CN102766688A

CN102766688A - Method for testing gene sequences

Info

Publication number: CN102766688A
Application number: CN2012102326016A
Authority: CN
Inventors: 盛司潼
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-04-17
Filing date: 2012-07-06
Publication date: 2012-11-07
Anticipated expiration: 2032-07-06
Also published as: CN102766688B

Abstract

The invention relates to the field of biological genetic engineering, and provides a method for testing gene sequences. The method includes the following steps: a first anchor primer is anchored and combined on the first adaptor of a nucleic acid segment to be tested containing a gene sequence, and a connection sequencing method is used for reading the sequence information of M nucleotides after the terminal of the first anchor primer is extended; endonuclease is utilized to partially or completely excise the nucleotides with read sequence information, and the second adaptor is connected with enzyme-digested product, so that a new nucleic acid segment to be tested is obtained; a second anchor primer is then anchored on the second adaptor of the new nucleic acid segment to be tested, and the connection sequencing is utilized again to read the sequence information of N nucleotides after the terminal of the second anchor primer is extended; reagent is replaced, and the steps of enzyme digestion, adaptor connection, anchor primer bonding, connection sequencing and the like are repeated, so that the needed gene sequence information is obtained. By means of the enzyme digestion-extension sequencing method, the purpose of increasing the read lengths of tested gene sequences is achieved.

Description

A kind of method that detects gene order

Technical field

The present invention relates to the biological gene engineering field, more particularly, relate to a kind of method that detects gene order.

Background technology

At present, the gold standard that detects to gene order is a sequence measurement, commonly sanger PCR sequencing PCR and tetra-sodium PCR sequencing PCR (Pyrosequencing), and wherein the tetra-sodium PCR sequencing PCR is applicable to high throughput analysis.When utilizing the tetra-sodium PCR sequencing PCR to check order, be connected with the segmental magnetic bead of order-checking and be fixed in the aperture of etching optical fiber slide (PTP plate).Because aperture is big (55 μ m * 44 μ m); Therefore; The magnetic bead stationkeeping is constant when checking order in order to make, and need in aperture, fill the mixture that contains multiple protein, to guarantee carrying out smoothly of sequencing reaction and Cai Tu; Add the use of luciferase, these factors cause the cost of tetra-sodium PCR sequencing PCR very high.

For the order-checking cost is reduced, prior art adopts the connection PCR sequencing PCR to replace the tetra-sodium PCR sequencing PCR to check order.Existing a kind of PCR sequencing PCR that connects utilizes endonuclease digestion to extend to check order, and as shown in Figure 1, the step of this method comprises: (1) utilizes and contains enzyme and cut the double chain oligonucleotide joint one of recognition site and be connected with nucleic acid fragment, obtains the determined nucleic acid fragment; (2) restriction enzyme of cutting recognition site with the identification enzyme carries out enzyme to nucleic acid fragment to be measured to be cut, and obtains wherein the double-stranded product that a chain contains protruding terminus; (3) on double-stranded product, connect one group of corresponding specific position and contain fluorescently-labeled double-stranded joint two, obtain connecting product; Obtain this specific position corresponding nucleotide sequences information through detecting the fluorescent signal that connects product; Wherein, double-stranded joint two contains protruding terminus, also contains enzyme and cuts recognition site; Cut the Nucleotide number between recognition site and the restriction enzyme site according to enzyme, what a or several Nucleotide said protruding terminus calculate in advance; (4) the connection product in the separating step (3) obtains separated product; (5) utilize in can identification step (3) cut recognition site with enzyme enzyme separated product carried out enzyme cut, obtain containing one group of fragment that enzyme is cut recognition site; (6) operation of repeating step (3) to (5) is until recording all nucleotide sequences that can survey on the determined nucleic acid fragment; During wherein last repetitive operation, can omit step (5).

In above-mentioned connection PCR sequencing PCR, utilize and to contain fluorescently-labeled double chain oligonucleotide joint as detection probes, the change of cutting the recognition site position with institute's band enzyme on the joint realizes and controls the extension propelling of the position of checking order.If use this method to detect the nucleic acid fragment that contains gene order; Can be because the Nucleotide number restriction between double chain oligonucleotide joint Nucleotide number and employed restriction enzyme enzyme recognition site and the restriction enzyme site; Make that can detect the gene order information that obtains can only equal enzyme at most and cut the Nucleotide number between recognition site and the restriction enzyme site; It reads long seriously restricted, is unfavorable for containing the detection and the analysis of the nucleic acid fragment of gene order.

Therefore, need a kind of method of new detection gene order, the order-checking that can increase when gene order detected is read long.

Summary of the invention

The object of the present invention is to provide a kind of method that detects gene order, be intended to solve prior art and read long too short problem when PCR sequencing PCR detects gene order with connecting.

In order to realize goal of the invention, a kind of method that detects gene order may further comprise the steps:

A. the first anchor primer grappling is incorporated on segmental first joint of the determined nucleic acid that contains gene order;

B. at the first anchor primer extending end fluorescent probe of connecting band different positions mark respectively, and detect the corresponding fluorescent signal that connects product, obtain the sequence information of M Nucleotide behind the first anchor primer extending end;

C. utilize restriction endonuclease with having obtained the nucleotide segment of sequence information or excision fully among the step B, obtain containing the segmental enzyme of waiting to check order and cut product;

D. enzyme is cut product and is connected second joint and obtain the new determined nucleic acid fragment that contains gene order, and second anchor primer is incorporated on segmental second joint of the new determined nucleic acid that contains gene order;

E. at the second anchor primer extending end fluorescent probe of connecting band different positions mark respectively, and detect the corresponding fluorescent signal that connects product, obtain the sequence information of N Nucleotide behind the second anchor primer extending end;

F. change reagent, the product of last step is carried out enzyme is cut, joint connects, anchor primer combines, fluorescent probe connects and fluorescent signal detects;

G. repeating step F is until obtaining containing gene order information required in the determined nucleic acid fragment of gene order;

Wherein, M, N are positive integer; Said gene is allelotrope or antitumor drug genes involved.

Wherein, first anchor primer described in the steps A contains at least one enzyme and cuts recognition site.

Further, said step C can may further comprise the steps:

C1. with the fluorescent probe that connects among the step B and the first anchor primer wash-out, first anchor primer and carry out chain extension of resetting forms double chain acid molecule with the determined nucleic acid fragment that contains gene order;

C2. restriction endonuclease through discern on first anchor primer with enzyme cut recognition site and carry out enzyme and cut, with having obtained the nucleotide segment of sequence information or excision fully among the step B, obtain containing the segmental enzyme of waiting to check order and cut product.

Wherein, step C may further comprise the steps:

C1 '. the other end at first anchor primer connects the 3rd double-stranded joint, and the 3rd joint contains at least one enzyme and cuts recognition site;

C2 '. utilize restriction endonuclease discern the 3rd joint with enzyme cut recognition site, with having obtained the nucleotide segment of sequence information or excision fully among the step B, obtain containing the segmental enzyme of waiting to check order and cut product.

In above-mentioned arbitrary scheme, said first anchor primer contains at least one specificity residue and/or an end seals.

Further, said step B may further comprise the steps:

B1. at the fluorescent probe of the first anchor primer extending end connecting band specific position mark that contains the specificity residue, detect the corresponding fluorescent signal that connects product, obtain the nucleotide sequence information of correspondence position;

B2. with specificity cutting agent cleavage specificity residue, with the fluorescent probe that connects in the last step and the first anchor primer wash-out, first anchor primer of resetting;

B3. the first anchor primer extending end repeat with the connection of the fluorescent probe of specific position mark with detect the corresponding operation that is connected the fluorescent signal of product, obtain the sequence information of M Nucleotide behind the first anchor primer extending end.

In above-mentioned arbitrary scheme, the determined nucleic acid fragment that contains gene order described in the steps A is fixed in surface of solid phase carriers.

Further, before steps A, can also comprise step:

A0. utilize solid phase carrier that gene order is increased, obtain being fixed in the determined nucleic acid fragment that contains gene order of surface of solid phase carriers.

Wherein, said steps A 0 may further comprise the steps:

The nucleic acid fragment that contains gene order that A01. will be used to increase is fixed in surface of solid phase carriers, obtains the surface and contains the solid phase carrier that at least one contains the nucleic acid fragment of gene order;

A02. primer is incorporated into the primer binding site on the surface of solid phase carriers, obtains being fixed with the amplification vector of primer;

A03. the nucleic acid fragment on the amplification vector is increased, obtain being fixed in the determined nucleic acid fragment that contains gene order of surface of solid phase carriers.

Wherein, Primer described in the steps A 02 comprises and is used for upstream primer and/or downstream primer that the said determined nucleic acid fragment that contains gene order is increased; Said upstream primer is and the complementary bonded nucleotide sequence of the determined nucleic acid fragment that contains gene order 5 ' end that said downstream primer is and the identical nucleotide sequence of determined nucleic acid fragment 3 ' terminal sequence that contains gene order.

Wherein, the amplification described in the steps A 03 is the unit molecule amplification.

Wherein, primer is incorporated into the mode of surface of solid phase carriers and is in the said steps A 02: primer matches with the group that surface of solid phase carriers carries and is connected, and realizes directly combining; Or the group that carries through connexon matches with group that primer and surface of solid phase carriers carry respectively and is connected, and realizes combining indirectly.

Further, said pairing ways of connecting adopts at least a in biotin-avidin/Streptavidin, nanometer gold/iodacetyl-sulfydryl, amino-aldehyde radical/carboxyl/isothiocyano, the acrylic amide-siloyl group/SEPIGEL 305.

Wherein, said allelotrope comprises at least one among F8, F9, FGFR3, IDS, GALT, HBB, HBA1, HBA2, ATP7B, PHEX, GJB2, COL4A5, the LMNA.

Wherein, the genes involved of antitumor drug shown in comprises at least one among CYP2C9, VKORC1, KIT, PDGFRA, CYP2C19, DHFR, GSTM1, MTHFR, RFC1, CYP19A1, UGT1A1, UGT1A7, UGT1A9, ABCB1, CYP2D6, CYP3A5, SULT1A1, UGT2B15, GSTA1, SOD2, CYP2B6, GSTP1, MDR1, BCRP, p53, CAT, CBR1, CBR3, EGFR, KRAS, the BRAF.

By on can know; The method of detection gene order according to the invention; Utilize restriction endonuclease to contain and obtained the nucleotide segment of sequence information or excision fully on the determined nucleic acid fragment of gene order, and then connect new joint, the anchor primer that grappling is new also carries out the connection of fluorescent probe and the detection of fluorescent signal; Extend forward and read nucleotide sequence, thereby the order-checking that has increased when detecting gene order is read long.

Description of drawings

Fig. 1 is a kind of connection PCR sequencing PCR synoptic diagram that utilizes endonuclease digestion to extend order-checking in the prior art.

Fig. 2 is the method flow diagram that detects gene order in the one embodiment of the invention.

Fig. 3 is the structural representation of a used anchor in the one embodiment of the invention.

Fig. 4 is the structural representation of an anchor in another embodiment of the present invention.

Fig. 5 is the structural representation of an anchor in another embodiment of the present invention.

Fig. 6 is the method synoptic diagram that obtains the sequence information of N Nucleotide behind the 2nd anchor extending end in the another embodiment of the present invention.

Fig. 7 utilizes enzyme to cut to extend the method synoptic diagram that order-checking detects gene order in the one embodiment of the invention.

Embodiment

In order to make the object of the invention, technical scheme and advantage clearer,, the present invention is further elaborated below in conjunction with accompanying drawing and embodiment.

Fig. 2 shows the method flow that detects gene order in the one embodiment of the invention, and this method may further comprise the steps:

S1. the first anchor primer grappling is incorporated on segmental first joint of the determined nucleic acid that contains gene order;

S2. at the first anchor primer extending end fluorescent probe of connecting band different positions mark respectively, and detect the corresponding fluorescent signal that connects product, obtain the sequence information of M Nucleotide behind the first anchor primer extending end;

S3. utilize restriction endonuclease with having obtained the nucleotide segment of sequence information or excision fully among the step S2, obtain containing the segmental enzyme of waiting to check order and cut product;

S4. enzyme is cut product and is connected second joint and obtain the new determined nucleic acid fragment that contains gene order, and second anchor primer is incorporated on segmental second joint of the new determined nucleic acid that contains gene order;

S5. at the second anchor primer extending end fluorescent probe of connecting band different positions mark respectively, and detect the corresponding fluorescent signal that connects product, obtain the sequence information of N Nucleotide behind the second anchor primer extending end;

S6. change reagent, the product of last step is carried out enzyme is cut, joint connects, anchor primer combines, fluorescent probe connects and fluorescent signal detects;

S7. repeating step S6 is until obtaining containing gene order information required in the determined nucleic acid fragment of gene order;

The technical scheme of the detection gene order that the present invention put down in writing; It is advantageous that; Carrying out sequencing reaction when detecting the gene order in the determined nucleic acid fragment contain gene order; Utilize restriction endonuclease to contain to have read on the determined nucleic acid fragment of gene order the nucleotide segment of sequence information or excision fully, and then on the nucleic acid fragment that contains the nucleotide sequence of waiting to check order, connect new joint, the anchor primer that grappling is new also carries out the connection of new fluorescent probe and the detection of corresponding fluorescent signal; Realize extending forward reading more nucleotide sequence, thereby the order-checking that has increased utilization connection PCR sequencing PCR detection gene order is read long.

Need to prove that allelotrope according to the invention is meant the gene of controlling the different shape of a certain proterties on the same position that is positioned at homologous chromosomes.If the type of gene of controlling a certain proterties on the homologous chromosomes on the same position is more than two, this equipotential gene just is called multiple allelomorphos.Only there are two different allelotrope in the multiple equipotential base at the most in any one diploid individuality.Exist to interact between the allelotrope, the effect of a kind of type decided biological character in allelotrope is better than another kind, and makes biologically when only showing the proterties of this type self, apparent recessive relation just occurred.Acting on strong is dominance, effect covered and can not show for recessive.The a pair of allelotrope that manifests recessive relation, what dominance was covered recessiveness fully is complete dominance, and both interact and marginal intermediate character occurred, are pink like safflower gene and the flower of spending the heterozygote of gene in vain, and this is an Incomplete dominance.In some cases, the effect of pair of alleles equates that do not give in mutually, heterozygote just shows the proterties that two allelotrope determine separately, and this is called codominance.For the more allelotrope of research,, can make prediction to some proterties of the organism of this equipotential gene source through detection to it.The allelotrope detection is combined with other molecular biology experiment (for example gene knockout, point mutation, RNA interference etc.); And further combine observation and Mathematical Statistics Analysis to a certain or some biological character, can between the genes all types of in the allelotrope, all types of gene and the relation between the corresponding biological character carry out more deep research.

The difference between individuals of drug reaction is a general phenomenon extremely clinically, and the reason that produces this species diversity mainly is divided into non-genetic factor and inherited genetic factors.The different of non-genetic factor and inherited genetic factors possibly cause same individuality to reaction appearance amount or even qualitative difference with a kind of medicine.Wherein, non-genetic factor comprises the multiple factor of sex, age, body weight, PD etc.; Inherited genetic factors mainly is meant the sequence information of the gene relevant with in vivo metabolism of medicine, conversion, signal transmission etc.Research to inherited genetic factors and non-genetic factor might realize personalized medicine.The sequence information of these genes has comprised a large amount of biological informations; The sequence information that obtains these genes is the first step to inherited genetic factors research; Also need combine further research, for example: the statistical study of biological test, clinical trial, clinical observation and integrated data etc., even also need set up various diseases model or mathematical model; The part biological that sequence information comprised that just might obtain these genes is learned information, promptly realizes the research to inherited genetic factors.

Antitumor drug genes involved according to the invention is meant and relevant genes such as in vivo metabolism of antitumor drug, conversion, signal transmission.

The method of detection gene order according to the invention, detecting the detected result that obtains is the nucleic acid sequence information of gene order, also is the nucleic acid sequence information of allelotrope or antitumor drug genes involved.When detection obtain be allelic nucleic acid sequence information the time; Can this method be combined with other molecular biology experiment (for example gene knockout, point mutation, RNA interference etc.); And further combine observation and Mathematical Statistics Analysis to a certain or some biological character, can between the genes all types of in the allelotrope, all types of gene and the relation between the corresponding biological character carry out more deep research.What obtain when detection is the nucleic acid sequence information of antitumor drug genes involved; Can the statistical study of this method and other molecular biology experiment (for example gene knockout, point mutation, RNA disturb etc.), clinical trial, clinical observation and integrated data etc. be combined; Even also need set up the model or the mathematical model of various effect of drugs, just might realize prediction to relevant antitumor drug curative effect.

The method of detection gene order according to the invention is to handle or detect to the tissue, body fluid or the excremental sample that break away from human body or animal body.That is, the said determined nucleic acid fragment that contains gene order derives from: blood, oral epithelium are scraped sampling, saliva, urine, paraffin-embedded tissue or puncture tissue.

The fluorescent probe of band different positions mark according to the invention can be divided into different groups according to the difference of the position of institute's tape label.Said position mark can be fluorescent mark.

That said fluorescently-labeled kind can be is a kind of, two kinds, four kinds or more.

When fluorescent mark has only when a kind of; In order to distinguish the different bases on the same position; To the base on the same position; Need extending end at anchor primer repeat 4 ligations and detect with fluorescent signal, at every turn in the ligation, fluorescent probe is a certain base (A, G, C or T) on the corresponding a certain specific position and contain fluorescently-labeled probe.In order to realize detection, then need repeat the detection of 4 * x time ligation and fluorescent probe to the base of x position.

When fluorescent mark has two kinds; In order to distinguish the different bases on the same position; To the base on the same position; Need extending end at anchor primer repeat 2 ligations and detect with fluorescent signal, at every turn in the ligation, fluorescent probe is certain two kinds of base (A, G, C or T) on the corresponding a certain specific position and contain fluorescently-labeled probe.In order to realize detection, then need repeat the detection of 2 * x time ligation and fluorescent probe to the base of x position.

When fluorescent mark has four kinds; In order to distinguish the different bases on the same position; To the base on the same position; Need extending end at anchor primer carry out 1 ligation and detect with fluorescent signal, at every turn in the ligation, fluorescent probe is certain four kinds of base (A, G, C or T) on the corresponding a certain specific position and contain fluorescently-labeled probe.In order to realize detection, then need repeat the detection of x ligation and fluorescent probe to the base of x position.

When fluorescently-labeled kind more for a long time, can carry out sequential detection with reference to such scheme.Preferably, fluorescently-labeled kind can be divided exactly or can divide exactly 4 by 4, with the design of simplifying fluorescent probe and follow-up test experience.

In addition, among the present invention, when the extending end of anchor primer connects fluorescent probe, mainly contain two kinds of ways of realization, the difference between them mainly is: whether each connect fluorescent probe before, grappling anchor primer again.If anchor primer again not, then after the connection of accomplishing each fluorescent probe and corresponding fluorescent signal detected, the fluorescent probe excision with connecting on the product kept anchor primer, connects new fluorescent probe then, and then carries out fluorescent signal and detect.If anchor primer again; Then after the connection of accomplishing each fluorescent probe and corresponding fluorescent signal detect; Whole connection product (connector of fluorescent probe and anchor primer) is removed; Again grappling anchor primer connects new fluorescent probe then, and further gathers the fluorescent signal that connects product.

First joint described in the step S1 is the one section known array that contains on the determined nucleic acid fragment of gene order, is positioned at segmental 3 ' end of determined nucleic acid or 5 ' end.

Various anchor primer of the present invention (anchor) is meant with the corresponding joint that contains on the determined nucleic acid fragment of gene order and carries out grappling bonded single stranded oligonucleotide.

The extending end of anchor of the present invention is meant can be used in and continues to connect and carry out the anchor end that nucleotide chain extends, and can be the 5 ' end of anchor, also can be the 3 ' end of anchor.

An anchor described in the step S1 is according to the base complementrity pair principle, carries out grappling bonded anchor primer with first joint on the determined nucleic acid fragment that contains gene order, is single stranded oligonucleotide, is used for connecting fluorescent probe at step S2.In addition, an anchor can also carry out different designs according to concrete needs.

In one embodiment of the invention, first joint of an anchor and determined nucleic acid fragment one end that contains gene order carries out grappling through the base complementrity pairing and combines.In this scheme, an anchor can not carry out sealing treatment, to reduce the synthetic cost of an anchor; Also can carry out sealing treatment, avoid interconnecting between the anchor, guarantee that an anchor is connected with the orientation of fluorescent probe at the end of an anchor.

The end of the one anchor is sealed, and can be the 3 ' end of an anchor, also can be 5 ' end, through sealing treatment both can control linkage direction, can avoid again interconnecting between the anchor.In an embodiment of this step; 3 ' the end of the one anchor is sealed; The method of sealing includes but not limited to two deoxidations, ammoxidation, amidation, and the 3 ' end that an anchor is closed can't continue to connect, i.e. the connection of an anchor can only occur in 5 ' end; And in another embodiment of this step, with an anchor 5 ' end seal, the method for sealing includes but not limited to dephosphorylation or amidation, i.e. the connection of an anchor can only occur in 3 ' end.

In another embodiment of the invention; The one anchor contains at least one specificity residue; Said specificity residue be meant itself or its with chemical bond energy by the residue of specificity cutting, it can make the nucleotide fragments at its place be easier to wash-out owing to being cut.The material that is used for the cleavage specificity residue is called the specificity cutting agent.Said specificity residue include but not limited to deoxyuridine acid (deoxy-Uracil, dU), Hypoxanthine deoxyriboside (deoxy Inosine, dI), contain the Nucleotide of phosphorothioate bond and the enzyme of nickase is cut recognition site.Its corresponding specificity cutting agent includes but not limited to uridylic-DNA glycosylase (Uracil-DNA Glycocasylase, UDG enzyme), Escherichia coli nucleic acid restriction endonuclease V, contains Ag, Hg, Cu, Mn, Zn or Cd ionic compound and nickase.Said nickase is meant that can discern in the double chain acid molecule the entrained enzyme of nucleotide chain cuts recognition site, and cuts and carry out enzyme on the nucleotide chain of recognition site and cut the Type II restriction enzyme that forms otch containing this enzyme; Nickase according to the invention includes but not limited to: Nt.Alw I restriction endonuclease, Nt.BsmA I restriction endonuclease, Nt.BspQ I restriction endonuclease, Nt.BstNB I restriction endonuclease.The one anchor design of this embodiment can be so that convenient more in follow-up wash-out replacing process.

In another embodiment of the invention; The one anchor contains at least one enzyme and cuts recognition site; This enzyme is cut recognition site and can directly be used in and partially or completely excise the nucleotide sequence that was read among the step S2, makes whole invention technical scheme steps simplify, and is easy and simple to handle.

First joint described in the step S1 is the one section known array that contains on determined nucleic acid fragment one end of gene order, is used to make an anchor grappling to be attached to the determined nucleic acid fragment that contains gene order.This first joint can be to obtain checking order after the sample to be checked order that design is synthetic voluntarily in the library construction process connects, and also can be that waiting of the obtaining sample copy body that checks order contains.

The said allelotrope of step S1 is meant the gene of controlling the different shape of a certain proterties on the same position that is positioned at homologous chromosomes.

The said antitumor drug genes involved of step S1 is meant and relevant genes such as in vivo metabolism of antitumor drug, conversion, signal transmission.

Wherein, said antitumor drug genes involved comprises at least one among CYP2C9, VKORC1, KIT, PDGFRA, CYP2C19, DHFR, GSTM1, MTHFR, RFC1, CYP19A1, UGT1A1, UGT1A7, UGT1A9, ABCB1, CYP2D6, CYP3A5, SULT1A1, UGT2B15, GSTA1, SOD2, CYP2B6, GSTP1, MDR1, BCRP, p53, CAT, CBR1, CBR3, EGFR, KRAS, the BRAF.

The determined nucleic acid fragment that contains gene order described in the step S1; Can be among DNA, RNA or the cDNA any; And joint is contained at its two ends at least, operation facility when checking order for making, and preferably wherein an end connector is connected with solid phase carrier; The said solid phase carrier that is used to connect can be unlike material and difform rigid, and its material includes but not limited to: glass, silicon, pottery, plastics and metal; Its shape includes but not limited to: flaggy shape, plate shaped, disc and sphere; For solid phase carrier, preferred magnetic bead of the present invention and slide.

The said determined nucleic acid fragment that contains gene order that contains joint, its source can be the determined nucleic acid fragments that contains gene order that connects joint that obtains, and can directly be used for order-checking; Also can be to obtain through the nucleic acid fragment that contains gene order is made up the order-checking library.If through making up the determined nucleic acid fragment that the order-checking library obtains containing gene order; Then need check order before the step S1 operation of library construction; The present invention preferably adopts following steps to carry out: S0. utilizes solid phase carrier that gene order is increased, and obtains being fixed in the determined nucleic acid fragment that contains gene order of surface of solid phase carriers.

Said step S0 specifically can may further comprise the steps:

The nucleic acid fragment that contains gene order that S01. will be used to increase is fixed in surface of solid phase carriers, obtains the surface and contains the solid phase carrier that at least one contains the nucleic acid fragment of gene order;

S02. primer is incorporated into the primer binding site on the surface of solid phase carriers, obtains being fixed with the amplification vector of primer;

S03. the nucleic acid fragment on the amplification vector is increased, obtain being fixed in the determined nucleic acid fragment that contains gene order of surface of solid phase carriers.

The utilization present technique scheme library construction that checks order; The determined nucleic acid fragment that will contain gene order is incorporated into surface of solid phase carriers simultaneously with the primer that is used for the amplification gene sequence; Can amplified production be fixed in surface of solid phase carriers; Improve the utilization ratio of solid phase carrier when amplification, improve the amplified production binding capacity of surface of solid phase carriers; The amplified production that utilizes the amplification of this method to obtain checks order, because the raising of the amplified production binding capacity of surface of solid phase carriers, can further strengthen the detection signal of order-checking, reduces the requirement to detecting instrument.

To the present technique scheme, need to prove the said solid phase carrier of present technique scheme; Can constitute by unlike material; Its material can adopt any one in glass, silica gel, pottery, plastics and the metal, and the surface of solid phase carrier does not have particular requirement, preferably contains the solid phase carrier of smooth-flat-surface; The particular type of solid phase carrier can be a solid phase carrier commonly used in the prior art, includes but not limited to plastic bead, granulated glass sphere, slide, magnetic bead and nm gold particles.The preferred magnetic bead that adopts is as solid phase carrier, so that the separation and purification of amplified reaction end back amplified production is convenient among the present invention.Above-mentioned just the present invention is for some specific embodiments of solid phase carrier, not in order to restriction protection scope of the present invention.

The nucleic acid fragment that contains gene order that is used to increase among the said step S01; Be meant and be fixed in surface of solid phase carriers; Being used for the single-chain nucleic acid sequence as amplification template, can be DNA, RNA or cDNA, and its source and the mode that is fixed in surface of solid phase carriers can have various ways; Can be to catch from biased sample through solid phase carrier to obtain, also can be to be incorporated into surface of solid phase carriers through the nucleic acid fragment sample that directly will contain gene order to obtain.

In a specific embodiments of the present invention; Directly before amplification to the gene order that will increase carry out fragmentation; The nucleic acid fragment two ends that utilize joint and fragmentation to obtain then are connected; And surface of solid phase carriers is modified processing accordingly, again the joint nucleic acid fragment that contains gene order afterwards is fixed in surface of solid phase carriers.

In another specific embodiments of the present invention, the nucleic acid fragment that contains gene order that is used for increasing is positioned at clinical blood sample to be measured at first.At first surface of solid phase carriers is carried out Streptavidin and modify, connect capture probe then, obtain containing the solid phase carrier of capture probe through biotin modification.The solid phase carrier that will contain capture probe directly mixes with clinical blood sample to be measured, therefrom contains the catching of nucleic acid fragment of gene order.After catching end, utilize spinning, can obtain the solid phase carrier that the surface is fixed with the nucleic acid fragment that contains gene order.

In another specific embodiments of the present invention, the nucleic acid fragment that contains gene order that is used for increasing is positioned at clinical disease patient's saliva.Be the nucleic acid fragment that contains gene order that obtains being used to increase; At first utilize corresponding primer to carry out general PCR, the nucleic acid fragment that contains gene order that will from patient's saliva, obtain amplifies, and carries out Separation and Recovery with gel electrophoresis then; Reclaiming product is connected with the biotinylation joint again; And the magnetic bead that solid phase carrier adopts Streptavidin to modify, both mix combination, can obtain the surface and be fixed with the magnetic bead that at least one contains the nucleic acid fragment of gene order.

Above-mentioned only is some specific embodiments that the surface of solid phase carriers fixed contains the nucleic acid fragment source of gene order among the present invention, not in order to restriction protection scope of the present invention.

Primer described in the step S02 is to be used for nucleotide sequence that the said determined nucleic acid fragment that contains gene order is increased, comprises at least a in upstream primer and the downstream primer.Said upstream primer is and the complementary bonded nucleotide sequence of the determined nucleic acid fragment that contains gene order 5 ' end that said downstream primer is and the identical nucleotide sequence of determined nucleic acid fragment 3 ' terminal sequence that contains gene order.In a specific embodiment of the present invention; Upstream primer or downstream primer are incorporated into the primer binding site of surface of solid phase carriers; Compare with the scheme that downstream primer is fixed in surface of solid phase carriers simultaneously with upstream primer, can when realizing goal of the invention, reduce the kind of reagent; In another specific embodiment of the present invention, upstream primer, downstream primer are incorporated into the primer binding site of surface of solid phase carriers simultaneously according to certain ratio, can when realizing goal of the invention, accelerate the speed of amplification.Wherein the blending ratio of upstream primer and downstream primer is variable as required, is preferably between the ratio of 1:2 to 2:1, more preferably 1:1.Above-mentioned is some used embodiments of the present invention for the blending ratio between upstream primer and the downstream primer, not in order to restriction protection scope of the present invention.

The said primer binding site of step S02 is meant that surface of solid phase carriers uses the site that combines with primer.

The described amplification vector of step S02; Be meant that the surface is fixed with the nucleic acid fragment that at least one contains gene order; And also be combined with the solid phase carrier of primer simultaneously, nucleic acid fragment and the primer that contain gene order this moment are fixed in surface of solid phase carriers simultaneously, can directly apply to amplification.

When primer is incorporated into surface of solid phase carriers described in the present invention, does not get rid of primer and combine with the nucleic acid fragment that contains gene order is complementary simultaneously yet, this does not influence the realization of goal of the invention of the present invention.

Primer described in the step S02 is incorporated into surface of solid phase carriers and can adopts multiple mode to realize.In a specific embodiments of the present invention, primer is connected through matching with group that surface of solid phase carriers carries, realizes directly combining, and can simplify the operation.In another specific embodiments of the present invention; Primer is through being connected with one of them group that connexon carries; Another group that carries through connexon again matches with the group that surface of solid phase carriers carries and is connected, thereby realizes that primer is connected with the indirect of solid phase carrier; In another specific embodiment of this embodiment, connexon is the existence that is similar to resin structure, uses this connexon except indirect connection that can realize primer and solid phase carrier, can also further improve surface of solid phase carriers bonded primer quantity.

Wherein, said connexon is used to connect primer and solid phase carrier.Said connexon can adopt multiple compound, includes but not limited to: alkane, strand nucleic acid molecule or comprise the compound of polymer part.

Above-mentioned pairing ways of connecting is varied, can adopt in biotin-avidin/Streptavidin, nanometer gold/iodacetyl-sulfydryl, amino-aldehyde radical/carboxyl/isothiocyano, the acrylic amide-siloyl group/SEPIGEL 305 any one.

In a specific embodiments of the present invention, primer contains biotin labeling, and solid phase carrier itself has been modified through Streptavidin, so both directly are connected with the Streptavidin pairing through vitamin H, and realization is connection directly.

In another specific embodiments of the present invention, surface of solid phase carriers contains amido modified, and primer is through carboxyl modified, and both match connections through amino-carboxyl, realize directly connection.

In another specific embodiments of the present invention; Adopt poly-compounds such as resin as connexon; Through amino respectively with primer with the aldehyde radical that carries of carboxyl and surface of solid phase carriers match and be connected, realize that primer combines with the indirect of surface of solid phase carriers.

In another specific embodiments of the present invention; With alkane molecule as connexon; Therefore contain amino and carboxyl on it, can match with the solid phase carrier of carboxylated primer and surface amination and is connected, the realization primer combines with the indirect of surface of solid phase carriers.

Above-mentioned only is some embodiments that primer is incorporated into surface of solid phase carriers among the present invention, not in order to restriction protection scope of the present invention.

The combination of primer and surface of solid phase carriers among the step S02, can with step S01 in to contain the combination of nucleic acid fragment and surface of solid phase carriers of gene order consistent, also can take different combination.In one embodiment of the invention, primer and the nucleic acid fragment that contains gene order all adopt with surface of solid phase carriers and directly match ways of connecting.Further, when adopting the identical combination mode, the nucleic acid fragment that contains gene order can also adopt identical or different group pairings to realize being connected with the direct or indirect pairing of solid phase carrier with primer.In a specific embodiment of this embodiment; Surface of solid phase carriers contains Streptavidin to be modified; And the nucleic acid fragment and the primer that contain gene order all contain biotin modification, and the nucleic acid fragment and the primer that contain gene order all are fixed in surface of solid phase carriers through the effect of the affine vitamin H of strepto-; In another specific embodiment of this embodiment; Surface of solid phase carriers contains amido modified; And the nucleic acid fragment that contains gene order contains carboxyl modified; Primer contains aldehyde group modified, and the nucleic acid fragment that contains gene order is realized being connected with the direct pairing of solid phase carrier through different group pairings with primer; In another specific embodiment of this embodiment; Surface of solid phase carriers contains amido modified; And the nucleic acid fragment that contains gene order contains carboxyl modified; Primer contains isothiocyano to be modified, and the nucleic acid fragment that contains gene order is realized being connected with the direct pairing of solid phase carrier through different group pairings with primer; In another specific embodiment of this embodiment; Solid phase carrier contains amino and Streptavidin after handling through different modifications; And the nucleic acid fragment that contains gene order contains carboxyl modified; Primer contains biotin modification, and the nucleic acid fragment that contains gene order is realized being connected with the direct pairing of solid phase carrier through different group pairings with primer; In another specific embodiment of this embodiment; Surface of solid phase carriers contains resin embedding; And the nucleic acid fragment and the primer that contain gene order match respectively at identical or different group entrained on the resin, thereby all adopt indirect ways of connecting to realize and being connected of solid phase carrier.

In another embodiment of the invention, the nucleic acid fragment that contains gene order adopts the different fixed mode to combine with surface of solid phase carriers with primer.In a specific embodiment of this embodiment; Surface of solid phase carriers contains Streptavidin to be modified; The nucleic acid fragment that contains gene order is realized being connected with the indirect pairing of solid phase carrier through capture probe; And the effect realization of primer through the affine vitamin H of strepto-is connected with the direct pairing of solid phase carrier, and both are connected with solid phase carrier through different combinations; In another specific embodiment of this embodiment; Surface of solid phase carriers contains amido modified; The nucleic acid fragment that contains gene order utilizes carboxyl modified to realize being connected with the direct pairing of solid phase carrier; Primer through on amino and the connexon with the aldehyde radical pairing is connected, and then be connected with the indirect of solid phase carrier through the realization of the aldehyde radical on the connexon, be connected with solid phase carrier through different combinations with primer thereby realization contains the nucleic acid fragment of gene order.

Above-mentioned embodiment and specific embodiment only are nucleic acid fragment and primer and some embodiments of surface of solid phase carriers bonded that contain gene order among the present invention, not in order to restriction protection scope of the present invention.

Among the step S03; Utilize amplification vector that the nucleic acid fragment that contains gene order is increased, be meant the signal increase that utilizes the feasible copy number that contains the nucleic acid fragment of gene order of certain method (like the method for chemistry, enzymatic or other types) to increase or cause containing the nucleic acid fragment existence of gene order.When the present technique scheme utilized amplification vector to increase, except that adding necessary amplifing reagent, the primer that also adds the small-amount free attitude was in order to the startup of acceleration amplification and the speed of amplification.The free state primer amount that adds can be adjusted according to fixed primer kind and the different of amount on the solid phase carrier accordingly.

In one embodiment of the invention, the surface of solid phase carriers fixed is a upstream primer, and in a specific embodiment, the free state primer of adding is a downstream primer; In another specific embodiment, can accelerate for making amplification rate, when adding a large amount of free state downstream primer, also added a small amount of upstream primer.

In another embodiment of the invention, the surface of solid phase carriers fixed is a downstream primer, and in a specific embodiment, the free state primer of adding is a upstream primer; In another specific embodiment, can accelerate for making amplification rate, when adding a large amount of free state upstream primer, also added a small amount of downstream primer.

In another specific embodiments of the present invention, upstream primer and downstream primer are incorporated into surface of solid phase carriers simultaneously, and in a specific embodiment, the free state primer of adding is wherein a kind of in upstream primer or the downstream primer; In another specific embodiment, add the upstream primer and the downstream primer of free state during amplification simultaneously, so that accelerate the speed of amplification, and the amount of upstream primer and downstream primer is good with 1:1, also can with other ratios.

The present invention can adopt multiple amplification mode; Include but not limited to the amplification of common polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcriptive intermediate, based on the amplification (NASBA) of nucleotide sequence, Q-Beta duplicates and rolling circle amplification (RCA); Preferred emulsion PCR (EPCR), bridge-type PCR, wherein bridge-type PCR can be divided into two kinds of water bridge-type PCR and emulsion bridge-type PCR again.

Unit molecule amplification according to the invention; Be meant containing the nucleic acid fragment of gene order; Form with denier (or even individual molecule) is spatially isolated (but these nucleic acid fragments that contain gene order still belong to same reaction system on the whole); In space separately, realize the amplification to the nucleic acid fragment that contains gene order, the amplified production of the homogeneous that obtains increasing is in order to promote the signal of the amplified production that obtains after the amplification.

Wherein, EPCR described in the present invention utilizes the separate space that each drop forms in the emulsion system; The nucleic acid fragment that contains gene order on the amplification vector carries out independent amplified reaction; In order to the unit molecule amplification technique of the amplified production that generates a large amount of homogeneous, its roughly operation steps be: the aqueous solution that will comprise all reacted constituents of PCR is injected into the MO surface of high speed rotating, and aqueous solution moment forms numerous by the little water droplet of MO parcel.These little water droplets have just constituted independently PCR reaction compartment.Under the perfect condition, each little water droplet only contains an amplification vector, includes other enough amplifing reagent (comprising archaeal dna polymerase, dNTP etc.).After the EPCR reaction, surface of solid phase carriers just is fixed with the nucleic acid fragment amplified production that contains gene order in the same source of copy huge amount.But EPCR concrete steps reference: BEAMing:single-molecule PCR on microparticles in water-in-oil emulsions, Frank Diehl, Meng Li; Yiping He, nature methods, Vol.3; No.7, July 2006.

Bridge-type PCR described in the present invention utilizes the nucleic acid fragment formation bridge-like structure be fixed in upstream primer or the downstream primer on the solid phase carrier with containing gene order to increase, thereby obtains the unit molecule amplification technique of a large amount of homologies, homogeneous amplified production.The ultimate principle of said bridge-type PCR is; The primer of bridge-type PCR is fixed on the solid phase carrier; Pcr amplification product can be fixed on the solid phase carrier in the PCR process; And pcr amplification product can with the primer complementary pairing on the solid phase carrier, Cheng Qiaozhuan, the primer of complementary pairing is that template increases with the amplified production with its Cheng Qiao then.Through the amount that the control original template adds, after bridge-type PCR reaction was accomplished, amplified production form with cluster bunch on solid phase carrier existed, and the amplified production of each bunch be with the dna profiling amplified production of originating.The key distinction of water bridge-type PCR and emulsion bridge-type PCR is that emulsion bridge-type PCR carries out bridge-type PCR in the isolated separate space in emulsion system, have the characteristic of water bridge-type PCR and EPCR simultaneously.Concrete principle and embodiment about bridge-type PCR can be with reference to following document: CN20061009879.X, US6227604 and Dual primer emulsion PCR for nextgeneration DNA sequencing; Ming Yan Xu et al Benchmarks Vol.48 No.5,2010.

In one embodiment of the invention, utilize PCR method that the nucleic acid fragment that contains gene order is increased, this method is simple to operate, but shortcoming is that the nucleic acid fragment that contains gene order that needs is more.

In another embodiment of the invention, utilize the method for RCA that the nucleic acid fragment that contains gene order is increased, the advantage of this embodiment is to form the unit molecule amplification, can obtain the amplified production of a large amount of homogeneous after amplification finishes.

In a preferred embodiment of the invention; Utilize EPCR that the nucleic acid fragment that amplification vector surface fixed contains gene order is increased; Can realize the unit molecule amplification; Accomplish amplification with minute quantity or even the single nucleic acid fragment that contains gene order, and obtain the amplified production of a large amount of homogeneous.

In another preferred embodiment of the present invention; Utilize bridge-type PCR that the nucleic acid fragment that amplification vector surface fixed contains gene order is increased; Can realize the unit molecule amplification equally; Accomplish amplification with minute quantity or even the single nucleic acid fragment that contains gene order, obtain the amplified production of a large amount of homogeneous.

In the specific embodiment in this embodiment, utilize water bridge-type PCR that the nucleic acid fragment that the surface of solid phase carriers fixed contains gene order is increased; And in another specific embodiment in this embodiment, utilize emulsion bridge-type PCR to realize surface of solid phase carriers fixed etc. is contained the amplification of the nucleic acid fragment of gene order.

Wherein, the amplified production described in the present invention refers to through behind the amplified reaction, and the surface is fixed with the solid phase carrier of the nucleotide sequence that a large amount of amplifications obtain.

Utilize EPCR and bridge-type PCR to carry out the unit molecule amplification in the above-mentioned preferred embodiment; Be fixed in the compared with techniques of solid phase carrier with the nucleic acid fragment that has only primer or only contain gene order; Can avoid to increase owing to lack wherein a kind of; Thereby can improve the efficient that solid phase carrier is used to increase, reduce the usage quantity of solid phase carrier, reduce cost.

Above-mentioned only is that the surface of solid phase carriers fixed that is used among the present invention to increase contains some embodiments of the nucleic acid fragment of gene order, not in order to restriction protection scope of the present invention.

After step S03 increases, comprise the solid phase carrier that surface bonding has the nucleic acid fragment that contains gene order of amplification in the amplified production that obtains, and the intact impurity of other unreacteds, therefore, carry out need carrying out purifying and recovering before the subsequent operations to amplified production.The purpose of purifying and recovering is that having the solid phase carrier of the nucleic acid fragment that contains gene order of amplification to separate with impurity surface bonding purifies, can use domestic method of the prior art, includes but not limited to spinning purification, column separating purification.

Among the step S2, the extending end of a said anchor is meant and behind an anchor, can continues to connect and carry out the end that nucleotide chain extends.

According to the different designs structure of an anchor described in the step S1, step S2 can adopt different implementations.

In one embodiment of the invention, an anchor contains the specificity residue, and in the specific embodiment in this embodiment, the structure of an anchor is as shown in Figure 3, and X shown in the figure is A, G, C or T, and Y represents the specificity residue, and n is a positive integer.In addition, the number of Y and the Y position changeable in X among the figure when having a plurality of Y, might not be to exist with the form that links to each other shown in Fig. 3 between Y and the Y, can be dispersed in the different positions of an anchor respectively.An above-mentioned anchor can be so that the realization of the wash-out of fluorescent probe and replacing be easier among the step S2.Utilize the method for an anchor performing step S2 of this structure may further comprise the steps:

S21. at the fluorescent probe of an anchor extending end connecting band specific position mark that contains the specificity residue, detect the corresponding fluorescent signal that connects product, obtain the nucleotide sequence information of correspondence position;

S22. with specificity cutting agent cleavage specificity residue, with a fluorescent probe that connects in the last step and an anchor wash-out, an anchor resets;

S23. repeat with the connection of the fluorescent probe of specific position mark and detect the operation that is connected the fluorescent signal of product accordingly at an anchor extending end, obtain the sequence information of M Nucleotide behind the anchor extending end.

Need to prove that in this technical scheme, specificity residue that an anchor contains described in the step S21 and corresponding specificity cutting agent thereof can comprise multiple.

In a preferred embodiment of the invention, as shown in Figure 4, the specificity residue that an anchor contains is the dU base, and its corresponding specificity cutting agent is the UDG enzyme.Utilize an anchor of structure as shown in Figure 4, step S2 can may further comprise the steps:

S21 '. at the fluorescent probe of an anchor extending end connecting band specific position mark that contains the dU base, detect the corresponding fluorescent signal that connects product, obtain the nucleotide sequence information of correspondence position;

S22 '. with UDG enzyme identification dU base and carry out enzyme and cut, with a fluorescent probe that connects in the last step and an anchor wash-out, an anchor resets;

S23 '. repeat with the connection of the fluorescent probe of specific position mark and detect the operation that is connected the fluorescent signal of product accordingly at an anchor extending end, obtain the sequence information of M Nucleotide behind the anchor extending end.

With this technical scheme performing step S2; It is advantageous that; The one anchor contains the dU base; The UDG enzyme that can directly utilize the specific recognition enzyme to cut the dU base cuts an anchor, forms short segments, thereby makes that the realization of wash-out and replacing of fluorescent probe among the step S2 is easier.

In another preferred embodiment of the present invention, as shown in Figure 5, an anchor with the specificity residue be that the enzyme of nickase Nt.Alw I is cut the identification base.Utilize an anchor of structure as shown in Figure 5, step S2 can may further comprise the steps:

S21 ' '. cut the fluorescent probe of an anchor extending end connecting band specific position mark of recognition site containing the nickase enzyme, detect the fluorescent signal of corresponding connection product, obtain the nucleotide sequence information of correspondence position;

S22 ' '. cut recognition site and carry out enzyme and cut with nickase identification nickase enzyme, with the fluorescent probe wash-out that connects in the last step;

S23 ' '. repeat with the connection of the fluorescent probe of specific position mark and detect the operation that is connected the fluorescent signal of product accordingly at an anchor extending end, obtain the sequence information of M Nucleotide behind the anchor extending end.

With this technical scheme performing step S2; It is advantageous that, when changing the different fluorescent probe in the corresponding base of fluorescent mark position, can utilize an anchor to go up entrained nickase enzyme and cut recognition site; Being connected between the fluorescent probe that directly will connect originally through corresponding nickase and the anchor interrupts; Thereby the fluorescent probe that easily will connect is originally washed off, connects new fluorescent probe, avoids resetting again after the anchor wash-out; The step that simplifies the operation is saved the cost of reagent simultaneously.

Wherein, M is positive integer described in the above-mentioned steps, and its numerical range is determined by ligase enzyme used in the present invention, is preferably 1 ~ 9, and more preferably 1 ~ 6.In one embodiment of the invention, utilize the T4 ligase enzyme to realize that enzyme of the present invention cuts the extension PCR sequencing PCR, the numerical value of M is preferably 1 ~ 6; In another embodiment of the invention, utilize the Tth ligase enzyme to realize that enzyme of the present invention cuts the extension PCR sequencing PCR, the numerical range of M is preferably 1 ~ 9.In above-mentioned preferable range; The method that the present invention utilizes enzyme to cut and extends order-checking detects gene order; Not only can increase the length of reading that checks order when detecting, can also further improve the accuracy that the fluorescent probe in the order-checking process is connected with anchor, and then improve the accuracy that nucleotide sequence information reads.Wherein, when utilizing the T4 ligase enzyme, when fluorescent probe was connected 3 ' end or the 5 ' end of anchor, 6 bases of that end that fluorescent probe is connected with anchor need and be waited the complete complementary pairing of fragment that checks order accordingly, could realize connection; When utilizing the Tth ligase enzyme, when fluorescent probe is connected 3 ' end or the 5 ' end of anchor, require 8 or 9 bases and the complete complementary pairing of fragment of waiting to check order accordingly of that end that fluorescent probe is connected with anchor respectively, could realize connection.

The fluorescent probe of employed band different positions mark is divided into different group types in the above-mentioned steps; With the different nucleotide sequence informations of the corresponding same specific position of different fluorescent marks in the set type, and the corresponding specific position difference of the fluorescent probe fluorescent mark of different set types.In each ligation, add the fluorescent probe of same set type,, can obtain this group fluorescent probe mark specific position corresponding nucleotide sequences information according to the fluorescent signal of being gathered; And the repetition of these operations of wash-out of the connection-collection fluorescent signal-fluorescent probe of the hybridization combination-fluorescent probe through an anchor can obtain the sequence information of M Nucleotide behind the anchor extending end accurately.

After step S2 detect to finish, contain the fluorescent probe that remains with bonded the one anchor and band specific position mark on the determined nucleic acid fragment of gene order.Carrying out enzyme for the ease of the restriction endonuclease among the step S3 cuts; Can be with an anchor extending end activation again; Add four kinds of Nucleotide then; Utilize archaeal dna polymerase, along the direction of an anchor extending end, the determined nucleic acid fragment that contains gene order that will be fixed on the solid phase carrier forms complete double chain acid molecule.

Restriction endonuclease described in the step S3 is meant that the specific enzymes that can discern on the double chain acid molecule to be contained cuts the identification base sequence, carries out the enzyme of double-strandednucleic acid cutting then in the position of cutting recognition site some amount base apart from enzyme.In the present invention; Can use enzyme to cut and to obtain flat terminal restriction endonuclease afterwards; The preferred enzyme that uses is cut the restriction endonuclease that can obtain the viscosity protruding terminus afterwards; It is selected for use principle to follow through the identification enzyme and cuts recognition site, can the restriction endonuclease of all or part of excision of nucleotide sequence of having obtained among the step S2 be got final product.When selecting restriction endonuclease for use, the preferred restriction endonuclease of optimal reactive temperature about 37 ℃, the sex change that the too high restriction endonuclease of optimal reactive temperature causes cutting the process double center chain at enzyme is easily dissociated, thereby causes the connection of follow-up fluorescent probe to be obstructed; Preferably to insensitive restriction endonuclease that methylates, so that the segmental nucleotide sequence of the determined nucleic acid that contains gene order is cut and obtained to the enzyme that can continue; Preferred enzyme is cut Nucleotide number between recognition site and the restriction enzyme site more than 4, and it is longer to make each enzyme extend the length of order-checking after cutting forward.

The used restriction endonuclease of the present invention can be an II type restriction endonuclease; Include but not limited to Acu I, Alw I, Bbs I, Bbv I, Bcc I, BceA I, BciV I, BfuA I, Bmr I, Bpm I, Bsa I, BseR I, Bsg I, BsmA I, BsmB I, BsmF I, BspM I, BspQ I, BtgZ I, Ear I, Eci I, Fau I, Fok I, Hga I, Hph I, HpyA V, Mbo II, Mly I, Mnl I, Ple I, Sap I, SfaN I, BpuE I, Mme I and NmeA III, wherein preferred Acu I, Bbv I, BceA I, Bpm I, BseR I, BspM I, Fok I, Hga I, Mbo II and Mnl I; Used restriction endonuclease also can be an III type restriction endonuclease, includes but not limited to Ecop1 and Ecop15 I.

Among the step S3, be used for being cut recognition site, can bring into, also can bring into through additive method through an anchor by the enzyme that restriction endonuclease is discerned.Cut the difference that recognition site is originated according to enzyme, step S3 also can realize through diverse ways.

In the embodiment in the present invention, enzyme is cut recognition site and is directly brought into by an anchor, and promptly an anchor contains at least one enzyme and cuts recognition site.

Utilize the enzyme that is contained on the anchor to cut recognition site, different ways performing step S3 can be arranged.In an embodiment of this programme; Step S3 can directly utilize restriction endonuclease discern on the anchor with enzyme cut recognition site; With having obtained the nucleotide segment of sequence information or excision fully among the step S2, obtain containing the segmental enzyme of waiting to check order and cut product.

Utilize the advantage of this embodiment performing step S3 to be, after step S2 obtains an anchor extending end after the sequence information of M Nucleotide, utilize the enzyme that is contained on the anchor to cut recognition site and directly carry out enzyme and cut, step can simplify the operation.

In another embodiment of this programme, utilize the enzyme that is contained on the anchor to cut recognition site, step S3 can comprise the steps:

S31. with a fluorescent probe that connects among the step S2 and an anchor wash-out, the anchor and carry out chain extension of resetting forms double chain acid molecule with the determined nucleic acid fragment that contains gene order;

S32. restriction endonuclease through discern on the anchor with enzyme cut recognition site, with having obtained the nucleotide segment of sequence information or excision fully among the step S2, obtain containing the segmental enzyme of waiting to check order and cut product.

Utilize the advantage of this technical scheme performing step S3 to be; At first utilize the extending end of an anchor to carry out chain extension; Extend to form double chain acid molecule with the determined nucleic acid fragment that contains gene order, can guarantee that enzyme cuts product and keep double-stranded state, be convenient to the connection of follow-up joint.

In a preferred embodiment of this embodiment; The survey nucleic acid fragment that contains gene order is connected and fixed on the microballon through 5 ' end; Contain enzyme on the anchor of first joint and cut recognition site sequence 5 ' and be incorporated into ... CTGAAG ... 3 '; Utilize this enzyme of II type restriction endonuclease Acu I identification to cut recognition site and carry out enzyme and cut among the step S3, the enzyme that the fragment 3 ' end that obtains waiting checking order contains two outstanding Nucleotide is cut product.

In another preferred embodiment of this embodiment; The determined nucleic acid fragment that contains gene order is connected and fixed on the microballon through 5 ' end; Contain enzyme on the anchor of first joint and cut recognition site sequence 5 ' and be incorporated into ... GCAGC ... 3 '; Utilize this enzyme of II type restriction endonuclease Bbv I identification to cut recognition site and carry out enzyme and cut among the step S3, the enzyme that the fragment 3 ' end that obtains waiting checking order contains two outstanding Nucleotide is cut product.

In another preferred embodiment of this embodiment; The determined nucleic acid fragment that contains gene order is connected and fixed on the microballon through 5 ' end; Contain enzyme on the anchor of first joint and cut recognition site sequence 5 ' and be incorporated into ... GAGTC ... 3 '; Utilize this enzyme of II type restriction endonuclease Mly I identification to cut recognition site and carry out enzyme and cut among the step S3, the fragment 3 ' end that obtains waiting to check order contains flat terminal enzyme and cuts product.

In a preferred embodiment of this embodiment; The determined nucleic acid fragment that contains gene order is connected and fixed on the microballon through 3 ' end; Contain enzyme on the anchor of first joint and cut recognition site sequence 5 ' and be incorporated into ... CATCC ... 3 '; Utilize this enzyme of Fok I enzyme identification to cut recognition site and carry out enzyme and cut among the step S3, the enzyme that the fragment 5 ' end that obtains waiting checking order contains 4 outstanding Nucleotide is cut product.

In a specific embodiment of this embodiment; The determined nucleic acid fragment that contains gene order is connected and fixed on the microballon through 5 ' end; Contain enzyme on the anchor of first joint and cut recognition site sequence 5 ' and be incorporated into ... CAGCAG ... 3 '; Utilize this enzyme of III type restriction endonuclease Ecop15 I enzyme identification to cut recognition site and carry out enzyme and cut among the step S3, the enzyme that the fragment 3 ' end that obtains waiting checking order contains two outstanding Nucleotide is cut product.

In another embodiment of the invention, enzyme is cut recognition site and is contained at least one enzyme and cut the 3rd joint of recognition site and bring into through connecting at the other end of an anchor.Utilize the 3rd joint, step S3 can may further comprise the steps:

S31 '. the other end at an anchor connects the 3rd double-stranded joint, and the 3rd joint contains at least one enzyme and cuts recognition site;

S32 '. utilize restriction endonuclease discern the 3rd joint with enzyme cut recognition site, with having obtained the nucleotide segment of sequence information or excision fully among the step S2, obtain containing the segmental enzyme of waiting to check order and cut product.

Need to prove that the other end of a said anchor is an anchor has been used to extend among the relative step S1 a end; If the other end of an anchor was closed, before step S3, need carry out activation earlier so before step S3; The activatory method can utilize of the prior art any; Only need the group that can be used in the other end connecting be come out and get final product; As in a preferred embodiment of the present invention; The one anchor itself contains the specificity residue, can directly utilize specificity cutting agent cleavage specificity residue to accomplish activation.Said the 3rd joint is to contain the nucleic acid molecule that at least one enzyme is cut recognition site, and the enzyme that is used to introduce restriction endonuclease is cut recognition site, so that in the subsequent step, will obtain the nucleotide segment of sequence information or excision fully through restriction endonuclease.Utilize the advantage of this technical scheme performing step S3 wide for suitability; Determined nucleic acid fragment to containing gene order does not have particular requirement; No matter contain on the determined nucleic acid fragment of gene order bonded the one anchor and whether contain enzyme and cut recognition site, all can realize through this technical scheme.

What need further specify is; The 3rd joint can be selected the corresponding different terminal joints that contain for use; Specifically can include but not limited to the joint of flat end fitting, protruding terminus joint, branch type joint and band loop-stem structure, the structure of these joints and characteristic can be referring to patent documentation: CN201110222952.4.The preferred joint that uses protruding terminus joint, branch type joint and band loop-stem structure among the present invention, the joint of these structures can be avoided occurring in connection procedure between a plurality of joints from the phenomenon that connects.To above-mentioned different implementation; The 3rd joint the above be used for endonuclease digestion recognition site that enzyme cuts can be above-mentioned restriction endonuclease pairing any one; The Nucleotide that only needs when design is synthetic, enzyme to be cut between recognition site and the restriction enzyme site calculates in advance; Make enzyme cut after just with read among the step S2 nucleotide segment of sequence information or fully excision get final product, therefore can select for use different enzymes to cut recognition site and corresponding restriction endonuclease realization thereof in the present invention to having obtained the excision of the Nucleotide of sequence information among the step S2.

Should be noted that; Above-mentioned embodiment only is to cut some specific embodiments of recognition site and corresponding restriction endonuclease thereof among the present invention about the enzyme of selecting for use; Not in order to restriction protection scope of the present invention; Use qualified other enzymes instead and cut recognition site and corresponding restriction endonuclease, can realize the object of the invention equally.

Cut product for obtaining containing the segmental enzyme of waiting to check order, can after the endonuclease reaction of step S3, carry out purifying and recovering, and the method for purifying and recovering can utilize multiple mode of the prior art to realize.In one embodiment of the invention, the determined nucleic acid fragment that contains gene order is fixed on the slide, directly cuts product and other separating substances purifying with the damping fluid flushing segmental enzyme of can realizing remaining to be checked order after enzyme is cut; In another specific embodiment of the present invention, the determined nucleic acid fragment that contains gene order is fixed on the magnetic bead, directly utilizes magnet absorption, slightly washes with damping fluid, can realize containing the segmental enzyme of waiting to check order and cut product and other separating substances purifying.

Among the step S4, before connecting second joint, cut the difference of product and the method for attachment of follow-up use, can optionally cut product and modify processing, like end-filling to enzyme according to the enzyme that obtains among the step S3; Also can directly utilize enzyme to cut the protruding terminus that obtains afterwards and carry out the connection of second joint.

In a specific embodiment of the present invention, according to utilize among the step S3 on the anchor cut recognition site with the enzyme of restriction endonuclease, carry out enzyme with the Fok I and cut, obtain the enzyme that 3 ' end contains 4 nucleotide sequence protruding terminuses and cut product.Enzyme is cut product carry out the end-filling processing earlier, and then connect second joint.The advantage of this embodiment is to avoid owing to long protruding terminus makes that the second joint synthetic kind is too much, thereby reduces the synthetic cost of second joint.

In another specific embodiment of the present invention, according to utilize among the step S3 on the anchor cut recognition site with enzyme and cut recognition site for the enzyme of Acu I, carry out enzyme with the Acu I and cut, obtain 3 ' contain 2 nucleotide sequence protruding terminuses enzyme cut product.Nucleotide to this protruding terminus is outstanding is less, only needs synthetic 4 ²Plant second joint and can realize connecting, therefore directly utilize enzyme to cut the protruding terminus that obtains afterwards and carry out the connection of second joint.The advantage of this embodiment is the step that can simplify the operation, and directly adds second joint and reacts and get final product.

Among the step S4, said second joint is to be used for and to contain the segmental enzyme of waiting to check order and cut product and be connected to form the segmental double chain acid molecule of new determined nucleic acid that contains gene order.Cut the mode of connection difference that product is terminal and select for use according to enzyme; Second joint can be selected the corresponding different terminal joints that contain for use; Include but not limited to the joint of flat end fitting, protruding terminus joint, branch type joint and band loop-stem structure, the structure of these joints and characteristic can be referring to patent documentation: CN201110222952.4.The preferred joint that uses protruding terminus joint, branch type joint and band loop-stem structure among the present invention, the joint of these structures can be avoided occurring in connection procedure between a plurality of joints from the phenomenon that connects.

Among the step S4; Complementary pairing for the ease of follow-up the 2nd anchor and second joint can carry out smoothly; Can cut double-strandednucleic acid that product forms after being connected with second joint to enzyme and connect product and handle, form the new determined nucleic acid fragment that contains gene order of single stranded form.Double-strandednucleic acid is connected on the product Nucleotide that complementary Nucleotide and the enzyme that is incorporated on the fragment of waiting to check order remain after cutting handle and form the segmental method of the new determined nucleic acid of strand, include but not limited to remove through the NaOH sex change annealed mode of dissociating or heat up.

Said the 2nd anchor be can with the second joint grappling bonded single stranded nucleic acid molecule, be used for connecting fluorescent probe at step S5; Said the 2nd anchor can be identical with an anchor or different.If the 2nd anchor is identical with an anchor; Then follow-up continuation enzyme can use identical restriction endonuclease to operate when cutting; Simplify the kind of reaction reagent; Simultaneously can also avoid causing follow-up enzyme to cut step and obtaining non-title product because of having identical restriction endonuclease sequence in the new determined nucleic acid fragment that contains gene order; If the 2nd anchor is different with an anchor, then can satisfy more actual needs in the new design of introducing in the 2nd anchor synthetic.The 2nd anchor is different with an anchor's, and it is different with kind to be that enzyme is cut the position of recognition site, also can be the difference of Nucleotide quantity.

The 2nd anchor can carry out other processing as required.In a specific embodiment of the present invention, 3 ' end or the 5 ' end with the 2nd anchor seals as required, in order to the control linkage direction, and avoids the 2nd anchor connecting certainly each other.In an embodiment of present embodiment, the method that 3 ' end is sealed includes but not limited to two deoxidations, ammoxidation, amidation, and it can't continue to connect, and the connection of controlling the 2nd anchor only occurs in 5 ' end; In another embodiment of present embodiment, the 5 ' end of the 2nd anchor is sealed, carry out dephosphorylation or amidation through 5 ' end and handle, thereby only keep 3 ' end as the end that connects with its sealing.

In another specific embodiment of the present invention, the 2nd anchor contains the specificity residue, can be so that the replacing of follow-up fluorescent probe and wash-out are easier.In a preferred implementation of the present invention; The 2nd anchor with the specificity residue be the dU base; Introduce several dU bases in its nucleotide sequence; The 2nd anchor of this structure can directly excise the dU base and form different short segments in the elution process of follow-up order-checking, be convenient to the realization of wash-out.

In another preferred embodiment of the present invention; The 2nd anchor with the specificity residue be the base that contains phosphorothioate bond; In its nucleotide sequence; Replace original P-O key with one or more phosphorothioate bonds (P-S), can directly utilize and contain the wash-out that Ag, Hg, Cu, Mn, Zn and Cd ionic compound cutting P-S key are realized the 2nd anchor.

In another preferred embodiment of the present invention; The 2nd anchor contains two enzymes and cuts recognition site; One of them is to be used to cut double-stranded digestion with restriction enzyme recognition site, and another nickase enzyme that is a strand being used for cutting two strands forms otch is cut recognition site.

Should be noted that the foregoing description only is some embodiments of the 2nd anchor among the present invention, not in order to restriction protection scope of the present invention.

Among the step S5, the sequence information of N Nucleotide can realize that this method may further comprise the steps through following method behind the 2nd anchor extending end:

S51. the fluorescent probe of connecting band specific position mark behind the 2nd anchor extending end detects the corresponding fluorescent signal that connects product, obtains the nucleotide sequence information of corresponding position;

S52. the fluorescent probe that last step is connected is removed, and the fluorescent probe of connecting band different positions mark detects the corresponding fluorescent signal that connects product, obtains the nucleotide sequence information of respective markers position;

S53. the operation of repeating step S52, the sequence information of N Nucleotide after obtaining the 2nd anchor extending end.

Connection and replacing that the fluorescent probe of technique scheme utilization band different positions mark carries out realize obtaining the sequence information of N Nucleotide behind the 2nd anchor extending end.Wherein, the removal of fluorescent probe described in the step S52 can have multiple mode to realize.

In an embodiment of this step; Utilize the NaOH sex change that fluorescent probe and the 2nd anchor are disintegrated down from new determined nucleic acid fragment; Then with the 2nd anchor recombine on second joint, carry out connection and the signal detection operation of the different fluorescent probe of follow-up mark position.This embodiment is simple, need not add other reagent.

In another preferred implementation of this step; The 2nd anchor contains several dU bases, therefore utilizes the UDG enzyme directly to carry out the excision of dU base, forms short segments; The intensification sex change is dissociated; Again obtain new determined nucleic acid fragment, then with the 2nd anchor recombine on second joint, carry out connection and the signal detection operation of the different fluorescent probe of follow-up mark position.

In another preferred implementation of this step; In the nucleotide sequence of the 2nd anchor; Several thiophosphoric acid keys (P-S) replace original P-O key, therefore directly utilize to contain the removal that Ag, Hg, Cu, Mn, Zn and Cd ionic compound cutting P-S key are realized the 2nd anchor and fluorescent probe.

In another preferred implementation of this step; The enzyme that a nickase is contained at the 2nd anchor extending end place is cut recognition site; Therefore directly utilize nickase with the removal that is connected between fluorescent probe and the 2nd anchor; Just can continue the different fluorescent probe in linkage flag position, realize the enforcement of follow-up order-checking step at the extending end of the 2nd anchor.This embodiment is directly removed fluorescent probe, and need the 2nd anchor not handled, and has both simplified the operation, and reduces reagent cost again.

Above-mentioned preferred implementation utilizes specific material with the excision of the connection among the 2nd anchor, forms short and small fragment, utilizes mild conditions can remove fluorescent probe then.

Wherein, N described in the above-mentioned steps is a positive integer, and its numerical range by ligase enzyme used in the present invention decision, is preferably 1 ~ 9 equally, more preferably 1 ~ 6.In one embodiment of the invention, utilize the T4 ligase enzyme to realize that enzyme of the present invention cuts the extension PCR sequencing PCR, the numerical value of N is preferably 1 ~ 6; In another embodiment of the invention, utilize the Tth ligase enzyme to realize that enzyme of the present invention cuts the extension PCR sequencing PCR, the numerical range of N is preferably 1 ~ 9.In above-mentioned preferable range, enzyme of the present invention is cut and is extended PCR sequencing PCR and not only can increase order-checking and read longly, can also further improve the accuracy that the fluorescent probe in the order-checking process is connected with anchor, and then the accuracy that reads of raising nucleotide sequence information.Wherein, when utilizing the T4 ligase enzyme, when fluorescent probe was connected 3 ' end or the 5 ' end of anchor, 6 bases of that end that fluorescent probe is connected with anchor need and be waited the complete complementary pairing of fragment that checks order accordingly, could realize connection; When utilizing the Tth ligase enzyme, when fluorescent probe is connected 3 ' end or the 5 ' end of anchor, require 8 or 9 bases and the complete complementary pairing of fragment of waiting to check order accordingly of that end that fluorescent probe is connected with anchor respectively, could realize connection.

Fig. 6 is for obtaining the method synoptic diagram of the sequence information of N Nucleotide behind the 2nd anchor extending end in specific embodiment among the step S5, this figure has represented the process that obtains the sequence information of N Nucleotide behind the 2nd anchor extending end intuitively.

Step 51. reads the 1st nucleotide sequence information behind the 2nd anchor extending end: under the effect of T4 ligase enzyme; The linkage flag position is the 1st a fluorescent probe behind the 2nd anchor extending end; Adopt figure then; Collect fluorescent signal, confirm behind the 2nd anchor extending end the 1st nucleotide sequence information according to the fluorescent signal that obtains;

Step 52. reads the 2nd nucleotide sequence information behind the 2nd anchor extending end: utilize the nickase enzyme of the 2nd anchor extending end place band to cut recognition site; Being connected between fluorescent probe and the 2nd anchor cut away; Dissociating mark position through the intensification sex change is the 1st fluorescent probe wash-out; Use mark position then instead and be the extending end that the 2nd fluorescent probe is connected to the 2nd anchor; Adopt figure imaging, collect fluorescent signal, confirm behind the 2nd anchor extending end the 2nd nucleotide sequence information according to the fluorescent signal that obtains;

Step 53. reads follow-up Nucleotide: the operation of repeating step 52, the fluorescent probe of using band different positions mark instead obtains the nucleotide sequence information of corresponding position, the sequence information of the 6th Nucleotide after obtaining the 2nd anchor extending end.

In the above-mentioned embodiment; The nickase enzyme that utilizes the 2nd anchor extending end place to contain is cut recognition site; Realize that directly the connection of the fluorescent probe of band different positions mark changes, thereby realize obtaining the sequence information of 6 Nucleotide behind the 2nd anchor extending end.

Should be noted that the foregoing description only is some specific embodiments that obtain the sequence information of N Nucleotide behind the 2nd anchor extending end among the performing step S5, not in order to restriction protection scope of the present invention.For example change the wherein numerical value of N,, get N=4, can realize that equally enzyme cuts the purpose of extending order-checking as in another specific embodiment of the present invention.

The reagent of changing described in the step S6 refers to the fluorescent probe of used restriction endonuclease among the step S3 to S5, joint, anchor and band different positions mark; Said replacing refers to generalized and changes, as change with reaction before in the different reagent of employed reagent or will before the reagent that reacted in reacting change the reagent that unreacted is crossed into.

After reagent is changed; The product of step S5 has been read the excision of nucleotide sequence with restriction endonuclease; And then make enzyme cut product to be connected with new joint, and combine new anchor, connect new fluorescent probe; Detect the fluorescent signal of this fluorescent probe, can obtain the nucleotide sequence of this fluorescent probe correspondence position.So carry out similar above-mentioned cyclical operation, can obtain containing gene order information required in the determined nucleic acid fragment of gene order.

Fig. 7 is the method synoptic diagram that detects gene order in the one embodiment of the invention, and this figure has represented the order-checking process that the method for utilizing enzyme to cut extension detects gene order information intuitively.

Step 1. anchor is hybridized combination: an anchor is incorporated on segmental first joint of the determined nucleic acid that contains gene order through base complementrity pairing hybridization, and the segmental 5 ' end of determined nucleic acid that wherein contains gene order is through being connected and fixed on bead surface; Wherein, to contain sequence be sequence 5 ' to an anchor ... CTGAAG ... 3 ' enzyme is cut recognition site, and contains the dU base in the nucleotide sequence of an anchor.

Step 2. is obtained the sequence information of 6 Nucleotide behind the anchor extending end: under the effect of T4 ligase enzyme; Extending end at an anchor connects the fluorescent probe that is used to detect; Detect the nucleotide sequence information that fluorescent signal obtains the corresponding position; And utilize UDG enzyme excision dU base to detect with the replacing of the fluorescent probe of the replacement that realizes an anchor and band different positions mark, and gather the fluorescent signal figure of correspondent probe, obtain the 1st to 6 nucleotide sequence information.

Step 3. enzyme is cut: the carrying out that cuts for ease of follow-up enzyme; After the sequence information of 6 Nucleotide behind the anchor extending end all is read; At first under the effect of archaeal dna polymerase, the determined nucleic acid fragment that will contain gene order extends to form complete double chain acid molecule; Utilize identification of Acu I enzyme spcificity and enzyme to cut enzyme entrained among the anchor then and cut recognition site; 6 nucleotide sequences excisions with an anchor extending end that had read before obtain the not order-checking fragment that 3 ' end contains two Nucleotide protruding terminuses; After the endonuclease reaction, utilize magnet absorption magnetic bead, will contain the segmental enzyme that do not check order and cut the product purification recovery.

Step 4. enzyme is cut product and connected second joint and combine the 2nd anchor: an end that utilizes the enzyme of step 3 to cut product contains two Nucleotide protruding terminuses, and is altogether 4 ²=16 kinds second joint connects under the effect of T4 ligase enzyme and obtains the double-stranded product that connects; After ligation finishes, two strands is connected the product sex change formation strand that dissociates, obtain being fixed in the new determined nucleic acid fragment that contains gene order on the magnetic bead; The 2nd anchor is incorporated on segmental second joint of new determined nucleic acid through the base complementrity pairing.Wherein, containing sequence on the 2nd anchor equally is sequence 5 ' ... CTGAAG ... 3 ' enzyme is cut recognition site, and also to contain sequence be 5 ' at distance 4 nucleotide position places, the 2nd anchor extending end place ... GGATC ... 3 ' enzyme is cut recognition site.

Step 5. is obtained the sequence information of 6 Nucleotide behind the 2nd anchor extending end: being connected between the fluorescent probe that utilizes nickase Nt.Alw I cutting belt specific position mark and the 2nd anchor; Realize the replacing of different mark position fluorescent probes; Thereby can read the nucleotide sequence information of different positions, obtain the sequence information of 6 Nucleotide behind the 2nd anchor extending end.

Step 6. is changed reagent; Operation in repeating step 3, step 4, the step 5; Obtain after the nucleotide sequence information of some amount (its numerical range is preferably 1 ~ 6), cut the nucleotide sequence that the means excision had been read through enzyme, and make up new determined nucleic acid fragment; Realize extending the purpose of order-checking with this, until obtaining containing gene order information required on the determined nucleic acid fragment of gene order.

To above-mentioned each technical scheme, by further specify the present invention the technique effect and the meliority of record technical scheme, the present invention provides a concrete operation embodiment.

Present embodiment is with a mono-clonal plasmid that includes one section nucleic acid fragment (SEQ ID NO:1) of F8 gene; With nucleic acid-templated as testing sample of the mono-clonal plasmid of the one section nucleic acid fragment that contains galt gene (SEQ ID NO:2); Design corresponding specificity amplification primer: F8F (SEQ ID NO:3) and F8R (SEQ ID NO:4), GALT (SEQ ID NO:5) and GALT (SEQ ID NO:6) respectively to above-mentioned two nucleic acid fragments; Utilize above-mentioned specificity amplification primer from above-mentioned two mono-clonal plasmids, to amplify the target nucleic acid fragment that is used for follow-up order-checking respectively then; Utilize the target nucleic acid fragment to make up corresponding order-checking library then, thereby contain the segmental detection of determined nucleic acid of gene order.

Segmental 5 ' the end connector of determined nucleic acid that wherein contains gene order combines with magnetic bead through the effect of the affine vitamin H of strepto-, and the determined nucleic acid segmental 3 ' that contains gene order is held and contained first joint sequence.For reducing the complicacy of operating process, two kinds of first joints that contain the nucleic acid fragment of gene order adopt identical sequence, and therefore both employed anchor also are the same in the present embodiment, and both operate simultaneously.The Pstar-II Plus order-checking platform that the Treatment Analysis of whole measuring program process and fluorescent signal view data adopts Shenzhen HYK Gene Technology Co., Ltd. to produce carries out.Said embodiment concrete operations are following.

One, the segmental amplification of determined nucleic acid.

Utilize above-mentioned nucleic acid-templated specificity amplification primer, the target nucleic acid fragment of F8 gene and galt gene is increased, obtain being used to make up the target nucleic acid fragment in order-checking library.Wherein, said amplification is carried out respectively, and reaction system is following: F primer (10 μ M), 2 μ L; R swims primer (10 μ M), 2 μ L; DNTP (each 2.5mM), 4 μ L; As nucleic acid-templated plasmid, 20ng; Ex Taq (5U/ μ L), 0.25 μ L; 10 * Ex Taq Buffer, 5 μ L; DdH ₂O adds to 50 μ L.

The PCR reaction conditions is following:

95℃?3min；

94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; Repeat 25 circulations;

72℃?7min。

Utilize PCR to reclaim test kit, the amplified production to each sample separates respectively, removes the primer and the dNTP of not amplification, sepharose purifying and recovering target nucleic acid fragment.

Two, the target nucleic acid fragment of utilizing amplification to obtain makes up the order-checking library.

The target nucleic acid product two ends that obtain of amplification connect first joint in step 1, carry out the unit molecule amplification then, the order-checking library that contains gene order of the usefulness that obtains checking order.

1. the target nucleic acid fragment is connected with first joint.

The protruding terminus joint that contains outstanding T base with end is as first joint; Its concrete sequence is SEQ ID NO:7 and SEQ ID NO:8; And the biotinylation that this first joint contains specific position is modified, so that follow-uply be connected with the magnetic bead that contains the Streptavidin modification.First joint is connected with the target nucleic acid fragment, obtains containing the target nucleic acid fragment of first joint.

Under the effect of T4 ligase enzyme; With two kinds of target nucleic acid products obtaining of amplification respectively with etc. mixed in molar ratio, be connected with first joint, obtain containing the target nucleic acid fragment of first joint; Linked system is: target nucleic acid mixture of products, 50 μ L (about 500ng); First joint, 2 μ L (about 3000ng); 10mM ATP, 5 μ L; T4 dna ligase (30U/ μ L), 1 μ L; 10 * T4 ligase enzyme damping fluid, 10 μ L; Add ddH ₂O to 100 μ L.

Hatch more than the 4h for 16 ℃, reaction utilizes the purification kit purifying and recovering after finishing.

2. utilize the target nucleic acid fragment that contains first joint to carry out the unit molecule amplification, make up the order-checking library.

Carry out the unit molecule amplification with the target nucleic acid fragment that contains first joint, make up the order-checking library, obtain containing the determined nucleic acid fragment of gene order, concrete operations are following.

The target nucleic acid fragment that 1) will contain biotin modification with contain Myone magnetic bead (1 μ m, the 10mg/mL that Streptavidin is modified; Invitrogen) combine, make magnetic bead surfaces be fixed with at least one target nucleic acid fragment, reaction system and reaction process are following: contain the target nucleic acid fragment of first joint, 0.018ng (10 ⁸Individual molecule); Myone magnetic bead (1 μ m, 10mg/mL; Invitrogen), 6 μ L; Spiral vibration mixing, reaction 30min is with an amount of TE damping fluid (10mM Tris-HCl, pH8.0; 1mM EDTA) clean twice, spinning, with the magnetic bead that obtains with 6 μ L binding buffer liquid (10mM Tris-HCl, pH7.5; 1mM EDTA; 1M NaCl; 0.01% Triton X-100) resuspended preservation.

2) the unit molecule amplimer is incorporated into magnetic bead surfaces.

Product and 5 ' end that step 1) is obtained contain biotin labeling and contain amidized unit molecule amplimer (F3, R3) reaction apart from the 6th Nucleotide of 5 ' end; The target nucleic acid fragment that makes biotinylated F3, R3 and contain first joint is combined in magnetic bead surfaces simultaneously; Wherein, The sequence of F3, R3 is SEQ ID NO:9 and SEQ ID NO:10, with the target nucleic acid fragment complementation that contains first joint.Reaction system and process are following: biotinylation and amidized primers F 3 (100 μ M), 0.3 μ L; Biotinylation and amidized primer R3 (100 μ M), 0.3 μ L; The bead suspension that obtains in the step 1), 6 μ L; Under the room temperature condition (18 ~ 25 ℃), 1h is hatched in the spiral vibration; An amount of TE buffer solution for cleaning 2 times, centrifugal, with the resuspended magnetic bead of 6 μ L TE damping fluids, obtain bead suspension, 4 ℃ of preservations are subsequent use.

In this step; Be used for combining with magnetic bead; And roughly the same as the quantity of the segmental mole number of target nucleic acid of unit molecule amplification template sequence and magnetic bead, therefore, a magnetic bead surfaces in the bead suspension of step 1) gained has only combined minute quantity or even single template sequence.

3) preparation is used for the emulsion system of unit molecule amplification.

Adopt the KE001 emulsion of Shenzhen HYK Gene Technology Co., Ltd. to prepare test kit, prepare emulsion system according to operation instruction.At first oil phase is prepared reagent and carry out mixing, place room temperature 30min, obtain being used to prepare the oil phase system of emulsion system with the spiral thermal agitation.

Utilize step 2) bead suspension that obtains prepares PCR Mix aqueous phase system, is example with 150 μ L, and this aqueous phase system is following: ddH ₂O, 113 μ L; 10 * PCR buffer (650mM Tris-HCl, pH8.0; 160mM (NH ₄) ₂SO ₄10mM DTT; 11mM MgCl ₂), 15 μ L; 50mM MgSO ₄, 3 μ L; 10mM dNTP, 3 μ L; Biotinylation and do not have amidized F3 (10 μ M), 0.5 μ L not; Biotinylation and do not have amidized R3 (10 μ M), 0.5 μ L not; Step 2) bead suspension that obtains, 6 μ L; 5U/ μ L DNA Taq enzyme, 9 μ L; With the mentioned component mixing, be prepared into PCR Mix aqueous phase system.

The oil phase system for preparing is put into the EP pipe with aqueous phase system according to the ratio of 4:1 mixes; Add the steel ball of assisting mixing simultaneously, the EP pipe is placed on the emulsion preparing instrument clamp, according to 15HZ; 10s; Change 17HZ again, the 8s mixing that vibrates is prepared into the emulsion system that is used for the unit molecule amplification.

4) utilize the emulsion system for preparing to carry out the unit molecule amplification.

The emulsion system that utilization prepares carries out the amplification of EPCR unit molecule, and reaction system and reaction process are as follows: 4min, 94 ℃;

30s，94℃，

55s，64℃，

45s, 72 ℃, cycle number is 3;

30s，94℃，

55s，61℃，

45s, 72 ℃, cycle number is 3;

30s，94℃，

55s，58℃，

45s, 72 ℃, cycle number is 3;

30s，94℃，

55s，57℃，

45s, 72 ℃, cycle number is 100;

6min，72℃；

Reaction finishes back 10 ℃ of preservations.

3. breakdown of emulsion discharges amplified production, and separating purifies obtains the library of checking order.

Add an amount of Virahol in the reaction product after the EPCR reaction finishes, 4000rpm behind the spiral concussion mixing, supernatant is removed in the 3min spinning, and amplified production adsorbs with magnet.

Add an amount of extraction buffer (Extraction buffer) in the amplified production, the centrifugal 3min layering of 4000rpm behind the spiral vibration mixing is adsorbed magnetic bead with magnet, with alveolar fluid clearance; Repeat this operation for several times.

Add an amount of TE then, the repeated washing number is all over amplified production, and is last with the resuspended magnetic bead of an amount of TE, obtains containing the segmental magnetic bead of determined nucleic acid of gene order.

Three, utilize the order-checking library to carry out enzyme and cut the extension order-checking.

1. an anchor grappling is incorporated on segmental first joint of the determined nucleic acid that contains gene order that contains gene order.

The segmental magnetic bead of determined nucleic acid that will contain gene order mixes with the order-checking damping fluid, on the slide that contains the activated carboxylic base group modification, carries out point sample and fixes, and forms the order-checking array.With combining through base complementrity pairing hybridization between an anchor (SEQ ID NO:11) and segmental first joint of the determined nucleic acid that contains gene order; The enzyme that wherein contains Acu I enzyme on the anchor extending end is cut recognition site; This reaction process and system are: 28 ℃; 400 μ L, 2 * SSPE (saline sodium phosphate EDTA) [175.32g/L NaCl, 31.202g/L NaH ₂PO ₄.2H ₂O, 0.01M EDTA, pH7.4] hybridization buffer carries out rinse to the determined nucleic acid fragment that contains gene order that is fixed in surface of glass slide; Add an anchor (15 μ M), be warming up to 65 ℃ under 2 * SSPE environment, keep 30s; Be cooled to 42 ℃, hybridization 1min; With magnet absorption magnetic bead, 30 ℃, 900 μ L cleaning buffer solutions [50mM KCl, 10mM Tris-HCl (pH7.4), 0.1mM EDTA] clean, and a unreacted anchor is separated remove.

2. obtain the sequence information of 6 Nucleotide behind the anchor extending end.

1) connects fluorescent probe.

The used fluorescent probe of present embodiment is divided into different group types, with the different IPs nucleotide sequence of the corresponding same specific position of different fluorescent marks in the set type, and the corresponding specific position difference of the fluorescent probe fluorescent mark of different set types.In each ligation, add the fluorescent probe of same set type,, can read this group fluorescent probe mark specific position corresponding nucleotide sequences information according to the fluorescent signal of being gathered.The structure of said fluorescent probe is 5 '-NNNXNNNNN-3 ', and wherein N is the degeneracy base, and X is any one among A, T, G and the C, X can 5 ' terminal number rise the 1st to the 6th in any position.

Under the effect of T4 ligase enzyme, be connected to after the anchor extending end after the different fluorescently-labeled one group of four kinds of fluorescent probe of No. 1 bit strip of correspondence are mixed, ligation process and system are: 30 ℃, connect damping fluid [100mM MgCl ₂.6H ₂O, 10mM Tris-HCl (pH7.4)] carry out rinse to hybridizing isolating product; Add 0.2U/ μ L T4 ligase enzyme, fluorescent probe (concentration is 2.5 μ M) connects in the damping fluid, and 30 ℃, reaction 20min; After ligation finished, with magnet absorption magnetic bead, 30 ℃, 900 μ L cleaning buffer solutions cleaned along the slide plane, with unreacted fluorescent probe be connected product and separate.

2) gather fluorescent signal, read the nucleotide sequence information of corresponding position.

The connection product that is connected with fluorescent probe is put into sequenator, under fluorescent microscope, excite, gather fluorescent signal, judge according to fluorescent signal and read the nucleotide sequence of this position.

3) wash-out the one anchor and fluorescent probe

On reading, in the step behind the nucleotide sequence information of corresponding position, can utilize diverse ways to carry out wash-out.

In the present embodiment, utilize the direct sex change of NaOH to dissociate, reaction process and system are: add NaOH (0.05M) in the sequencing reaction system, sex change 30s; With magnet absorption magnetic bead, 30 ℃, 900 μ L elution buffers clean, and the dissociated anchor of sex change and fluorescent probe and NaOH are cleaned separation.

In another embodiment of the present invention, utilize the UDG enzyme that the dU base among the one anchor is cut the formation small segment, with the direct wash-out of small segment, reaction process and system are then: enzyme cutting buffering liquid, 20 μ L; The UDG enzyme, 10 μ L; Elution buffer adds to 200 μ L; 37 ℃, 5min carries out enzyme and cuts; After enzyme is cut end,, add 900 μ L elution buffers and carry out the wash-out separation, do not connect the determined nucleic acid fragment that contains gene order of an anchor with magnet absorption magnetic bead.

4) repeat above-mentioned steps, connect, obtain behind the anchor extending end the 1st to 6 nucleotide sequence information thereby read through the fluorescent probe of changing different mark positions.

3.Acu the I enzyme is cut, and obtains containing the segmental enzyme that do not check order and cuts product.

1) extends to form double chain acid molecule.

The carrying out that cuts for ease of follow-up enzyme; After 6 nucleotide sequence informations all are read behind the anchor extending end; Described NaOH sex change dissociating method is removed a fluorescent probe and an anchor before utilizing; And recombine the one anchor is on first joint, and under the effect of archaeal dna polymerase, the determined nucleic acid fragment that will contain gene order extends to form complete double chain acid molecule then; Extension system and process are: under 30 ℃ of conditions, with Klenow enzyme reaction buffer solution rinse the one anchor and the segmental binding substances of determined nucleic acid that contains gene order; Klenow enzyme (BioLabs

Inc. that adds dNTP and 0.1U/ μ L; Article No. M0210L); In 1 * NEBuffer2 reaction buffer; 37 ℃, hatch 10min; After extension finished, with magnet absorption magnetic bead, 30 ℃, 900 μ L cleaning buffer solutions cleaned, and separated the complete double chain acid molecule that the determined nucleic acid fragment that obtains containing gene order extends to form.

2) Acu I enzyme is cut.

After obtaining double chain acid molecule, carry out enzyme with the Acu I and cut, obtain containing the segmental enzyme of waiting to check order and cut product, reaction system that enzyme is cut and process are: 30 ℃, add 400 μ L, 1 * NEBuffer, 2,40 μ M SAM in the double chain acid molecule; Add the Acu I enzyme (BioLabs Inc.) of 0.05U/ μ L, hatch 2h for 37 ℃; After endonuclease reaction finished, with magnet absorption magnetic bead, 30 ℃, 900 μ L cleaning buffer solutions cleaned, and separated to obtain containing the segmental enzyme of waiting to check order and cut product, wherein contained the protruding terminus that the segmental nucleotide chain 3 ' end of waiting to check order contains two Nucleotide.

4. enzyme is cut product and is connected second joint, and combines the 2nd anchor.

1) enzyme is cut product and is connected second joint.

Utilize enzyme cut product with protruding terminus, be connected with second joint (SEQ ID NO:12 and SEQ ID NO:13) of structure as shown in Figure 7, wherein the base of protruding terminus is A or G or C or T, second joint is to be altogether 4 with kind ²The form of=16 kinds of mixed in molar ratio such as joint adds in the ligation, and the system of ligation and process are: under 30 ℃ of conditions, cut at the enzyme that reclaims and to add 1 * connection damping fluid in the product; Adding concentration is 16 kinds of joint blended second joints of 10 μ M, and the T4 ligase enzyme of 0.2U/ μ L, hatches 1h under 16 ℃ of conditions; After ligation finished, with magnet absorption magnetic bead, 30 ℃, 900 μ L cleaning buffer solutions cleaned, and separate the new determined nucleic acid fragment that contains gene order that obtains the double chain acid molecule form.

2) combination of the 2nd anchor.

With the dissociated method of NaOH sex change, the new determined nucleic acid fragment that contains gene order of double chain acid molecule form is transformed into contains the new determined nucleic acid fragment that the segmental strand that do not check order contains gene order.The hybridization articulated system and the process of the 2nd anchor (SEQ ID NO:14) and second joint are: 28 ℃, and, strand adds 400 μ L, 2 * SSPE in containing the new determined nucleic acid fragment of gene order, add the 2nd anchor (10 μ M), keep 30s for 60 ℃; Be cooled to 42 ℃ then, 2min is hatched in hybridization; After hybridization finished, with magnet absorption magnetic bead, 30 ℃, 900 μ L cleaning buffer solutions cleaned, and unreacted the 2nd anchor is separated remove.

5. obtain the sequence information of 6 Nucleotide behind the 2nd anchor extending end.

Obtain the same procedure and the similar operations of 6 nucleotide sequence informations behind the anchor extending end in the refer step 2; Fluorescent probe through changing different mark positions connects, and reads to obtain behind the 2nd anchor extending end the 1st to 6 nucleotide sequence information.

6. obtain follow-up nucleotide sequence information.

The anchor that more renews, joint and restriction endonuclease; The operation of repeating step 3 to step 5; Cut the nucleotide sequence that the means excision had been read through enzyme; And make up the new determined nucleic acid fragment that contains gene order, realize extending forward the purpose of order-checking with this, until obtaining containing the complete nucleotide sequence information that can read on the determined nucleic acid fragment of gene order.

To F8 gene nucleic acid fragment and the formed determined nucleic acid fragment that contains gene order of the galt gene nucleic acid fragment resulting fluorescent signal view data that checks order; Utilize with Pstar II-Plus order-checking platform and analyze; Obtain the nucleotide sequence that sequence number is respectively SEQ ID NO:15 and SEQ ID NO:16, more concrete analytical results is as shown in table 1.

Table 1. fluorescent signal analysis of image data is figure as a result

Project	Preceding 100bases accuracy rate	100 ~ 200bases accuracy rate	200 ~ 300bases accuracy rate	The average accuracy rate of preceding 300bases
					F8 gene nucleic acid fragment	99.96%（Q30）	99.82%（Q20）	99.20%（Q20）	99.75%（Q20）
The galt gene nucleic acid fragment	99.96%（Q30）	99.87%（Q20）	99.17%（Q20）	99.76%（Q20）

Wherein, Q30 described in the table refers in the order-checking process read error rate only at millesimal Nucleotide; Q20 refers in the order-checking process read error rate only at centesimal Nucleotide.

According to table 1 data; Can reach 99% with Q20 in the order-checking process is that trust data is as judgement criteria; Through the analytical data that obtains in the foregoing description; Utilize technical scheme that the present invention puts down in writing in the detection that contains gene order at present embodiment, can reach the high-quality order-checking of 300bp at least and read long.

Simultaneously; In order to verify the accuracy of present embodiment sequencing result; The mono-clonal plasmid that includes F8 gene nucleic acid fragment at first and include the galt gene nucleic acid fragment is carried out sequencing with the sanger PCR sequencing PCR; The result of nucleotide sequence result who obtains and this enforcement gained is compared comparison result shows: the sequencing result of the two is 100% on similarity.Therefore, the resulting result of present embodiment is credible result.

Should be noted that the technical scheme of being put down in writing with the present invention carries out the detection of gene order, should all nucleotide sequence informations of determined nucleic acid fragment that contain gene order be read out in theory.But according to the data that obtain among the said embodiment; The high quality that obtains in the present embodiment is read the long 300bp that is at least; And along with reading long increase; It reads accuracy rate and presents progressively downward trend, its reason possibly be since continuous round-robin enzyme cut with attended operation in, the concatenation ability that the enzyme of restriction endonuclease is cut ability and ligase enzyme descends to some extent and causes.Therefore, strengthen as if the concatenation ability of cutting ability and ligase enzyme for the enzyme of restriction endonuclease, the technical scheme of utilizing the present invention to put down in writing detects gene order, can reach the longer length of reading.

Secondly; The foregoing description only is a specific embodiments of technical scheme that the present invention puts down in writing, and one or more genes in the different testing samples utilize corresponding amplimer; Detect through the foregoing description identical operations and step, can obtain similar result equally.

The order-checking platform that Treatment Analysis adopted of order-checking process in addition, and fluorescent signal view data can also be Pstar-II e, the Pstar-II that Shenzhen HYK Gene Technology Co., Ltd. produces.

The source of testing sample according to the invention can be varied, and its source includes but not limited to that blood, oral epithelium scrape sampling, saliva, paraffin-embedded tissue and puncture tissue etc.In testing process, when the technical scheme of utilizing the present invention to put down in writing detected gene order, the kind and the number of the gene order of detection were unrestricted, can arbitrary combination.

Above-mentioned practical implementation just detects a certain particular segment in F8 gene and the galt gene; If need detect to other zones of these two genes; Only need the corresponding specificity amplification primer of design to get final product, corresponding specificity amplification primer is to including but not limited to: F8 gene: SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; Galt gene: SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.

Certainly; If need be to F9, FGFR3, IDS, HBB, HBA1, HBA2, ATP7B, PHEX, GJB2, COL4A5, when isogenic some zone of LMNA is detected; Also only need the corresponding specificity amplification primer of design to get final product, corresponding specificity amplification primer is to including but not limited to: F9:SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32; FGFR3:SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36; IDS:SEQ ID NO:37 and SEQ ID NO:38; HBA1:SEQ ID NO:39 and SEQ ID NO:40; HBA2:SEQ ID NO:41 and SEQ ID NO:42; HBB:SEQ ID NO:43 and SEQ ID NO:44; ATP7B:SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48; PHEX:SEQ ID NO:49 and SEQ ID NO:50; GJB2:SEQ ID NO:51 and SEQ ID NO:52; COL4A5:SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56; LMNA:SEQ ID NO:57 and SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:60.

Certainly; If need be to CYP2C19, VKORC1, CYP2C9, KIT, PDGFRA, DHFR, GSTM1, MTHFR, RFC1, CYP19A1, UGT1A1, UGT1A7, UGT1A9, ABCB1, CYP2D6, CYP3A5, SULT1A1, UGT2B15, GSTA1, SOD2, CYP2B6, GSTP1, MDR1, BCRP, p53, CAT, CBR1, CBR3, EGFR, KRAS, when isogenic some zone of BRAF is detected; Also only need the corresponding specificity amplification primer of design to get final product; Corresponding specificity amplification primer is to including but not limited to: CYP2C19:SEQ ID NO:61 and SEQ ID NO:62; SEQ ID NO:63 and SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66; VKORC1:SEQ ID NO:67 and SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72; CYP2C9:SEQ ID NO:73 and SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:76; KIT:SEQ ID NO:77 and SEQ ID NO:78, SEQ ID NO:79 and SEQ ID NO:80; PDGFRA:SEQ ID NO:81 and SEQ ID NO:82; DHFR:SEQ ID NO:83 and SEQ ID NO:84; GSTM1:SEQ ID NO:85 and SEQ ID NO:86; MTHFR:SEQ ID NO:87 and SEQ ID NO:88; RFC1:SEQ ID NO:89 and SEQ ID NO:90; CYP19A1:SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:93 and SEQ ID NO:94; UGT1A1:SEQ ID NO:95 and SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100; UGT1A7:SEQ ID NO:101 and SEQ ID NO:102; UGT1A9:SEQ ID NO:103 and SEQ ID NO:104; ABCB1:SEQ ID NO:105 and SEQ ID NO:106; CYP2D6:SEQ ID NO:107 and SEQ ID NO:108; CYP3A5:SEQ ID NO:109 and SEQ ID NO:110; SULT1A1:SEQ ID NO:111 and SEQ ID NO:112; UGT2B15:SEQ ID NO:113 and SEQ ID NO:114; GSTA1:SEQ ID NO:115 and SEQ ID NO:116; SOD2:SEQ ID NO:117 and SEQ ID NO:118; CYP2B6:SEQ ID NO:119 and SEQ ID NO:120; GSTP1:SEQ ID NO:121 and SEQ ID NO:122; MDR1:SEQ ID NO:123 and SEQ ID NO:124; BCRP:SEQ ID NO:125 and SEQ ID NO:126; P53:SEQ ID NO:127 and SEQ ID NO:128; CAT:SEQ ID NO:129 and SEQ ID NO:130; CBR1:SEQ ID NO:131 and SEQ ID NO:132; CBR3:SEQ ID NO:133 and SEQ ID NO:134.

The above is merely preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of within spirit of the present invention and principle, being done, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.

SEQUENCE?LISTING

< 110>Sheng Sitong

< 120>a kind of method that detects gene order

<130>

<160> 134

<170> PatentIn?version?3.3

<210> 1

<211> 458

<212> DNA

< 213>artificial sequence

<400> 1

gtccattctg?tcctagcaag?tgttttccat?ttcagattct?ctacttcata?gccataggtg 60

tcttattcct?actttacagg?tctgattgga?tgccacagga?aatcagtcta?ttggcatgtg 120

attggaatgg?gcaccactcc?tgaagtgcac?tcaatattcc?tcgaaggtca?cacatttctt 180

gtgaggaacc?atcgccaggc?gtccttggaa?atctcgccaa?taactttcct?tactgctcaa 240

acactcttga?tggaccttgg?acagtttcta?ctgttttgtc?atatctcttc?ccaccaacat 300

ggtaatatct?tggatcttta?aaatgaatat?tataaatatc?cagtcctact?tttaataaaa 360

tttactggac?aatgtacagg?aatatagtgt?gttgctgaag?gggacattat?agagttactg 420

tattagacct?cagccaagat?cccctagttt?ccagcttc 458

<210> 2

<211> 268

<212> DNA

< 213>artificial sequence

<400> 2

ggacggagaa?agtgaaaggt?gaggcacggc?cctgcagatt?ttccagcgga?tcccccggtg 60

gcctcatgtc?gcgcagtgga?accgatcctc?agcaacgcca?gcaggcgtca?gaggcggacg 120

ccgcagcagc?aaccttccgg?gcaaacggta?actgcaccgc?ggcagggact?cgctggggcg 180

cggagccgag?ccctcccctt?ccttaggaag?ctttcgtccc?ctccgaaggt?tggaacgctc 240

atcccgagcc?agaccgacaa?ggcgtaca 268

<210> 3

<211> 22

<212> DNA

< 213>artificial sequence

<400> 3

gtccattctg?tcctagcaag?tg 22

<210> 4

<211> 21

<212> DNA

< 213>artificial sequence

<400> 4

gaagctggaa?actaggggat?c 21

<210> 5

<211> 22

<212> DNA

< 213>artificial sequence

<400> 5

ggacggagaa?agtgaaaggt?ga 22

<210> 6

<211> 20

<212> DNA

< 213>artificial sequence

<400> 6

tgtacgcctt?gtcggtctgg 20

<210> 7

<211> 21

<212> DNA

< 213>artificial sequence

<400> 7

gccggaagtc?cgccacttca?g 21

<210> 8

<211> 22

<212> DNA

< 213>artificial sequence

<400> 8

ctgaagtggc?ggacttccgg?ct 22

<210> 9

<211> 27

<212> DNA

< 213>artificial sequence

<400> 9

ttttttgccg?gaagtccgcc?acttcag 27

<210> 10

<211> 27

<212> DNA

< 213>artificial sequence

<400> 10

ttttttctga?agtggcggac?ttccggc 27

<210> 11

<211> 20

<212> DNA

< 213>artificial sequence

<400> 11

ctgaaguggc?ggacutccgg 20

<210> 12

<211> 24

<212> DNA

< 213>artificial sequence

<400> 12

gactgatagc?ttcaggactg?ctga 24

<210> 13

<211> 26

<212> DNA

< 213>artificial sequence

<220>

<221> misc_feature

<222> (25)..(26)

<223> n?is?a,?c,?g,?or?t

<400> 13

tcagcagtcc?tgaagctatc?agtcnn 26

<210> 14

<211> 20

<212> DNA

< 213>artificial sequence

<400> 14

cagucctgaa?gctaucagtc 20

<210> 15

<211> 458

<212> DNA

< 213>artificial sequence

<400> 15

gtccattctg?tcctagcaag?tgttttccat?ttcagattct?ctacttcata?gccataggtg 60

tcttattcct?actttacagg?tctgattgga?tgccacagga?aatcagtcta?ttggcatgtg 120

attggaatgg?gcaccactcc?tgaagtgcac?tcaatattcc?tcgaaggtca?cacatttctt 180

gtgaggaacc?atcgccaggc?gtccttggaa?atctcgccaa?taactttcct?tactgctcaa 240

acactcttga?tggaccttgg?acagtttcta?ctgttttgtc?atatctcttc?ccaccaacat 300

ggtaatatct?tggatcttta?aaatgaatat?tataaatatc?cagtcctact?tttaataaaa 360

tttactggac?aatgtacagg?aatatagtgt?gttgctgaag?gggacattat?agagttactg 420

tattagacct?cagccaagat?cccctagttt?ccagcttc 458

<210> 16

<211> 268

<212> DNA

< 213>artificial sequence

<400> 16

ggacggagaa?agtgaaaggt?gaggcacggc?cctgcagatt?ttccagcgga?tcccccggtg 60

gcctcatgtc?gcgcagtgga?accgatcctc?agcaacgcca?gcaggcgtca?gaggcggacg 120

ccgcagcagc?aaccttccgg?gcaaacggta?actgcaccgc?ggcagggact?cgctggggcg 180

cggagccgag?ccctcccctt?ccttaggaag?ctttcgtccc?ctccgaaggt?tggaacgctc 240

atcccgagcc?agaccgacaa?ggcgtaca 268

<210> 17

<211> 20

<212> DNA

< 213>artificial sequence

<400> 17

tttatggttt?tgcttgtggg 20

<210> 18

<211> 20

<212> DNA

< 213>artificial sequence

<400> 18

tggaaaggca?agaactcacc 20

<210> 19

<211> 19

<212> DNA

< 213>artificial sequence

<400> 19

ctgggaatgg?gagagaacc 19

<210> 20

<211> 20

<212> DNA

< 213>artificial sequence

<400> 20

tccctgattc?ctctaccctc 20

<210> 21

<211> 20

<212> DNA

< 213>artificial sequence

<400> 21

cccccaactg?taagggtcac 20

<210> 22

<211> 21

<212> DNA

< 213>artificial sequence

<400> 22

tcctgacaca?agcaaccatt?c 21

<210> 23

<211> 20

<212> DNA

< 213>artificial sequence

<400> 23

tgacaaagcg?agactccatc 20

<210> 24

<211> 20

<212> DNA

< 213>artificial sequence

<400> 24

ctgcccataa?ccaaacttcc 20

<210> 25

<211> 17

<212> DNA

< 213>artificial sequence

<400> 25

cctgctggtg?ggtgaga 17

<210> 26

<211> 19

<212> DNA

< 213>artificial sequence

<400> 26

gcagggctct?acaggctta 19

<210> 27

<211> 20

<212> DNA

< 213>artificial sequence

<400> 27

ttgacttggt?gtcttttggc 20

<210> 28

<211> 20

<212> DNA

< 213>artificial sequence

<400> 28

ctttacctcc?aagcctccac 20

<210> 29

<211> 21

<212> DNA

< 213>artificial sequence

<400> 29

catgttccat?ttgccaatga?g 21

<210> 30

<211> 22

<212> DNA

< 213>artificial sequence

<400> 30

gctccagttt?tgacacacca?tc 22

<210> 31

<211> 23

<212> DNA

< 213>artificial sequence

<400> 31

taggtcagtg?gtcccaagta?gtc 23

<210> 32

<211> 23

<212> DNA

< 213>artificial sequence

<400> 32

tgattagtta?gtgagaggcc?ctg 23

<210> 33

<211> 18

<212> DNA

< 213>artificial sequence

<400> 33

tccggcagac?gtacacgc 18

<210> 34

<211> 19

<212> DNA

< 213>artificial sequence

<400> 34

cccaaatcct?cacgcaacc 19

<210> 35

<211> 20

<212> DNA

< 213>artificial sequence

<400> 35

tgcgagaccc?tccagacaag 20

<210> 36

<211> 20

<212> DNA

< 213>artificial sequence

<400> 36

agcagagacg?aggagagggg 20

<210> 37

<211> 21

<212> DNA

< 213>artificial sequence

<400> 37

aacctagcac?ctaccatgag?g 21

<210> 38

<211> 21

<212> DNA

< 213>artificial sequence

<400> 38

cagctagagg?ttcccagaca?t 21

<210> 39

<211> 18

<212> DNA

< 213>artificial sequence

<400> 39

aaaccccacc?cctcactc 18

<210> 40

<211> 17

<212> DNA

< 213>artificial sequence

<400> 40

tacacctccc?gcaaccc 17

<210> 41

<211> 17

<212> DNA

< 213>artificial sequence

<400> 41

agggcagagg?atcacgc 17

<210> 42

<211> 17

<212> DNA

< 213>artificial sequence

<400> 42

tgcaaggagg?ggaggag 17

<210> 43

<211> 21

<212> DNA

< 213>artificial sequence

<400> 43

tagcaacctc?aaacagacac?c 21

<210> 44

<211> 21

<212> DNA

< 213>artificial sequence

<400> 44

gtcagtgcct?atcagaaacc?c 21

<210> 45

<211> 20

<212> DNA

< 213>artificial sequence

<400> 45

tgtgaaactg?gtggaagagg 20

<210> 46

<211> 20

<212> DNA

< 213>artificial sequence

<400> 46

aaccaccata?tagcccaagg 20

<210> 47

<211> 23

<212> DNA

< 213>artificial sequence

<400> 47

ctcaggagtg?tgactatgga?agc 23

<210> 48

<211> 23

<212> DNA

< 213>artificial sequence

<400> 48

gacatggtga?ggaataaaag?agc 23

<210> 49

<211> 21

<212> DNA

< 213>artificial sequence

<400> 49

agcagtttat?cttggctttc?c 21

<210> 50

<211> 20

<212> DNA

< 213>artificial sequence

<400> 50

cagcaagaca?cggtgaaaag 20

<210> 51

<211> 20

<212> DNA

< 213>artificial sequence

<400> 51

tcctgtgttg?tgtgcattcg 20

<210> 52

<211> 21

<212> DNA

< 213>artificial sequence

<400> 52

cattgtggca?tctggagttt?c 21

<210> 53

<211> 22

<212> DNA

< 213>artificial sequence

<400> 53

gaaggggtaa?actggagaga?ag 22

<210> 54

<211> 22

<212> DNA

< 213>artificial sequence

<400> 54

tgggtgttta?tctcagcatc?ag 22

<210> 55

<211> 22

<212> DNA

< 213>artificial sequence

<400> 55

atccatctaa?tcccatgttt?gc 22

<210> 56

<211> 23

<212> DNA

< 213>artificial sequence

<400> 56

tctatcaaca?ccagcctctt?ttc 23

<210> 57

<211> 21

<212> DNA

< 213>artificial sequence

<400> 57

actccgagca?gtctctgtcc?t 21

<210> 58

<211> 20

<212> DNA

< 213>artificial sequence

<400> 58

ccttgacact?gccagtcacc 20

<210> 59

<211> 20

<212> DNA

< 213>artificial sequence

<400> 59

gttcccacca?aagttcaccc 20

<210> 60

<211> 18

<212> DNA

< 213>artificial sequence

<400> 60

gcctcgtcca?gcaagcag 18

<210> 61

<211> 22

<212> DNA

< 213>artificial sequence

<400> 61

atggatcctt?ttgtggtcct?tg 22

<210> 62

<211> 22

<212> DNA

< 213>artificial sequence

<400> 62

gactggaaaa?ggcaacaaaa?gc 22

<210> 63

<211> 22

<212> DNA

< 213>artificial sequence

<400> 63

tgcttttaag?ggaattcata?gg 22

<210> 64

<211> 22

<212> DNA

< 213>artificial sequence

<400> 64

aaatgtactt?cagggcttgg?tc 22

<210> 65

<211> 23

<212> DNA

< 213>artificial sequence

<400> 65

ccttttccat?cagttcttac?ttg 23

<210> 66

<211> 23

<212> DNA

< 213>artificial sequence

<400> 66

tatagtccca?aactggaatc?aac 23

<210> 67

<211> 19

<212> DNA

< 213>artificial sequence

<400> 67

tgctccgtgg?ctggttttc 19

<210> 68

<211> 20

<212> DNA

< 213>artificial sequence

<400> 68

gcacacctgg?aggagaagac 20

<210> 69

<211> 19

<212> DNA

< 213>artificial sequence

<400> 69

ttctcgggca?gggtccaag 19

<210> 70

<211> 20

<212> DNA

< 213>artificial sequence

<400> 70

cggagccact?cacctaacaa 20

<210> 71

<211> 18

<212> DNA

< 213>artificial sequence

<400> 71

tggagtgttc?gggaggtg 18

<210> 72

<211> 20

<212> DNA

< 213>artificial sequence

<400> 72

aaagcaaagc?agatgaggtc 20

<210> 73

<211> 20

<212> DNA

< 213>artificial sequence

<400> 73

tctttcttgc?ctgggatctc 20

<210> 74

<211> 21

<212> DNA

< 213>artificial sequence

<400> 74

ggtcagtgat?atggagtagg?g 21

<210> 75

<211> 21

<212> DNA

< 213>artificial sequence

<400> 75

tcagctaaag?tccaggaaga?g 21

<210> 76

<211> 22

<212> DNA

< 213>artificial sequence

<400> 76

attacccagt?caaagatatt?cc 22

<210> 77

<211> 20

<212> DNA

< 213>artificial sequence

<400> 77

aaagtatgcc?acatcccaag 20

<210> 78

<211> 22

<212> DNA

< 213>artificial sequence

<400> 78

tatggtagac?agagcctaaa?ca 22

<210> 79

<211> 22

<212> DNA

< 213>artificial sequence

<400> 79

attaggaact?ctgtgaaagg?ac 22

<210> 80

<211> 21

<212> DNA

< 213>artificial sequence

<400> 80

ctgtcaagca?gagaatgggt?a 21

<210> 81

<211> 21

<212> DNA

< 213>artificial sequence

<400> 81

aactgtctcc?ctccttcctt?g 21

<210> 82

<211> 21

<212> DNA

< 213>artificial sequence

<400> 82

cagaacagcc?caaataagca?g 21

<210> 83

<211> 19

<212> DNA

< 213>artificial sequence

<400> 83

atttcgcgcc?aaacttgac 19

<210> 84

<211> 20

<212> DNA

< 213>artificial sequence

<400> 84

agaaaagggg?aatccagtcg 20

<210> 85

<211> 19

<212> DNA

< 213>artificial sequence

<400> 85

cttttgtccg?agggtggtg 19

<210> 86

<211> 19

<212> DNA

< 213>artificial sequence

<400> 86

aggtaacgga?acaaggggc 19

<210> 87

<211> 19

<212> DNA

< 213>artificial sequence

<400> 87

catccctcgc?cttgaacag 19

<210> 88

<211> 18

<212> DNA

< 213>artificial sequence

<400> 88

aggacggtgc?ggtgagag 18

<210> 89

<211> 22

<212> DNA

< 213>artificial sequence

<400> 89

ttcgtgaatc?tcctgttttt?tg 22

<210> 90

<211> 20

<212> DNA

< 213>artificial sequence

<400> 90

ttgcaccact?gcactacagc 20

<210> 91

<211> 23

<212> DNA

< 213>artificial sequence

<400> 91

tgcacccaga?tgagactaaa?aac 23

<210> 92

<211> 21

<212> DNA

< 213>artificial sequence

<400> 92

ttggtgacaa?cccataggag?g 21

<210> 93

<211> 19

<212> DNA

< 213>artificial sequence

<400> 93

attcggcagc?aaacttggg 19

<210> 94

<211> 19

<212> DNA

< 213>artificial sequence

<400> 94

tgggaggcgg?aggttacag 19

<210> 95

<211> 22

<212> DNA

< 213>artificial sequence

<400> 95

cctgctacct?ttgtggactg?ac 22

<210> 96

<211> 18

<212> DNA

< 213>artificial sequence

<400> 96

ctcctgccag?aggttcgc 18

<210> 97

<211> 22

<212> DNA

< 213>artificial sequence

<400> 97

ccatgctggg?aagatactgt?tg 22

<210> 98

<211> 19

<212> DNA

< 213>artificial sequence

<400> 98

cctccctttg?gaatggcac 19

<210> 99

<211> 22

<212> DNA

< 213>artificial sequence

<400> 99

gatagggcac?cagtcagagt?tg 22

<210> 100

<211> 20

<212> DNA

< 213>artificial sequence

<400> 100

tcacctgaag?ccagcaagtc 20

<210> 101

<211> 18

<212> DNA

< 213>artificial sequence

<400> 101

acggcaccat?tgcgaagt 18

<210> 102

<211> 20

<212> DNA

< 213>artificial sequence

<400> 102

agaccacaga?ggggagggag 20

<210> 103

<211> 20

<212> DNA

< 213>artificial sequence

<400> 103

ccccaaggca?aagaccataa 20

<210> 104

<211> 20

<212> DNA

< 213>artificial sequence

<400> 104

accagcacaa?aacctgaccc 20

<210> 105

<211> 23

<212> DNA

< 213>artificial sequence

<400> 105

tgaagttttt?ttctcactcg?tcc 23

<210> 106

<211> 21

<212> DNA

< 213>artificial sequence

<400> 106

tctgtggggt?catagagcct?c 21

<210> 107

<211> 20

<212> DNA

< 213>artificial sequence

<400> 107

gggtgtccca?gcaaagttca 20

<210> 108

<211> 20

<212> DNA

< 213>artificial sequence

<400> 108

cccgttctgt?cccgagtatg 20

<210> 109

<211> 20

<212> DNA

< 213>artificial sequence

<400> 109

accacccagc?ttaacgaatg 20

<210> 110

<211> 19

<212> DNA

< 213>artificial sequence

<400> 110

cacacccagg?aagccagac 19

<210> 111

<211> 22

<212> DNA

< 213>artificial sequence

<400> 111

tcctgaatca?gtaatccgag?cc 22

<210> 112

<211> 20

<212> DNA

< 213>artificial sequence

<400> 112

aagggggaga?tgctgtggtc 20

<210> 113

<211> 22

<212> DNA

< 213>artificial sequence

<400> 113

ctgtgttgac?atcttcggct?tc 22

<210> 114

<211> 23

<212> DNA

< 213>artificial sequence

<400> 114

catctgccag?aatgacatca?aac 23

<210> 115

<211> 20

<212> DNA

< 213>artificial sequence

<400> 115

caacagtcgg?agcctctttc 20

<210> 116

<211> 22

<212> DNA

< 213>artificial sequence

<400> 116

taatcagtgc?tggaagaagt?gg 22

<210> 117

<211> 18

<212> DNA

< 213>artificial sequence

<400> 117

agcccagcct?gcgtagac 18

<210> 118

<211> 18

<212> DNA

< 213>artificial sequence

<400> 118

cgtggtgctt?gctgtggt 18

<210> 119

<211> 19

<212> DNA

< 213>artificial sequence

<400> 119

aggcaaacct?caccacccc 19

<210> 120

<211> 20

<212> DNA

< 213>artificial sequence

<400> 120

tttcctctcc?cagaccccac 20

<210> 121

<211> 19

<212> DNA

< 213>artificial sequence

<400> 121

gaatgacggc?gtggaggac 19

<210> 122

<211> 19

<212> DNA

< 213>artificial sequence

<400> 122

gggtgagggc?acaagaagc 19

<210> 123

<211> 21

<212> DNA

< 213>artificial sequence

<400> 123

cttggggtaa?attgaggctc?g 21

<210> 124

<211> 20

<212> DNA

< 213>artificial sequence

<400> 124

agagggctga?gagcagggac 20

<210> 125

<211> 24

<212> DNA

< 213>artificial sequence

<400> 125

tatagacatg?aatacatcag?cgga 24

<210> 126

<211> 23

<212> DNA

< 213>artificial sequence

<400> 126

cacccatagg?caagactaaa?gac 23

<210> 127

<211> 20

<212> DNA

< 213>artificial sequence

<400> 127

gtccccggac?gatattgaac 20

<210> 128

<211> 19

<212> DNA

< 213>artificial sequence

<400> 128

agaagcccag?acggaaacc 19

<210> 129

<211> 19

<212> DNA

< 213>artificial sequence

<400> 129

aaggaagggt?cccgctctg 19

<210> 130

<211> 22

<212> DNA

< 213>artificial sequence

<400> 130

gtattccgtc?tgcaaaactg?gc 22

<210> 131

<211> 18

<212> DNA

< 213>artificial sequence

<400> 131

gggctcacag?ctccatcc 18

<210> 132

<211> 22

<212> DNA

< 213>artificial sequence

<400> 132

acaatttttc?ctctcctgca?tc 22

<210> 133

<211> 20

<212> DNA

< 213>artificial sequence

<400> 133

ggcggcattg?acactagctg 20

<210> 134

<211> 20

<212> DNA

< 213>artificial sequence

<400> 134

tgagcaccac?atccccagag 20

Claims

1. a method that detects gene order is characterized in that, may further comprise the steps:

C. utilize restriction endonuclease with having obtained the nucleotide segment of sequence information or excision fully among the step B, obtain having and contain the segmental enzyme of waiting to check order and cut product;

2. the method for detection gene order according to claim 1 is characterized in that, first anchor primer described in the steps A has and contains at least one enzyme and cut recognition site.

3. the method for detection gene order according to claim 2 is characterized in that, said step C may further comprise the steps:

4. the method for detection gene order according to claim 1 is characterized in that, step C may further comprise the steps:

C1 '. connect the 3rd double-stranded joint at the other end of first anchor primer, the 3rd joint has and contains at least one enzyme and cut recognition site;

5. according to the method for each described detection gene order in the claim 1 to 4, it is characterized in that said first anchor primer has and contains at least one specificity residue and/or an end seals.

6. the method for detection gene order according to claim 5 is characterized in that, said step B may further comprise the steps:

B1. at the fluorescent probe that has the first anchor primer extending end connecting band specific position mark that contains the specificity residue, detect the corresponding fluorescent signal that connects product, obtain the nucleotide sequence information of correspondence position;

7. the method for detection gene order according to claim 1 is characterized in that, the determined nucleic acid fragment that contains gene order described in the steps A is fixed in surface of solid phase carriers.

8. the method for detection gene order according to claim 7 is characterized in that, before steps A, also comprises step:

9. the method for detection gene order according to claim 8 is characterized in that, said steps A 0 may further comprise the steps:

The nucleic acid fragment that contains gene order that A01. will be used to increase is fixed in surface of solid phase carriers, obtains the surface and has and contain the solid phase carrier that at least one contains the nucleic acid fragment of gene order;

10. the method for detection gene order according to claim 9; It is characterized in that; Primer described in the steps A 02 comprises and is used for upstream primer and/or downstream primer that the said determined nucleic acid fragment that contains gene order is increased; Said upstream primer is and the complementary bonded nucleotide sequence of the determined nucleic acid fragment that contains gene order 5 ' end that said downstream primer is and the identical nucleotide sequence of determined nucleic acid fragment 3 ' terminal sequence that contains gene order.

11. the method for detection gene order according to claim 9 is characterized in that, the amplification described in the steps A 03 is the unit molecule amplification.

12. the method for detection gene order according to claim 9 is characterized in that, primer is incorporated into the mode of surface of solid phase carriers and is in the said steps A 02:

Primer matches with the group that surface of solid phase carriers carries and is connected, and realizes directly combining;

Or the group that carries through connexon matches with group that primer and surface of solid phase carriers carry respectively and is connected, and realizes combining indirectly.

13. the method for detection gene order according to claim 12; It is characterized in that said pairing ways of connecting adopts at least a in biotin-avidin/Streptavidin, nanometer gold/iodacetyl-sulfydryl, amino-aldehyde radical/carboxyl/isothiocyano, the acrylic amide-siloyl group/SEPIGEL 305.

14. method according to each described detection gene order in the claim 1 to 4,7 to 13; It is characterized in that; Said allelotrope comprises at least one among F8, F9, FGFR3, IDS, GALT, HBB, HBA1, HBA2, ATP7B, PHEX, GJB2, COL4A5, the LMNA, shown in the antitumor drug genes involved comprise at least one among CYP2C9, VKORC1, KIT, PDGFRA, CYP2C19, DHFR, GSTM1, MTHFR, RFC1, CYP19A1, UGT1A1, UGT1A7, UGT1A9, ABCB1, CYP2D6, CYP3A5, SULT1A1, UGT2B15, GSTA1, SOD2, CYP2B6, GSTP1, MDR1, BCRP, p53, CAT, CBR1, CBR3, EGFR, KRAS, the BRAF.