CN101065483B - Method of identifying nucleotide polymorphisms - Google Patents
Method of identifying nucleotide polymorphisms Download PDFInfo
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- CN101065483B CN101065483B CN200580040391.9A CN200580040391A CN101065483B CN 101065483 B CN101065483 B CN 101065483B CN 200580040391 A CN200580040391 A CN 200580040391A CN 101065483 B CN101065483 B CN 101065483B
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
Abstract
The invention provides a method of analyzing nucleic acid sequences which is excellent in easily and simultaneously detecting multiple types of nucleotide polymorphisms contained in a sample. Multiple types of nucleotide polymorphisms contained in a sample are simultaneously identified by hybridizing the sample having multiple types of nucleotide polymorphisms to be measured with multiple types of first oligonucleotides and multiple types of second oligonucleotides, linking the first oligonucleotides with the second oligonucleotides by using an enzyme having a nuclease activity to form linked oligonucleotides, and identifying the types of these linked oligonucleotides by using detection probes having sequences complementary to the linked oligonucleotides.
Description
Technical field
The present invention relates to the analytical procedure of nucleotide sequence.More detailed theory relates to the nucleic acid sequence analysis method of the multiple nucleotide polymorphisms that contains in the test sample simultaneously.Nucleic acid sequence analysis method of the present invention is applicable to genetically engineered or molecular biology, and relevant industrial field therewith.
Background technology
Among the present invention, so-called nucleotide polymorphisms refers to have and the not homotactic gene form of wild-type, Genetic polymorphism in drug metabolism side effect and owing to play an important role in the treatment failure that individual difference causes.Known in addition also is reason as the individual difference of basal metabolism known to the physique etc.And these work with a plurality of ill genetic markers.Therefore, find out that these sudden changes are important clinically, in clinical study, conventional phenotype classification is subject to praising highly (for example referring to non-patent literature 1 and 2) for mental patient and spontaneous volunteer especially.For this reason, be accompanied by the evaluation as the Genetic polymorphism of its root, just become a kind of needs for detection of the nucleic acid sequence analysis method of range gene type.
Nucleic acid sequence analysis method is in the past determined method (Sequencing method) just like nucleotide sequence.Nucleotide sequence determines that method can detect the nucleotide polymorphisms that is included in the nucleotide sequence, and identified, but carry out preparation, polymerase reaction, the polyacrylamide gel electrophoresis of template nucleic acid, the operations such as parsing of nucleotide sequence, essential a lot of labour and time.And the automatic sequence analyser that uses in recent years can be saved the labour, but the expensive high such problem of equipment of needs is arranged.
Additive method also has Southern hybrid method (for example referring to non-patent literature 3).If adopt this method, can identify the DNA zone that has with the base sequence of labeled DNA probe complementation.When namely adopting the Southern hybrid method, nucleic acid fragment is carried out electrophoresis at the flat board of agarose or polyacrylamide gel, after clip size (length) separation, make its sex change become strand, again the films such as soluble cotton are tiled on this flat board, nucleic acid fragment is shifted with the electrophoresis former state, after fixing, with with the marks such as RI (radio isotope) the specific dna probe of having of nucleotide polymorphisms is formed crossbred, then detect on the film nucleic acid fragment with the probe complementation with methods such as radioactive automatic developings.
If adopt the Southern hybrid method, can determine electrophoresis position and the molecular weight of purpose nucleic acid fragment, but have following problem: in the operations such as electrophoresis or radioactive automatic developing, need to expend for a long time, can not analyze fast; Because measure and whether only to depend on and the specific dna probe of having of nucleotide polymorphisms is formed crossbred, so the specificity of probe and imprecision, be difficult to identify the similar sequence for detection of having or not nucleotide polymorphisms.
In addition, as utilizing enzyme to identify the method for nucleotide sequence, the method for known employing ligase enzyme (such as referring to patent documentation 1 and 2), adopt the method (such as referring to patent documentation 3) of nuclease etc.In as the known oligonucleotide ligation assay of method (OLA) that adopts ligase enzyme, cross over two probes in institute definite purpose zone or probe member and hybridize in that purpose is regional.Probe member is in case form base pair with the purpose base of adjacency, and the relative end of probe member is by ligation, for example with the ligase enzyme processing, can in conjunction with.Detect the probe member that connects, can understand the existence of aim sequence.
Demand degree for the method that whether has separately (for example in the gene screening field) for detection of a plurality of sequences in the goal gene increases in recent years.For example there is the variation that reaches 400 kinds relevant with the cyst cystic fibrosis.In the examination of carrying out for the gene corresponding to this disease is carried out pre-treatment, for more effective evaluation " cyst cystic fibrosis ", preferably check whole contingent aberrant gene sequence variations in the people's to be detected genomic dna.Desirable detection be one-time detection go out the position that might morph existence whether.
The method of the multiple nucleotide polymorphisms of this parsing is that the oligonucleotide probe of multiple detection usefulness is combined on the solid phase, adopts to be called dna microarray (DNA chip) as the hybridization analysis method of solid phase.But, in the hybridization analysis method of solid phase, must adopt repeatedly the liquid treatment operation, in order to preserve for the necessary preciseness of the single Nucleotide mispairing of identification, must prudent insulation and the wash temperature of controlling each step.Because the suitableeest hybridization conditions alters a great deal because probe sequence is different, the complicated process of this method is its difficult point.
For this problem, proposed to use tens of oligonucleotide through dna microarray for a kind of polymorphic sequence, thereby resolved the method (for example referring to patent documentation 4) of polymorphism.If adopt this method, then adopt a kind of form according to various polymorphisms and the array of special one-tenth imbricate (tiled) probe is analyzed.Imbrication mode (tiling) can be used one group of relevant immobilization probe, and wherein several probe performances are to the characteristic of canonical sequence complete complementary, other then performance and canonical sequence generation mispairing (for example referring to patent documentation 5).But this method be owing to will use tens of oligonucleotide with respect to a kind of polymorphism, thereby is very expensive method.
A kind of polymorphism of known promising detection Cytochrome P450 and adopt the technology of 240 probes, this method must be made many probes, costs a lot of money.In addition, identify in this way multiple polymorphism, just must use more multiprobe, beyond thought non-specific responding appears in difficult guarantor.In order to reduce non-specific responding, just must design tight probe, this just has to study huge permutation and combination.
For multiple polymorphism, also disclose and utilized ligase enzyme, the oligonucleotide special to polymorphism in the connection carries out method for measuring (for example referring to patent documentation 6) with dna microarray again.Although the oligonucleotide in this method on the dna microarray can reduce, owing to only connect oligonucleotide with ligase enzyme, therefore must use combine endways the phosphate oligonucleotide, expense is higher, and phosphate is when being connected with oligonucleotide, do not know actually and which oligonucleotide generation ligation, non-specific connection occurs easily.Therefore when being specifically designed to the parsing polymorphism, polymorphism is more, and is dangerous just higher.
Patent documentation 1 Japanese JP 6-44880 number
No. the 2622255th, patent documentation 2 Japanese Patents
Patent documentation 3 Japanese kokai publication hei 3-43098 numbers
Patent documentation 4 TOHKEMY 2002-514091 numbers
Patent documentation 5EP730663 number
Patent documentation 6 TOHKEMY 2000-511060 numbers
Non-patent literature 1 European Consensus Conference onPharmacogenetics .Commission of the EuropeanCommunities, Luxembourg 87-96 page or leaf, (nineteen ninety distribution)
Non-patent literature 2 European Journal of Clinical Pharmacology, the 36th volume, 551-554 page or leaf, (distribution in 1989)
Non-patent literature 3 laboratory manuals, genetically engineered, [Dan, the 70th～75 page, (distribution in 1996)
The simple declaration of accompanying drawing
Fig. 1 is the figure of the part example of expression reaction mechanism of the present invention.
Summary of the invention
Invent problem to be solved
In the technology in the past, in order to resolve the polymorphism of base, all have the essential high price instrument that uses, the specificity of probe is essential rigorous, the problem that the manipulation require time is long.In addition, resolve at the same time in the situation of multiple nucleotide polymorphisms, also have the necessary significant care of operation, the problems such as non-specific ligation can occur.Therefore, purpose of the present invention is to provide a kind of superior nucleic acid sequence analysis method, in order to easily detect simultaneously the kind that is contained in the multiple nucleotide polymorphisms in the sample.
The means that adopt for dealing with problems
Because collective of the present invention studies intensively, adopt the following stated means, solved above-mentioned problem, realized the present invention.Namely the present invention relates to identify simultaneously the method for multiple nucleotide polymorphisms.
1. the authentication method of a plurality of nucleotide polymorphisms, to identify simultaneously the method that is contained in the multiple nucleotide polymorphisms in the sample, it is characterized in that, sample with multiple nucleotide polymorphisms kind to be measured, hybridize with multiple the first oligonucleotide and multiple the second oligonucleotide, with the enzyme that contains nuclease and the enzyme that contains the ligase enzyme activity the first oligonucleotide is connected with the second oligonucleotide, make its oligonucleotide that form to connect, adopt the kind of identifying the oligonucleotide of this connection with the detection that oligonucleotide after this is connected has a complementary sequence with probe.
2. the authentication method of above-mentioned 1 described a plurality of nucleotide polymorphisms is characterized in that, contain with the first oligonucleotide in the sequence of nucleotide polymorphisms Sequence complementation.
3. the authentication method of above-mentioned 1 described a plurality of nucleotide polymorphisms is characterized in that, contain with the second oligonucleotide in infer the sequence that Sequence that nucleotide polymorphisms is arranged is not complementary.
4. the authentication method of each described a plurality of nucleotide polymorphisms in above-mentioned 1～3 is characterized in that, contains 5 ' the terminal sequence of inferring that Sequence that nucleotide polymorphisms is arranged is not complementary with the second oligonucleotide.
5. the authentication method of each described a plurality of nucleotide polymorphisms in above-mentioned 1～3, it is characterized in that, with the first oligonucleotide and the second oligonucleotide when containing the purpose nucleic acid of inferring the Sequence that nucleotide polymorphisms is arranged and contact, have in the Sequence of nucleotide polymorphisms in supposition, be designed to have at least more than one base overlapping.
6. the authentication method of each described a plurality of nucleotide polymorphisms in above-mentioned 1～3 is characterized in that the first oligonucleotide is labeled in advance.
7. the authentication method of above-mentioned 6 described a plurality of nucleotide polymorphisms, it is characterized in that any method that mark is selected from a group material that comprises enzyme, vitamin H, fluorescent substance, heparin, antigen, antibody, radioactive substance, luminophore and specific nucleotide sequence.
8. above-mentioned 6 or the authentication method of 7 described a plurality of nucleotide polymorphisms, it is characterized in that, carry out mark with the fluorescent substance with different wavelength of fluorescence for detection of the first oligonucleotide of wild-type respectively with the first oligonucleotide for detection of nucleotide polymorphisms.
9. the authentication method of each described a plurality of nucleotide polymorphisms in above-mentioned 1～3 is characterized in that, take the method that detects the oligonucleotide that connects with probe in detecting as hybridization.
10. the authentication method of each described a plurality of nucleotide polymorphisms in above-mentioned 1～3 is characterized in that, detects with probe to contain complementary with the part of the second oligonucleotide at least sequence.
11. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, detects with probe to contain complementary with the part of the first oligonucleotide and the second oligonucleotide at least sequence.
12. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, detects a part that contains polymorphic sequence with probe.
13. the authentication method of above-mentioned 12 described a plurality of nucleotide polymorphisms is characterized in that, the polymorphic sequence that detects with probe partly is to mix base.
14. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, has more than one detection to be fixed on the solid phase with probe.
15. the authentication method of above-mentioned 14 described multiple nucleotide polymorphisms is characterized in that, being fixed with the solid phase that detects with probe is dna microarray.
16. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, detects make the oligonucleotide amplification of connection by amplified reaction after.
17. the authentication method of above-mentioned 16 described a plurality of nucleotide polymorphisms is characterized in that, amplification method is any selected from a group method that comprises PCR, NASBA, LCR, SDA, RCR and TMA method.
18. the authentication method of each described multiple nucleotide polymorphisms is characterized in that in above-mentioned 1～3, sample is the nucleic acid that increases in advance.
19. the authentication method of above-mentioned 18 described a plurality of nucleotide polymorphisms is characterized in that amplification method is any selected from a group method that comprises PCR, NASBA, LCR, SDA, RCR and TMA method.
20. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, adopts in turn and/or simultaneously the enzyme that contains nuclease and the enzyme that contains the ligase enzyme activity.
21. the authentication method of each described multiple nucleotide polymorphisms is characterized in that in above-mentioned 1～3, the enzyme that contains nuclease is the same enzyme with the enzyme that contains the ligase enzyme activity.
22. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, the enzyme that contains nuclease is at least a enzyme selected from a group enzyme that comprises mung-bean nuclease, S1 nuclease, exonuclease I～VII.
23. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, the enzyme that contains the ligase enzyme activity is at least a enzyme selected from a group enzyme that comprises T4DNA ligase enzyme, Ecoli dna ligase, RNA ligase enzyme.
24. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, the enzyme that contains nuclease is the thermotolerance enzyme with the enzyme that contains the ligase enzyme activity.
25. the authentication method of above-mentioned 24 described a plurality of nucleotide polymorphisms is characterized in that, the thermotolerance enzyme is from Tth, Taq, KOD or Pfu.
26. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, the enzyme that contains nuclease is archaeal dna polymerase.
27. the authentication method of above-mentioned 26 described multiple nucleotide polymorphisms, it is characterized in that archaeal dna polymerase be from comprise Taq DNA polymerase from Tth, from the Taq DNA polymerase of Taq, from the Taq DNA polymerase of KOD with from selected one kind or two or more archaeal dna polymerase a group enzyme of the Taq DNA polymerase of Pfu
28. the authentication method of each described multiple nucleotide polymorphisms in above-mentioned 1～3, nucleotide polymorphisms comprise polymorphism, insertion, deletion polymorphism any of a base.
29. the authentication method of a plurality of nucleotide polymorphisms is to identify simultaneously the method that is contained in the multiple nucleotide polymorphisms in the sample, its feature is as follows:
(1) preparation contains multiple the first oligonucleotide of this nucleotide polymorphisms Sequence, and preparation contains multiple the second oligonucleotide of sequence adjacent with this first oligonucleotide and that do not hybridize with this nucleotide polymorphisms Sequence;
(2) this first oligonucleotide and this second oligonucleotide are hybridized with the karyomit(e) or the nucleic acid fragment that are contained in the sample;
(3) when hybridization has occured in various oligonucleotide, remove the nucleotide polymorphisms Sequence does not form base pair at this second oligonucleotide base sequence with the enzyme that contains nuclease, then with the enzyme effect that contains the ligase enzyme activity, this oligonucleotide is connected, forms the oligonucleotide that connects;
(4) adopt to contain at least to detect with the complementary multiple base of the part of this second oligonucleotide and use probe, be connected the complementary multiple base of a part with the first oligonucleotide with this second oligonucleotide and detect with probe in detecting and be somebody's turn to do the oligonucleotide that be connected or/and contain at least.
30. a test kit is to identify simultaneously the test kit that is contained in the multiple nucleotide polymorphisms in the sample, it is characterized in that comprising at least following (i)～(v):
(i) the two or more the first oligonucleotide that comprise the nucleotide polymorphisms Sequence;
(ii) the two or more the second oligonucleotide that comprise sequence adjacent with the first oligonucleotide and that do not hybridize with this nucleotide polymorphisms Sequence;
(iii) at least a enzyme that contains nuclease;
(iv) at least a enzyme that contains the ligase enzyme activity;
(v) complementary with the part of the second oligonucleotide at least detection probe, and/or complementary with the part of the second oligonucleotide and the first oligonucleotide at least detection probe.
31. a test kit is to identify simultaneously the test kit that is contained in the multiple nucleotide polymorphisms in the sample, it is characterized in that comprising at least following (i)～(vi):
(i) have in the Sequence of nucleotide polymorphisms in supposition, contain the two or more polymorphism the first oligonucleotide with the sequence of the base complementrity that shows polymorphism;
(ii) have in the Sequence of nucleotide polymorphisms in supposition, contain the two or more wild-type the second oligonucleotide with the sequence of the base complementrity that shows wild-type;
(iii) two or morely contain with polymorphism and/or wild-type the first oligonucleotide is adjacent and with this supposition the second oligonucleotide of the sequence that the nucleotide polymorphisms Sequence do not hybridize is arranged;
(iv) at least a enzyme that contains nuclease;
(v) at least a enzyme that contains the ligase enzyme activity;
(vi) complementary with the part of the second oligonucleotide at least detection probe, and/or complementary with the part of the second oligonucleotide and the first oligonucleotide at least detection probe.
32. above-mentioned 30 or 31 described test kits contain to be fixed with in advance and detect the solid phase of using probe.
33. above-mentioned 30 or 31 described test kits is characterized in that, solid phase is dna microarray.
Effect of the present invention
By adopting in turn and/or simultaneously the enzyme that contains nuclease and the enzyme that contains the ligase enzyme activity, with the special oligonucleotide that is connected of nucleotide polymorphisms, the specific detection gene can detect multiple nucleotide polymorphisms expediently.
The best mode that carries out an invention
Below describe the present invention in detail.Contained containing has karyomit(e) or its fragment at specific nucleotide polymorphisms position in the sample, gets final product so long as comprising the purpose nucleic acid with nucleotide polymorphisms that carries the gene information that will detect, and is not particularly limited.This purpose nucleic acid, gene extron or intron, the promotor etc. that can lift Alu sequence, coded protein are example.More specifically, can lift with the various diseases that comprises inherited disease and the gene relevant with living habit disease (hypertension, diabetes etc.) with drug metabolism is example.The ACE gene relevant with hypertension for example.
Among the present invention, the polymorphism nucleic acid of so-called karyomit(e) or nucleic acid fragment, refer to have at least in the wild-type nucleic acid Nucleotide point mutation to occur and by the nucleic acid of other nucleotide subsitution, perhaps in the part of wild-type nucleic acid, contained the nucleic acid of insertion sequence or deletion sequence.
Can illustrate physique difference by this nucleotide polymorphisms, method of the present invention is the alkali the polymorphism whether nucleic acid in the test sample exists supposition.
The method of the detection nucleotide polymorphisms among the present invention is to adopt in turn and/or simultaneously the enzyme that contains nuclease and to contain the enzyme of ligase enzyme activity as feature.This activity can be passed through single enzyme, also can realize by the enzyme that possesses simultaneously two kinds of activity.
Particularly, the enzyme that contains nuclease can be lifted mung-bean nuclease, S1 nuclease, exonuclease I～VII etc.The enzyme that contains the ligase enzyme activity can be lifted T4DNA ligase enzyme, e. coli dna ligase, RNA ligase enzyme etc.The enzyme that contains the enzyme of nuclease and contain the ligase enzyme activity because of be the thermotolerance enzyme preferably.For example anti-Hot enzyme can be the enzyme from Tth, Taq, KOD, Pfu.And the enzyme that contains nuclease also can be archaeal dna polymerase.For example can use the Taq DNA polymerase from Tth, Taq, KOD, Pfu.
The specific form of detection nucleotide polymorphisms method of the present invention is to identify the multiple nucleotide polymorphisms method in the sample that is contained in:
(1) preparation comprises the first oligonucleotide of the Sequence of nucleotide polymorphisms, by complementary with the nucleic acid of the chain of this first oligonucleotide hybridization, the first oligonucleotide is adjacent hybridizes with this, and preparation contains the second oligonucleotide that does not form the sequence of complementary strand with any sequence of this nucleotide polymorphisms Sequence.
(2) this first oligonucleotide and the second oligonucleotide and nucleic acid are hybridized
(3) in the nucleotide polymorphisms Sequence that when oligonucleotide has carried out hybridization separately, has produced, remove the base that does not form the base pair that the second oligonucleotide has with the enzyme that contains nuclease.At this moment, residual have the base of phosphate to be removed.
That is, the effect of the enzyme by containing nuclease begins to produce phosphate, and the effect of the enzyme by containing the ligase enzyme activity couples together the first oligonucleotide and the second oligonucleotide.By detecting the oligonucleotide of this connection, identify the method for the nucleotide polymorphisms in the specific nucleic acid sequence that is contained in the sample.
Particularly, the first oligonucleotide is to contain the oligonucleotide of base that the Sequence complementation of nucleotide polymorphisms is arranged with supposition.The supposition part of this nucleotide polymorphisms is 3 ' end of the first Nucleotide preferably.
The second oligonucleotide is more prone to the position of 3 ' one side than the first oligonucleotide.The second oligonucleotide is characterised in that it better is to contain the oligonucleotide that the not complementary base of the Sequence of nucleotide polymorphisms is arranged with supposition.Preferably there is the not complementary base of the Sequence of nucleotide polymorphisms to be present in 5 ' end of the second oligonucleotide with supposition.
Now the method for design of the first oligonucleotide and the second oligonucleotide is described in more detail.Inferring when in the purpose nucleic acid that the nucleotide polymorphisms Sequence is arranged the second oligonucleotide being contacted with the first oligonucleotide containing, better is to be designed to have at least more than one base to overlap in supposition has the Sequence of nucleotide polymorphisms.In order to make it overlapping, can infer have the Sequence of nucleotide polymorphisms to hybridize to 3 ' tip designs Cheng Nengyu of the first oligonucleotide; And 5 ' tip designs of the second oligonucleotide is become can not have the Sequence of nucleotide polymorphisms to hybridize with supposition.At 5 ' end of the second oligonucleotide, can not there be the Sequence of nucleotide polymorphisms that the length of the structure of hybridization occurs with supposition, have at least a base to get final product.This is at 5 ' end of the second oligonucleotide, when removing with nuclease can not have the nucleotide polymorphisms Sequence that the base of hybridization occurs with supposition the time, be designed to by the enzyme that contains the ligase enzyme activity the first oligonucleotide and the second oligonucleotide to be coupled together get final product.
The length of the first oligonucleotide and the second oligonucleotide among the present invention is 13～35 bases, preferably 16～30 bases.For example, 3 ' terminal bases of the first oligonucleotide is designed to infer the oligonucleotide that the nucleotide polymorphisms position is arranged, the first oligonucleotide of wild-type then disposes the base with this base complementrity of this wild-type nucleic acid, simultaneously, the first oligonucleotide of polymorphism then disposes the base with this base complementrity of polymorphism nucleic acid.And again 5 ' tip designs of the second oligonucleotide is become when with the first oligonucleotide a base overlaid being arranged, the overlapping base of this second oligonucleotide is then selected not complementary base, no matter be wild-type or polymorphism.By selection and wild-type or polymorphism not complementary base, when detecting a kind of nucleotide polymorphisms, can only prepare a kind of the second oligonucleotide.
When having carried out such design, only in the combination of the first oligonucleotide of wild-type/the second oligonucleotide/wild-type nucleic acid and polymorphism the first Nucleotide/the second oligonucleotide/polymorphism nucleic acid, the lap of the second oligonucleotide is removed, because in full accord, the overlapping base that can remove the second oligonucleotide with the enzyme that contains nuclease.This moment can be by acting on it with the enzyme that contains the ligase enzyme activity in order to remove residual phosphate, and the first oligonucleotide is connected with the second oligonucleotide becomes a chain; And in the combination of the first oligonucleotide of wild-type/the second oligonucleotide/polymorphism nucleic acid and polymorphism the first Nucleotide/the second oligonucleotide/wild-type nucleic acid, 3 ' end of the first oligonucleotide can not couple together with the enzyme effect that contains the ligase enzyme activity owing to can not be complementary.
Contain the enzyme of nuclease and contain the enzyme of ligase enzyme activity
Among the present invention, make simultaneously it act on above-mentioned wild-type the first oligonucleotide and polymorphism the first oligonucleotide.
I) make above-mentioned the first oligonucleotide and the second oligonucleotide with contain nucleotide polymorphisms karyomit(e) or nucleic acid fragment hybridize.Hybridization conditions can be general conditions.For example can carry out according to the described method of " molecular cloning " (version in 1989).
Ii) then cut off with the lap of the enzyme that contains nuclease with the second oligonucleotide.Operable enzymic activity as mentioned above.Exonuclease activity preferably, the also exonuclease such as archaeal dna polymerase preferably.
In the situation that 5 ' end and the first oligonucleotide of the second oligonucleotide overlaps, adopt 5 ' exonuclease, with the lap cut-out of the second oligonucleotide, and expose phosphate.
Iii) simultaneously, with the enzyme that contains the ligase enzyme activity, the phosphate that will be exposed to the second oligonucleotide 5 ' end is connected with 3 ' end of the first oligonucleotide, becomes an oligonucleotide.
In the present invention, if not complementary corresponding to the nucleotide polymorphisms position in the first oligonucleotide, it is not complementary corresponding to the position of nucleotide polymorphisms namely to use endonuclease to cut off yet, so use the ligase enzyme active function, can not connect into a Nucleotide.
When to having used wild-type the first oligonucleotide sample nucleic acid to adopt to contain the enzyme of nuclease and contained in the situation of enzyme of ligase enzyme activity, sample nucleic acid is if wild-type then forms a chain, if polymorphism, then do not react.On the contrary, when to having used polymorphism the first oligonucleotide test sample nucleic acid to adopt to contain the enzyme of nuclease and contained in the situation of enzyme of ligase enzyme activity, sample nucleic acid is if polymorphism then forms a chain, if wild-type, then do not react.The higher organism take the mankind as representative particularly, a kind of gene have respectively from each one of father's gene and mother's gene, if adopt this method, can distinguish sample gene is that wild-type homology or polymorphism are same, or allos.
The present invention also provides a kind of method that makes the enzyme that contains nuclease and the enzyme that contains the ligase enzyme activity act on multiple nucleotide polymorphisms to be detected in same reaction system.Multiple among the present invention means to detect at least a or two above nucleotide polymorphisms.When identifying multiple nucleotide polymorphisms, also can adopt the enzyme effect that in different reaction systems, makes the enzyme that contains nuclease and contain the ligase enzyme activity, but in same reaction system, then more easy, also more be conducive to reduce expenses.The enzyme that contains nuclease and the enzyme that contains the ligase enzyme activity are acted in the situation of multiple nucleotide polymorphisms simultaneously, be used for wild-type and be preferably different marks with the first oligonucleotide that is used for polymorphism, for example add the fluorescent mark that to be distinguished with wavelength of fluorescence.It is different with the fluorescent mark of the first oligonucleotide that is used for polymorphism that mark is used for wild-type, when identifying multiple nucleotide polymorphisms, even the first oligonucleotide and the second oligonucleotide mix, also can not make troubles at least.
In the inadequate situation of purpose nucleic acid content to be detected, before above-mentioned hybridization, can pass through in advance following amplified reaction, the above-mentioned nucleic acid fragment that contains polymorphic sequence is increased.Above-mentioned amplification method is not particularly limited, such as PCR (Polymerase chain reaction; Japan JP 4-67960 communique, Japanese JP 4-67957 communique), NASBA (Nucleic acid sequence-based amplification method; Nature the 350th volume, the 91st page (1991)), LCR (WO89/12696, Japanese kokai publication hei 2-2934 number etc.), SDA (Strand Displacment Amplification; Nucleic Acids Res. the 20th volume, the 691st page (1992)), RCR (WO90/01069), TMA (Transcription Mediatedamplification Method; J.Clin.Microbiol. the 31st volume, the 3270th page (1993)) etc. can adopt.
In addition, make the enzyme that contains nuclease and contain the enzyme effect of ligase enzyme activity, and after oligonucleotide connected, also can increasing with amplified reaction shown in above-mentioned, it detects in conjunction with product.3 ' end side of 5 ' end side of the first oligonucleotide and the second oligonucleotide can add the sequence of undertaking the promotor effect for amplified reaction at this moment.
Detect
Adopt to detect and to use probe, detect by the oligonucleotide of the following stated to the connection that obtains by above-mentioned reaction.
The first oligonucleotide preferably adds mark in advance.In this case, can when synthetic, import the Nucleotide that vitamin H is arranged, synthesize linking agent, fluorescent substance etc., or add the modification groups such as oligomerization dGTP, oligomerization dATP, oligomerization dTTP, oligomerization dCTP at 5 ' end.Perhaps can synthesize by the Nucleotide of replacing in the oligonucleotide sequence with the Nucleotide that vitamin H, synthetic linking agent, fluorescent substance etc. are arranged, to import modification group.Can also be behind synthesizing ribonucleotide, to wherein importing
32P,
35The marker of these known oligonucleotide of fluorescent substance such as the enzymes such as the radioactive substances such as S, ALP, POD, FITC, HEX, 6-FAM, TET.Can also be at wild-type the first oligonucleotide oligonucleotide sequence different from difference affix in nucleotide polymorphisms the first oligonucleotide.
It is as follows to attempt the concrete grammar example: make the specific nucleic acid that adopted the second oligonucleotide and hybridize through the first oligonucleotide of the marks such as fluorescent substance, after using in turn and/or simultaneously the enzyme that contains ligase enzyme and containing the enzyme effect of nuclease activity, make it to dissociate, again with in conjunction with the detection probe special to polymorphism on the solid phase again, and to wild-type special detection is hybrid with it with probe, wash out the not nucleic acid of hybridization, whether hybridization has occured according to the marker detection with the first oligonucleotide of the nucleic acid of various detections after with probe hybridization, detect detection signal ratio with probe by each again, identify whether wild-type of nucleotide polymorphisms, polymorphism or mixed state type.
With the first oligonucleotide that is used for polymorphism not isolabeling is arranged owing to be used for wild-type, for example add the fluorescent mark of different colours, can adopt the detection that mixes base in the nucleotide polymorphisms sequence, to carry out the detection of wild-type and polymorphism with probe with a kind of.
Be used for wild-type and be attached with different marks on the first oligonucleotide of polymorphism, for example can distinguish the fluorescent mark of wavelength of fluorescence, can be at least a reactive system, the enzyme that makes simultaneously the enzyme that contains nuclease and contain the ligase enzyme activity acts on multiple nucleotide polymorphisms.At this moment, if mark is different for the fluorescent mark on the first oligonucleotide of wild-type and polymorphism at least, during then for the identification of multiple nucleotide polymorphisms, exist the first oligonucleotide and the second oligonucleotide also not to have problem even mix.Mark is used for wild-type and is used for the fluorescent mark of the first oligonucleotide of polymorphism, as long as can distinguish wavelength of fluorescence, adopts generation different colours fluorochrome then better.This point is not particularly limited, for example can be at 5 ' end mark Cy3 of the first oligonucleotide that is used for wild-type, and 5 ' end mark Cy5 at the first oligonucleotide that is used for polymorphism also can make opposite mark.
With figure a part of example of reaction mechanism of the present invention can be described, but the present invention is not limited by this figure.Among Fig. 1, expression is take purpose nucleic acid as polymorphism nucleic acid, adopts the first oligonucleotide of 5 ' terminal wild-type of having modified with Cy3 and the first oligonucleotide of 5 ' terminal polymorphism of having modified with Cy5.
In the situation of this example, the 5 ' end of setting in the second oligonucleotide is " supposition has the nucleotide polymorphisms Sequence ", sets simultaneously the base not complementary with " supposition has the nucleotide polymorphisms Sequence ".In addition, in the case, 5 ' the terminal base that can also be positioned at the second oligonucleotide also can be set as when purpose nucleic acid is wild-type also not and this base complementrity.Simultaneously, in this case, the G of 3 ' terminal A and 5 ' end of the second oligonucleotide that also is designed to the first oligonucleotide of polymorphism overlaps.The G of 3 ' terminal C and 5 ' end of the second oligonucleotide that also can be designed to the first oligonucleotide of wild-type overlaps.
The first oligonucleotide of polymorphism and the situation that the second oligonucleotide is had an effect in (1) expression polymorphism nucleic acid of Fig. 1.In this case, 3 ' terminal A of the first oligonucleotide of polymorphism is hybridized at the polymorphism position, on the position that is adjacent, hybridizes from second T that 5 ' end of the second oligonucleotide is counted.And the G of 5 ' end of the second oligonucleotide just is in the state that does not occur to hybridize owing to not complementary with the polymorphism position.Use the enzyme effect that contains nuclease this moment, then 5 ' of the second oligonucleotide terminal G is cut off, and generates simultaneously phosphate.Act on this with the enzyme that contains the ligase enzyme activity, then two oligonucleotide connect.
The first oligonucleotide of wild-type and the situation that the second oligonucleotide is had an effect in (2) expression polymorphism nucleic acid of Fig. 1.In this case, therefore 3 ' end of the first oligonucleotide of wild-type does not hybridize owing to not complementary with the polymorphism position.And the G of 5 ' end of the second oligonucleotide is in the state that does not occur to hybridize also owing to not complementary with the polymorphism position.The enzyme that contains nuclease this moment can not cut off the G of 5 ' end of the second oligonucleotide.Therefore the enzyme that contains the ligase enzyme activity can not work, and two oligonucleotide do not connect yet.
Do not represent on the figure that purpose nucleic acid is the situation of wild-type nucleic acid, the result who represents with Fig. 1 is opposite, and the first oligonucleotide of polymorphism is not connected with the second oligonucleotide, and wild-type the first oligonucleotide is connected with the second oligonucleotide.
In situation shown in Figure 1, when purpose nucleic acid during only with polymorphism nucleic acid homology, can have to the first oligonucleotide of polymorphism and the connector of the second oligonucleotide; When purpose nucleic acid during only with the wild-type nucleic acid homology, then have to the first oligonucleotide of wild-type and the connector of the second oligonucleotide; When purpose nucleic acid is the polymorphism nucleic acid of allos and wild-type nucleic acid, then obtain ratio near the mixture of two kinds of connectors of 1: 1.
Problem for convenience of explanation, Fig. 1 is illustrated a kind of situation of nucleotide polymorphisms, and the present application provides measuring method at least a or nucleotide polymorphisms more than 2 kinds.Namely, if mark is different for the fluorescent mark with the first oligonucleotide that is used for polymorphism of wild-type at least, to mix with the second oligonucleotide for the identification of the first oligonucleotide of multiple nucleotide polymorphisms, in same reactive system, make the enzyme that contains nuclease and the enzyme that contains the ligase enzyme activity act on them.Like this, when same reactive system carries out relevant multiple nucleotide polymorphisms detection, can detect as follows.
Detect multiple nucleotide polymorphisms, for detection of the multiple detection probe of these nucleotide polymorphisms, can adopt the solid phase of every kind of each self-retaining of probe and the method that do not interfere with each other.To act on the resultant of reaction that multiple nucleotide polymorphisms produces with the enzyme that contains nuclease and the enzyme that contains the ligase enzyme activity simultaneously in order being determined in few extremely a kind of reactive system, can to adopt and fix the dna microarray that oligonucleotide is used in above-mentioned detection.Consider from the aspect of cost and ease-to-operate, adopt dna microarray that its advantage is also arranged.
The invention provides facility and spend the detection system of utilizing dna microarray that also possesses some good points.In order to prepare this dna microarray, now be illustrated with regard to the method for design that detects with probe.The nucleotide polymorphisms part that probe of the present invention contains must have continuous 15 more than the base at least, is more preferably the base sequence by at least 18 continuous based compositions.Can contain the sequence with the partial sequence complementation of the second oligonucleotide, or contain simultaneously the sequence with the partial sequence complementation of the partial sequence at the nucleotide polymorphisms position of containing the first oligonucleotide and the second oligonucleotide.If detect with probe and specific nucleic acid sequence and form complementary strand, no matter be DNA, RNA or PNA, can prepare with chemical synthesis process or biological method.For example utilize DNA sensitizer 391 property of perkinelmer company to synthesize by phosphoramidite method.Purifying then can carry out with MONO-Q post or anti-phase post at the high performance liquid phase instrument.Other synthetic methods also have phosphotriester method, H-phosphoric acid ester method, the inferior phosphinylidyne method of sulfo-etc.
Even detect with the polymorphic sequence part mixing base of probe also harmless.The so-called base of mixing means to include the base of base that can both be complementary with wild-type or polymorphism.Specifically, in the polymorphic sequence part, when wild-type is C, polymorphism be A the time, the part of polymorphic sequence can be T or G base.It is as follows now to illustrate concrete inflation method: when synthetic with phosphoramidite method etc., T and G can be synthesized.Detection exists ratio different and different with the mixture synthesis rate with the base in the probe, and the ratio of the signal that selection hope obtains gets final product.Its ratio was advisable with 1: 1.Can lift wild-type is C, and polymorphism is that the situation of A is that example illustrates, but when polymorphism when being multiple, though mix base have more than 3 kinds also no problem.
Use when employed detection prepares dna microarray with probe in the present invention, can use existing method.The preparation method of dna microarray, known have the method (on-chip method) at the direct synthesising probing needle of surface of solid phase carriers and the probe for preparing in advance be fixed on the method on solid-phase matrix surface, and dna microarray of the present invention is then preferably with rear a kind of method preparation.When the probe for preparing in advance being fixed on the solid-phase matrix surface, then synthesize the probe that has imported the function base, again with probe points on the surface treated solid-phase matrix surface, make it that covalent attachment (Lamture for example occur, J.B.et al.Nucl.Acids Res.22:2121-2125,1994; Guo, Z.et al.Nucl.Acids Res.22:5456-5465,1994).Probe generally covalently bind on the surperficial treated solid-phase matrix by spacer or linking agent.Also know to have and make small polyacrylamide gel proper alignment at glass surface, again with probe covalent attachment method (Yershov in the above, G.et al.Proc.Natl.Acad.Sci.USA 94:4913,1996) be combined in the method (TOHKEMY 2001-186880 communique) on the solid-phase matrix that is covered by polylysine or with probe.In addition, also know the array that has at siliceous microarray preparation microelectrode, electrode be provided with contain Streptavidin the agarose pervious course as reactive site, make this position become positively charged lotus and fix biotinylated probe, electric charge by control a part, might the tight method of hybridizing (Sosnowski, R.G.et al.Proc.Natl.Acad.Sci.USA 94:1119-1123,1997) of high speed.Dna microarray of the present invention can prepare with above-mentioned any method.In addition, when probe being dropped in the solid-phase matrix surface and carry out point sample, can carry out by pin mode (pin mode), also can adopt the ink-jetting style of in TOHKEMY 2001-116750 communique or TOHKEMY 2001-186881 communique, delivering.Behind the point sample, by cooling, add moisture (under the humidity about 80%, keeping certain hour) at drop, dryout dry being fixed processing etc., each point is fixed on the solid substrate, can finish the preparation of dna microarray.In addition, the solid-phase matrix of dna microarray the glass (slide glass) that dna microarray uses, can also use plastics, silica gel and pottery etc. except common.
Dna microarray provided by the invention and the first that is connected be connected ligase enzyme and the second oligonucleotide be owing to have at least about 26 to 70 bases, so it has reactive good with detection probes, and on dna microarray maneuverable characteristics.
According to an example of past method therefor, in order to detect the polymorphism of a base, must 240 probes of preparation.And adopt method of the present invention, for identifying the polymorphism of a base, if be ready to the first oligonucleotide that is used for wild-type of the fluorescent substance mark that different wavelength of fluorescence are arranged and be used for the first oligonucleotide of polymorphism and the second oligonucleotide each is a kind of, at least aly contain the detection that mixes base with probe as long as only prepare.Therefore, when measuring multiple nucleotide polymorphisms at the same time, used method is superior to in the present invention far away.Simultaneously, because number of probes reduces, reduced in addition the advantage of the occurrence probability of non-specific responding.Non-specific responding usually becomes the obstacle of industrialization, and the present invention has made larger contribution to the industrial field of the industry at aspects such as high speed, high efficiencies.
Test kit
Among the present invention, test kit can comprise:
(i) contain the first oligonucleotide more than 2 kinds of the Sequence of nucleotide polymorphisms;
(ii) contain with the first oligonucleotide and be connected, and the second oligonucleotide more than 2 kinds of the sequence of not hybridizing with this nucleotide polymorphisms Sequence;
(iii) contain at least a enzyme of nuclease;
(iv) contain at least a enzyme of ligase enzyme activity;
(v) complementary with the part of the second oligonucleotide at least two or more detection probe, and/or at least with the two or more detection probe of some complementation of the second oligonucleotide and the first oligonucleotide.
In addition, among the present invention, test kit also can comprise:
(i) the first oligonucleotide of two or more polymorphisms, they contain the sequence with the base complementrity that shows polymorphism in this nucleotide polymorphisms Sequence of inferring;
(ii) the second oligonucleotide of two or more wild-types, they contain the sequence with the base complementrity that shows wild-type in this nucleotide polymorphisms Sequence of inferring;
(iii) two or more the second oligonucleotide, the first oligonucleotide of they and polymorphism and/or wild-type sequence adjacent and that have the Sequence of this nucleotide polymorphisms not hybridize with supposition;
(iv) contain at least a enzyme of nuclease;
(v) contain a kind of at least a enzyme that contains the ligase enzyme activity;
(vi) complementary with the part of the second oligonucleotide at least two or more detection probe, and/or complementary with the part of the second oligonucleotide and the first oligonucleotide at least two or more detection probe
Detection has following feature with the detection probes on the dna microarray: with nuclease remove those when the oligonucleotide hybridization after the nucleotide polymorphisms Sequence does not form the base sequence of base pair, detect again the first oligonucleotide and the second oligonucleotide conjugates that connects into the ligase enzyme active function.Simultaneously, the first and the second oligonucleotide also can be in advance with marks such as above-mentioned enzyme, vitamin H, fluorescent substance, heparin, antigen, antibody, radioactive substance and luminophores.
Embodiment
Below specify the present invention with embodiment, but the present invention is not limited to embodiment.
(1)
Synthesizing of the PCR primer that gene amplification is used and probe
Gene and SNP (single nucleotide polymophism, the single nucleotide polymorphism) position measured are illustrated in the table 1.In order to resolve simultaneously this 5 kinds of single nucleotide polymorphism, synthesized the oligonucleotide (hereinafter to be referred as few 1～30) of the base sequence shown in the sequence numbering 1～30 (referring to table 2) by the synthetic authorized company (companies such as Japanese Bioservice, サ ワ デ イ one, GENSET KK, AmershamPharmacia, Biotech) of DNA.
Table 1
| SNP No. | The gene name | The SNP position | dbs SNP No. |
| 1 | Nitricoxide synthase 3 | Glu298Asp | rs 1799983 |
| 2 | Proangiotensin | T704C(M235T) | Jrs699 |
| 3 | Angiotensin II 1 receptor | A1166C | rs5186 |
| 4 | Glycoprotein I IIa | C1565T | rs5918 |
| 5 | Factor VII | Arg 353Gln | rs1801020 |
Table 2
| The oligonucleotide name | Sequence | Characteristic |
| Few 1 | AACGGTCGCTTCGAC GTGCT | The just side primer of SNP No.1 |
| Few 2 | GCACCTCAAGGACCA GCTC | The antisense side primer of SNP No.1 |
| Few 3 | GGCTGTGACAGGATG GAAGACT | The just side primer of SNP No.2 |
| Few 4 | TGGAAGTGGACGTAGG TGTTGA | The antisense side primer of SNP No.2 |
| Few 5 | GGAGCAAGAGAACAT TCCTCTG | The just side primer of SNP No.3 |
| Few 6 | TCGGTTCAGTCCACAT AATGC | The antisense side primer of SNP No.3 |
| Few 7 | AGCTCTGATTGCTGG ACTTCTC | The just side primer of SNP No.4 |
| Few 8 | GGGCACAGTTATCCTT CAGCAG | The antisense side primer of SNP No.4 |
| Few 9 | CAGTCCTATTGATCTG GACTCCTG | The just side primer of SNP No.5 |
| Few 10 | AAGTGTTGACTCCAA GCTCACC | The antisense side primer of SNP No.5 |
| Few 11 | AGAAGGAAGAGTTCT GGGGGC | The first oligonucleotide Cy3 mark of SNP No.1 |
| Few 12 | CAGAAGGAAGAGTTC TGGGGGA | The first oligonucleotide Cy5 mark of SNP No.1 |
| Few 13 | GTCATCTGGGGCCTG CAGCAG | The second oligonucleotide of SNP No.1 |
| Few 14 | TGCTGTCCACACTGGC TCCCG | The first oligonucleotide Cy3 mark of SNP No.2 |
| Few 15 | GTGCTGTCCACACTG GCTCCCA | The first oligonucleotide Cy5 mark of SNP No.2 |
| Few 16 | CTCAGGGAGCAGCCA GTCTTCCA | The second oligonucleotide of SNP No.2 |
| Few 17 | AGCACTTCACTACCA AATGAGCC | The first oligonucleotide Cy3 mark of SNP No.3 |
| Few 18 | CAGCACTTCACTAC C AAATGAGCA | The first oligonucleotide Cy5 mark of SNP No.3 |
| Few 19 | GTTAGCTACTTTTCAG AATTGAAGGAG | The second oligonucleotide of SNP No.3 |
| Few 20 | GGTCACAGCGAGGTG AGCCCG | The first oligonucleotide Cy3 mark of SNP No.4 |
| Few 21 | AGGTCACAGCGAGGT GAGCCCA | The first oligonucleotide Cy5 mark of SNP No.4 |
| Few 22 | CGAGGCAGGGCCTGT AAGTCAGG | The second oligonucleotide of SNP No.4 |
| Few 23 | AGCAGCAGAGCCCTC ATGGCG | The first oligonucleotide Cy3 mark of SNP No.5 |
| Few 24 | GAGCAGCAGAGCCCT CATGGCA | The first oligonucleotide Cy5 mark of SNP No.5 |
| Few 25 | CTCCGTCCGTTGGTCC AGCTG | The second oligonucleotide of SNP No.5 |
| Few 26 | TTTTTCCCAGATGAKC CCCCAGAA | The detection of SNP No.1 has amido modified with probe at 5 ' end |
| Few 27 | TTTTTCTGGCTGCTCC CTGAYGGG | The detection of SNP No.2 has amido modified with probe at 5 ' end |
| Few 28 | TTTTTATTCTGAAAAG TAGCTAAKGCTCATTT | The detection of SNP No.3 has amido modified with probe at 5 ' end |
| Few 29 | TTTTTCCCTGCCTCYG GGCTCACC | The detection of SNP No.4 has amido modified with probe at 5 ' end |
| Few 30 | TTTTTACGGACGGAY GCCATGAGG | The detection of SNP No.5 has amido modified with probe at 5 ' end |
(2) use repeatedly PCR (Multi-PCR) amplification gene
Dna solution with the extracting from people's white cell of benzene phenol-chloroform method is used as sample, adds following reagent, carries out repeatedly the nucleic acid fragment that pcr amplification contains 5 kinds of gene pleiomorphism positions by following condition.
Reagent
Preparation contains 25 μ l solution of following reagent.
BTaq dna polymerase reaction liquid
Few 1～10 each 2.5pmol
* 10 damping fluids 2.5 μ l
2mM dNTP 2.5μl
BTaqDNA polysaccharase (Japan weaving (strain) 1.25U
The dna solution 20ng that extracts
Amplification condition
94 ℃-5 minutes
94 ℃ of-30 seconds, 60 ℃ of-30 seconds, 72 ℃ of-30 seconds (40 circulations)
72 ℃-2 minutes
(3) nuclease/ligase enzyme reaction
In step (2), among the reaction solution 20 μ l of amplification, add following reagent 10 μ l anabolic reaction mixed solutions.
Few 11～25 each 2.5pmol
* 10 bTaq damping fluids 1 μ l
200mM MgCl
2 1.2μl
100mM NAD 0.3μl
Tth dna ligase (Epicentre Technology) 5U
RTaq archaeal dna polymerase (Japan's weaving (strain)) 0.625U
Alkaline phosphatase (Japan's weaving (strain)) 3U
Reaction conditions
37 ℃-30 minutes
94 ℃-5 minutes
94 ℃ of-30 seconds, 60 ℃-2 minutes (15 circulations)
(4) detect
(a) preparation of dna microarray
With widow 16～20 respectively with sampling liquid (Song Bo glass industry corporation: DSP0050) mixed with 1: 1.With wave carrier piece (the Song Bo glass industry corporation: SDA0011) on of mixed oligonucleotide solution point at the preparation dna microarray.In constant humidity cabinet, place a night.After the drying, carefully wash with water, in the 1%BSA aqueous solution, place a night.Repeatedly after the careful washing of twice water, with whizzer (2000rpm, 2 minutes) centrifugal drying.
(b) hybridization
In the reaction solution 2.5 μ l of step (3), add 0.6N NaOH solution 2.5 μ l mixings.In mixed solution, add 20 μ l hybridization solution [(200mM citric acid-phosphoric acid buffer (pH6.0), 2%SDS (sodium lauryl sulphate), 750mM NaCl, 0.1%NaN
3), fully mixing, the dna microarray for preparing in step (a) adds cover glass, and mixed solution is injected from the gap of two slides.55 ℃ are incubated 2 hours.
(c) clean
Be incubated after 2 hours, in the washings 1 (2 * SSC (pH 7.0), 1%SDS) that is incubated in advance in 55 ℃, placed 20 minutes.In washings 2 (250 μ l 50mM Tris-hydrochloride buffers (pH7.5), 0.025%Tween20 solution), under room temperature, placed 15 minutes again.Use at last whizzer (2000rpm, 2 minutes) centrifugal drying.
(d) measure
After the ア レ イ ス キ ヤ Na FLA8000 mensuration with Fuji Photo Film Co., Ltd.'s product.Analyze with ア レ イ ゲ イ ジ software (Fuji Photo Film Co., Ltd.'s product), measure the fluorescent value of spot.By as seen from Table 3, can resolve simultaneously 5 kinds of SNP, and judge its nucleotide polymorphisms.
Table 3
| SNP No. | The Cy3 signal | The Cy5 signal | Log(Cy3/Cy5) |
| 1 | 128608 | 470 | 2.44 |
| 2 | 80780 | 201784 | -0.40 |
| 3 | 27991 | 205552 | -0.87 |
| 4 | 5400 | 163314 | -1.48 |
| 5 | 112254 | 149547 | -0.12 |
Application possibility on the industry
The invention provides a kind of method of judging the nucleotide polymorphisms Sequence, the method is to make the first and the second oligonucleotide and specific nucleic acid hybridization, after active with ligase enzyme in nuclease act in turn or simultaneously, whether there are the first of connection and the method that the second oligonucleotide is judged with combining the array detection that detects with probe.Owing to by the activity of nuclease, 5 ' terminal phosphate of the second oligonucleotide is exposed, when the preparation oligonucleotide, can omit the reaction that makes the phosphate combination.Simultaneously, therefore the second oligonucleotide generation ligation that only goes out phosphate in 5 ' ends exposed owing to the effect of nuclease has reduced non-specific ligation.The present invention also provides a method of using simultaneously nuclease and the multiple nucleotide polymorphisms of ligase enzyme active function at least a reactive system.More since the first and the second oligonucleotide that ligase enzyme connects have at least about 26 to 70 bases, so have the reactive good of detection probes, characteristics that can convenient operation on array.Can be easy to detect multiple nucleotide polymorphisms by the present invention, being expected has larger contribution in industrial community.
Claims (19)
1. the authentication method of a plurality of nucleotide polymorphisms is to identify simultaneously the method that is contained in the multiple nucleotide polymorphisms in the sample, it is characterized in that,
With the purpose nucleic acid samples that contains multiple nucleotide polymorphisms to be measured, hybridize with the multiple oligonucleotide that is consisted of by the second oligonucleotide shown in the first oligonucleotide shown in following (1) and following (2), with the enzyme that contains nuclease and contain the enzyme of ligase enzyme activity will be with the first oligonucleotide of sample hybridization be connected and be connected with the second oligonucleotide that sample is hybridized, make it form the oligonucleotide that connects, adopt the kind of identifying the oligonucleotide of this connection with the detection that oligonucleotide after this is connected has a complementary sequence with probe, wherein detect with probe be contained polymorphic sequence partly respectively with the mixture of two kinds of probes of wild-type and polymorphism complementation
The combination of the first oligonucleotide that (1) is consisted of by wild-type the first oligonucleotide and polymorphism the first oligonucleotide, described wild-type the first oligonucleotide contains the sequence complementary with the wild-type sequence part, and described polymorphism the first oligonucleotide contains the sequence complementary with the polymorphic sequence part;
(2) adjacent with the first oligonucleotide and the supposition with purpose Nucleotide have the corresponding part of sequence of nucleotide polymorphisms contain with wild-type and polymorphism in any equal the second oligonucleotide of not complementary base, adjacent referring to wherein, infer when in the purpose nucleic acid that the nucleotide polymorphisms Sequence is arranged the second oligonucleotide being contacted with the first oligonucleotide containing, be designed in supposition has the Sequence of nucleotide polymorphisms, to have at least more than one base to overlap.
2. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, more than one detection is fixed on the solid phase with probe.
3. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 2 is characterized in that, being fixed with the solid phase that detects with probe is dna microarray.
4. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, detects make the oligonucleotide amplification of connection by amplified reaction after.
5. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 4 is characterized in that, amplification method is any selected from a group method that is comprised of PCR, NASBA, LCR, SDA, RCR and TMA method.
6. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, sample is the nucleic acid that increases in advance.
7. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 6 is characterized in that, amplification method is any selected from a group method that is comprised of PCR, NASBA, LCR, SDA, RCR and TMA method.
8. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, adopts in turn and/or simultaneously the enzyme that contains nuclease and the enzyme that contains the ligase enzyme activity.
9. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, the enzyme that contains nuclease is the same enzyme with the enzyme that contains the ligase enzyme activity.
10. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, the enzyme that contains nuclease is at least a enzyme selected from a group enzyme that is comprised of mung-bean nuclease, S1 nuclease, exonuclease I～VII.
11. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, the enzyme that contains the ligase enzyme activity is at least a enzyme selected from a group enzyme that is comprised of T4DNA ligase enzyme, e. coli dna ligase, RNA ligase enzyme.
12. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, the enzyme that contains nuclease is the thermotolerance enzyme with the enzyme that contains the ligase enzyme activity.
13. the authentication method of the described a plurality of nucleotide polymorphisms of claim 12 is characterized in that, the thermotolerance enzyme is from Tth, Taq, KOD or Pfu.
14. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1 is characterized in that, the enzyme that contains nuclease is archaeal dna polymerase.
15. the authentication method of the described a plurality of nucleotide polymorphisms of claim 14, it is characterized in that archaeal dna polymerase is from by from the Taq DNA polymerase of Tth, from selected one kind or two or more archaeal dna polymerase the Taq DNA polymerase of Taq, a group enzyme that forms from the Taq DNA polymerase of KOD with from the Taq DNA polymerase of Pfu.
16. the authentication method of a plurality of nucleotide polymorphisms claimed in claim 1, nucleotide polymorphisms are polymorphism, insertion, deletion polymorphism any of a base.
17. a test kit is the test kit of the multiple nucleotide polymorphisms of identifying that simultaneously purpose nucleic acid in the sample is contained, it is characterized in that comprising at least following (i)～(vi):
(i) infer to have in the Sequence of nucleotide polymorphisms at purpose nucleic acid, contain the two or more polymorphism the first oligonucleotide with the sequence of the base complementrity that shows polymorphism;
(ii) infer to have in the Sequence of nucleotide polymorphisms at purpose nucleic acid, contain the two or more wild-type the first oligonucleotide with the sequence of the base complementrity that shows wild-type;
(iii) contain with polymorphism and/or wild-type the first oligonucleotide is adjacent and two or more the second oligonucleotide of the sequence that the nucleotide polymorphisms Sequence do not hybridize is arranged in this supposition, adjacent referring to wherein, infer when in the purpose nucleic acid that the nucleotide polymorphisms Sequence is arranged the second oligonucleotide being contacted with the first oligonucleotide containing, be designed in supposition has the Sequence of nucleotide polymorphisms, to have at least more than one base to overlap;
(iv) at least a enzyme that contains nuclease;
(v) at least a enzyme that contains the ligase enzyme activity;
(vi) complementary with the part of the second oligonucleotide and the first oligonucleotide at least detection probe, and detect with probe be contained polymorphic sequence partly respectively with the mixture of two kinds of probes of wild-type and polymorphism complementation.
18. the described test kit of claim 17 is characterized in that, detecting provides with solid phase form with probe.
19. the described test kit of claim 18 is characterized in that, solid phase is dna microarray.
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