzyme site; Every pair of bigeminy label of even number molecule, a side adds different R sequences in advance, opposite side adds and Odd complementary Even sequence and E2 restriction enzyme site; (e) every pair of bigeminy label is cut at random through the E2 enzyme and is connected, or the complementation of Odd-Even sequence is extended and generation tetrad label after the E2 enzyme is cut.Use F/R primer amplification 4 labels again, the PCR product is cut further series connection insertion cloning vector of back, clone, positive plasmid order-checking with the default E3 enzyme in the F/R outside.Identify the kind and the quantity of each code tag.

11. the reagent of label series connection, order-checking in the tag molecule micro-sphere array analysis storehouse of the preparation of the step (1) in the arbitrary detection method of claim 1-10 and the step (3).

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