CN101065483A - Method of identifying nucleotide polymorphisms - Google Patents
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- CN101065483A CN101065483A CN200580040391.9A CN200580040391A CN101065483A CN 101065483 A CN101065483 A CN 101065483A CN 200580040391 A CN200580040391 A CN 200580040391A CN 101065483 A CN101065483 A CN 101065483A
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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 therewith industrial field.
Background technology
Among the present invention, so-called nucleotide polymorphisms is meant to have and the not homotactic gene form of wild-type, the polymorphism gene 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 subjected 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 polymorphism gene of its root, the nucleic acid sequence analysis method that is used to detect the range gene type just becomes a kind of needs.
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 labeled DNA probe complementary base sequence.When promptly adopting the Southern hybrid method, nucleic acid fragment is carried out electrophoresis on the flat board of agarose or polyacrylamide gel, after clip size (length) separation, make its sex change become strand, again 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 RI marks such as (radio isotope) the specific dna probe of having of nucleotide polymorphisms is formed crossbred, detect on the film and probe complementary nucleic acid fragment with methods such as radioactive automatic developings then.
If adopt the Southern hybrid method, can determine the electrophoresis position and the molecular weight of purpose nucleic acid fragment, but have following problem: in 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 discern and be used to detect the similar sequence that has or not nucleotide polymorphisms.
In addition, as utilizing enzyme to discern the method for nucleotide sequence, the method for known employing ligase enzyme (for example referring to patent documentation 1 and 2), adopt the method (for example 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 probe member opposing ends 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.
Whether exist the demand degree of the method in (for example in the gene screening field) to increase separately for being used for a plurality of sequences of testing goal gene 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,, preferably check whole contingent aberrant gene sequence variations in the people's to be detected genomic dna for more effective evaluation " cyst cystic fibrosis ".Ideal detect 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 the hybridization analysis method of dna microarray (DNA chip) as 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 of probe sequence is different, the complicated process of this method is its difficult point.
At this problem, proposed to use tens of oligonucleotide through dna microarray, thereby resolved the method (for example referring to patent documentation 4) of polymorphism at a kind of polymorphic sequence.If adopt this method, then adopt a kind of according to various polymorphisms form 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 probes show the complete complementary characteristic of canonical sequence, 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 multiple polymorphism in this way, just must use more multiprobe, cannot say for sure to occur beyond thought non-specific responding.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.Though the oligonucleotide in this method on the dna microarray can reduce, owing to only connect oligonucleotide with ligase enzyme, therefore must use combine the phosphate oligonucleotide endways, expense is higher, and phosphate is when being connected with oligonucleotide, do not know actually and which oligonucleotide generation ligation, non-specific connection takes place easily.Therefore when being specifically designed to the parsing polymorphism, polymorphism is many more, and is dangerous just high more.
Patent documentation 1 Japan special fair 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 1European Consensus Conference onPharmacogenetics.Commission of the EuropeanCommunities, Luxembourg 87-96 page or leaf, (nineteen ninety distribution)
Non-patent literature 2European Journal of Clinical Pharmacology, the 36th volume, 551-554 page or leaf, (distribution in 1989)
Non-patent literature 3 laboratory manuals, genetically engineered, [, 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 under the situation of multiple nucleotide polymorphisms, also have the necessary significant care of operation, problems such as non-specific ligation can take place.Therefore, purpose of the present invention is to provide a kind of superior nucleic acid sequence analysis method, so that easily detect the kind that is contained in the multiple nucleotide polymorphisms in the sample simultaneously.
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.Promptly the present invention relates to identify simultaneously the method for multiple nucleotide polymorphisms.
1. the authentication method of a plurality of nucleotide polymorphisms, be to identify the method that is contained in the multiple nucleotide polymorphisms in the sample simultaneously, it is characterized in that, sample with multiple nucleotide polymorphisms kind to be measured, hybridize with multiple first kind of oligonucleotide and multiple second kind of oligonucleotide, with the enzyme that contains nuclease and contain the active enzyme of ligase enzyme first kind of oligonucleotide is connected with second kind of 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 first kind of oligonucleotide in nucleotide polymorphisms sequence part complementary sequence.
3. the authentication method of above-mentioned 1 described a plurality of nucleotide polymorphisms is characterized in that, contain with second kind of oligonucleotide in infer the sequence part complementary sequence not that nucleotide polymorphisms is arranged.
4. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, contains with 5 ' of second kind of oligonucleotide terminal to infer the sequence part complementary sequence not that nucleotide polymorphisms is arranged.
5. the authentication method of each described a plurality of nucleotide polymorphisms in above-mentioned 1～3, it is characterized in that, with first kind of oligonucleotide and second kind of oligonucleotide when containing the purpose nucleic acid of inferring the sequence part that nucleotide polymorphisms is arranged and contact, have in the sequence part 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 is characterized in that in above-mentioned 1～3, and first kind of oligonucleotide is labeled in advance.
7. the authentication method of above-mentioned 6 described a plurality of nucleotide polymorphisms, it is characterized in that mark is any selected method 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 the first kind of oligonucleotide that is used to detect wild-type carries out mark with the fluorescent substance with different wavelength of fluorescence respectively with the first kind of oligonucleotide that is used to detect nucleotide polymorphisms.
9. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in above-mentioned 1～3, is hybridization with the method that detects the oligonucleotide that connects with probe in detecting.
10. 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 at least a part of complementary sequence with second kind of oligonucleotide.
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 at least a part of complementary sequence with first kind of oligonucleotide and second kind of oligonucleotide.
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 the enzyme that contains nuclease in turn and/or simultaneously and contains the active enzyme of ligase enzyme.
21. the authentication method of each described multiple nucleotide polymorphisms is characterized in that in above-mentioned 1～3, containing the enzyme of nuclease and containing the active enzyme of ligase enzyme is the same enzyme.
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, contains the active enzyme of ligase enzyme and 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, containing the enzyme of nuclease and containing the active enzyme of ligase enzyme is the thermotolerance enzyme.
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 an 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 the selected archaeal dna polymerase more than a kind or 2 kinds 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 the method that is contained in the multiple nucleotide polymorphisms in the sample simultaneously, its feature is as follows:
(1) preparation contains multiple first kind of oligonucleotide of this nucleotide polymorphisms sequence part, and preparation contains multiple second kind of oligonucleotide of sequence adjacent with this first kind of oligonucleotide and that do not hybridize with this nucleotide polymorphisms sequence part;
(2) this first kind of oligonucleotide and this second kind of oligonucleotide are hybridized with the karyomit(e) or the nucleic acid fragment that are contained in the sample;
(3) when hybridization has taken place in various oligonucleotide, remove the nucleotide polymorphisms sequence does not partly form base pair on this second kind of oligonucleotide base sequence with the enzyme that contains nuclease, then with containing the active enzyme effect of ligase enzyme, this oligonucleotide is connected, forms the oligonucleotide that connects;
(4) adopt to contain to detect with the multiple base of a part of complementary of this second kind of oligonucleotide at least and use probe, detect this oligonucleotide that be connected with the multiple base of a part of complementary of this second kind of oligonucleotide and first kind of oligonucleotide with probe in detecting or/and contain at least.
30. a test kit is to identify the test kit that is contained in the multiple nucleotide polymorphisms in the sample simultaneously, it is characterized in that comprising at least following (i)～(v):
(i) the two or more first kind of oligonucleotide that comprises nucleotide polymorphisms sequence part;
The (ii) two or more second kind of oligonucleotide that comprises sequence adjacent with first kind of oligonucleotide and that do not hybridize with this nucleotide polymorphisms sequence part;
The (iii) at least a enzyme that contains nuclease;
The (iv) at least a active enzyme of ligase enzyme that contains;
(v) detect with a part of complementary of second kind of oligonucleotide at least and use probe, and/or at least with a part of complementary detection probe of second kind of oligonucleotide and first kind of oligonucleotide.
31. a test kit is to identify the test kit that is contained in the multiple nucleotide polymorphisms in the sample simultaneously, it is characterized in that comprising at least following (i)～(vi):
(i) have in the sequence part of nucleotide polymorphisms in supposition, contain first kind of oligonucleotide of two or more polymorphisms with the sequence of the base complementrity that shows polymorphism;
(ii) have in the sequence part of nucleotide polymorphisms, contain second kind of oligonucleotide of two or more wild-types with the sequence of the base complementrity that shows wild-type in supposition;
(iii) two or morely contain with polymorphism and/or first kind of oligonucleotide of wild-type is adjacent and with this supposition second kind of oligonucleotide of the sequence that nucleotide polymorphisms sequence part do not hybridize arranged;
The (iv) at least a enzyme that contains nuclease;
(the v) at least a active enzyme of ligase enzyme that contains;
(vi) detect with a part of complementary of second kind of oligonucleotide at least and use probe, and/or at least with a part of complementary detection probe of second kind of oligonucleotide and first kind of oligonucleotide.
32. above-mentioned 30 or 31 described test kits, containing predetermined fixed has the solid phase that detects with probe.
33. above-mentioned 30 or 31 described test kits is characterized in that, solid phase is a dna microarray.
Effect of the present invention
By adopting in turn and/or simultaneously the enzyme that contains nuclease and contain the active enzyme of ligase enzyme, with the special oligonucleotide that is connected of nucleotide polymorphisms, special 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 various diseases that comprises inherited disease and the gene relevant with living habit disease (hypertension, diabetes etc.) with drug metabolism is example.For example relevant ACE gene with hypertension.
Among the present invention, the polymorphism nucleic acid of so-called karyomit(e) or nucleic acid fragment, be meant and have at least a Nucleotide point mutation to take place and, perhaps in the part of wild-type nucleic acid, contained the nucleic acid of insertion sequence or deletion sequence in the wild-type nucleic acid by the nucleic acid of other nucleotide subsitution.
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 a feature to adopt the enzyme that contains nuclease in turn and/or simultaneously and to contain the active enzyme of ligase enzyme.This activity can be passed through single enzyme, also can realize by possessing two kinds of active enzymes simultaneously.
Particularly, the enzyme that contains nuclease can be lifted mung-bean nuclease, S1 nuclease, exonuclease I～VII etc.Contain the active enzyme of ligase enzyme and can lift T4DNA ligase enzyme, e. coli dna ligase, RNA ligase enzyme etc.Contain the enzyme of nuclease and contain the active enzyme of ligase enzyme because of be the thermotolerance enzyme preferably.For example the thermotolerance enzyme can be the enzyme from Tth, Taq, KOD, Pfu.And the enzyme that contains nuclease also can be an archaeal dna polymerase.For example can use 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 first kind of oligonucleotide of the sequence part of nucleotide polymorphisms, by with the nucleic acid complementation of the chain of this first kind of oligonucleotide hybridization, with this first kind of adjacent hybridization of oligonucleotide, preparation contains the second kind of oligonucleotide that does not form the sequence of complementary strand with any sequence of this nucleotide polymorphisms sequence part.
(2) this first kind of oligonucleotide and second kind of oligonucleotide and nucleic acid are hybridized
(3) in the nucleotide polymorphisms sequence part that when oligonucleotide has carried out hybridization separately, has produced, remove the base that does not form second kind of base pair that oligonucleotide had 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, by the effect that contains the active enzyme of ligase enzyme first kind of oligonucleotide and second kind of oligonucleotide is coupled together.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, first kind of oligonucleotide is to contain and the oligonucleotide of inferring the sequence part complementary base that nucleotide polymorphisms is arranged.The supposition part of this nucleotide polymorphisms is 3 ' end of first kind of Nucleotide preferably.
Second kind of oligonucleotide is more prone to the position of 3 ' one side than first kind of oligonucleotide.Second kind of oligonucleotide is characterised in that it better is to contain and infer the sequence part oligonucleotide of complementary base not that nucleotide polymorphisms is arranged.Preferably the sequence part that nucleotide polymorphisms is arranged with supposition not the complementary base be present in 5 ' end of second kind of oligonucleotide.
Now the method for design to first kind of oligonucleotide and second kind of oligonucleotide is described in more detail.Inferring when in the purpose nucleic acid that nucleotide polymorphisms sequence part is arranged second kind of oligonucleotide being contacted with first kind of oligonucleotide containing, better is to be designed to have at least more than one base to overlap in sequence that supposition has a nucleotide polymorphisms in partly.In order to make it overlapping, can infer have the sequence of nucleotide polymorphisms partly to hybridize to 3 ' tip designs Cheng Nengyu of first kind of oligonucleotide; And 5 ' tip designs of second kind of oligonucleotide is become can not have the sequence of nucleotide polymorphisms partly to hybridize with supposition.At 5 ' end of second kind of oligonucleotide, the length of the structure that the sequence of nucleotide polymorphisms partly hybridizes can not be arranged with supposition, have at least a base to get final product.This is at 5 ' end of second kind of oligonucleotide, when removing with nuclease can not the base that the nucleotide polymorphisms sequence partly hybridizes be arranged with supposition the time, be designed to and first kind of oligonucleotide and second kind of oligonucleotide to be coupled together get final product by containing the active enzyme of ligase enzyme.
The length of first kind of oligonucleotide and second kind of oligonucleotide among the present invention is 13～35 bases, preferably 16～30 bases.For example, 3 ' terminal bases of first kind of oligonucleotide is designed to infer the oligonucleotide that the nucleotide polymorphisms position is arranged, first kind of oligonucleotide of wild-type then disposes the base with this base complementrity of this wild-type nucleic acid, simultaneously, first of polymorphism kind of oligonucleotide then disposes the base with this base complementrity of polymorphism nucleic acid.And again 5 ' tip designs of second kind of oligonucleotide is become when with first kind of oligonucleotide a base overlaid being arranged, the overlapping base of this second kind of oligonucleotide is then selected not complementary base, no matter be wild-type or polymorphism.By selection and wild-type or polymorphism complementary base not, when detecting a kind of nucleotide polymorphisms, can only prepare a kind of second kind of oligonucleotide.
When having carried out such design, only in the combination of first kind of oligonucleotide/second kind of oligonucleotide/wild-type nucleic acid of wild-type and first kind of Nucleotide/second of polymorphism kind of oligonucleotide/polymorphism nucleic acid, the lap of second kind of oligonucleotide is removed, because in full accord, the overlapping base that can remove second kind of oligonucleotide with the enzyme that contains nuclease.This moment can be by acting on it with containing the active enzyme of ligase enzyme in order to remove residual phosphate, and first kind of oligonucleotide is connected with second kind of oligonucleotide becomes a chain; And in the combination of first kind of oligonucleotide/second kind of oligonucleotide/polymorphism nucleic acid of wild-type and first kind of Nucleotide/second kind of oligonucleotide/wild-type nucleic acid of polymorphism, 3 ' end of first kind of oligonucleotide can not couple together with containing the active enzyme effect of ligase enzyme owing to can not be complementary.
Contain the enzyme of nuclease and contain the active enzyme of ligase enzyme
Among the present invention, make it act on first kind of oligonucleotide of above-mentioned wild-type and first kind of oligonucleotide of polymorphism simultaneously.
I) make above-mentioned first kind of oligonucleotide and second kind of 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) cut off with the lap of the enzyme that contains nuclease then second kind of oligonucleotide.Operable enzymic activity as mentioned above.Exonuclease activity preferably, also exonuclease such as archaeal dna polymerase preferably.
Under the situation that 5 ' end and first kind of oligonucleotide of second kind of oligonucleotide overlaps, adopt 5 ' exonuclease,, and expose phosphate the lap cut-out of second kind of oligonucleotide.
Iii) simultaneously, with containing the active enzyme of ligase enzyme, the phosphate that will be exposed to second kind of oligonucleotide, 5 ' end is connected with 3 ' end of first kind of oligonucleotide, becomes an oligonucleotide.
In the present invention, if not complementary corresponding to the nucleotide polymorphisms position in first kind of oligonucleotide, it is not complementary corresponding to the position of nucleotide polymorphisms promptly to use endonuclease to cut off yet, so use the ligase enzyme active function, can not connect into a Nucleotide.
When to having used first kind of oligonucleotide sample of wild-type nucleic acid to adopt to contain the enzyme of nuclease and contained under the situation of the active enzyme of ligase enzyme, sample nucleic acid is if wild-type then forms a chain, if polymorphism, then do not react.On the contrary, when to having used first kind of oligonucleotide test sample of polymorphism nucleic acid to adopt to contain the enzyme of nuclease and contained under the situation of the active enzyme of ligase enzyme, sample nucleic acid is if polymorphism then forms a chain, if wild-type, then do not react.Particularly be the higher organism of representative with the mankind, a kind of gene has 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 and has made the enzyme that contains nuclease and contain the method that the active enzyme of ligase enzyme acts on multiple nucleotide polymorphisms to be detected in same reaction system.Multiple among the present invention means and detects at least a or two above nucleotide polymorphisms.When identifying multiple nucleotide polymorphisms, also can adopt in different reaction systems, to make the enzyme that contains nuclease and contain the active enzyme effect of ligase enzyme, but in same reaction system, then more easy, also more help reducing expenses.In at least a reaction system, make the enzyme that contains nuclease and contain the active enzyme of ligase enzyme and act on simultaneously under the situation of multiple nucleotide polymorphisms, be used for wild-type and be preferably different marks, for example add the fluorescent mark that to be distinguished with wavelength of fluorescence with the first kind of oligonucleotide that is used for polymorphism.It is different with the fluorescent mark of the first kind of oligonucleotide that is used for polymorphism that mark is used for wild-type, when identifying multiple nucleotide polymorphisms, even first kind of oligonucleotide and second kind of oligonucleotide mix, also can not make troubles at least.
Under the inadequate situation of purpose nucleic acid content to be detected, before above-mentioned hybridization, can pass through following amplified reaction in advance, the above-mentioned nucleic acid fragment that contains polymorphic sequence is increased.Above-mentioned amplification method is not particularly limited, as PCR (Polymerase chain reaction; Japan special fair 4-67960 communique, Japan special fair 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 the volume, the 3270th page (1993)) etc. can adopt.
In addition, make the enzyme that contains nuclease and contain the active enzyme effect of ligase enzyme, and after oligonucleotide connected, also can increasing with amplified reaction shown in above-mentioned, it detects in conjunction with product.5 ' end side of first kind of oligonucleotide and 3 ' end side of second kind of oligonucleotide can add the sequence of undertaking the promotor effect that is used 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.
First kind of 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 modification groups such as oligomerization dGTP, oligomerization dATP, oligomerization dTTP, oligomerization dCTP at 5 ' end.Perhaps can synthesize, to import modification group by the Nucleotide of replacing in the oligonucleotide sequence with the Nucleotide that vitamin H, synthetic linking agent, fluorescent substance etc. are arranged.Can also be behind synthesizing ribonucleotide, to the marker that wherein imports these known oligonucleotide of fluorescent substance such as enzymes such as radioactive substances such as 32P, 35S, ALP, POD, FITC, HEX, 6-FAM, TET.Can also in first kind of oligonucleotide of first kind of oligonucleotide of wild-type and nucleotide polymorphisms, distinguish the different oligonucleotide sequence of affix.
It is as follows to attempt the concrete grammar example: make the specific nucleic acid that adopted second kind of oligonucleotide and hybridize through first kind of oligonucleotide of marks such as fluorescent substance, after using the enzyme that contains ligase enzyme in turn and/or simultaneously and containing the active enzyme effect of nuclease, make it to dissociate, again with in conjunction with the detection probe special on the solid phase again to polymorphism, and detection probe and its hybridization special to wild-type, wash out the not nucleic acid of hybridization, whether hybridization has taken place according to the marker detection with first kind of oligonucleotide of the nucleic acid of various detections after with probe hybridization, detect detection signal ratio by each again, identify whether wild-type of nucleotide polymorphisms with probe, polymorphism or mixed state type.
With the first kind of oligonucleotide that is used for polymorphism not isolabeling is arranged owing to be used for wild-type, for example added 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 used for additional on first kind of oligonucleotide of polymorphism different marks being arranged, for example can distinguish the fluorescent mark of wavelength of fluorescence, can at least a reactive system, make the enzyme that contains nuclease simultaneously and contain the active enzyme of ligase enzyme to act on multiple nucleotide polymorphisms.At this moment, if mark is used for fluorescent mark difference on first kind of oligonucleotide of wild-type and polymorphism at least, when then being used to identify multiple nucleotide polymorphisms, exist first kind of oligonucleotide and second kind of oligonucleotide also not to have problem even mix.Mark is used for wild-type and is used for the fluorescent mark of first kind of 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 kind of oligonucleotide that is used for wild-type, and 5 ' end mark Cy5 at the first kind of 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 a polymorphism nucleic acid with purpose nucleic acid, adopts first kind of oligonucleotide of 5 ' terminal wild-type of having modified with Cy3 and first kind of oligonucleotide of 5 ' terminal polymorphism of having modified with Cy5.
Under the situation of this example, set that 5 ' in second kind of oligonucleotide is terminal to be " supposition has nucleotide polymorphisms sequence part ", set simultaneously and " supposition has nucleotide polymorphisms sequence part " complementary base not.In addition, in the case, 5 ' the terminal base that can also be positioned at second kind of oligonucleotide also can be set at 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 second kind of oligonucleotide that also is designed to first kind of oligonucleotide of polymorphism overlaps.The G of 3 ' terminal C and 5 ' end of second kind of oligonucleotide that also can be designed to first kind of oligonucleotide of wild-type overlaps.
First kind of oligonucleotide and second kind of situation that oligonucleotide is had an effect of polymorphism in (1) expression polymorphism nucleic acid of Fig. 1.In this case, 3 ' terminal A of first kind of oligonucleotide of polymorphism is hybridized at the polymorphism position, on the position that is adjacent, hybridizes from second T that 5 ' end of second kind of oligonucleotide is counted.And the G of 5 ' end of second kind of oligonucleotide just is in the state that does not take place to hybridize owing to not complementary with the polymorphism position.Use the enzyme effect that contains nuclease this moment, then 5 ' of second kind of oligonucleotide terminal G is cut off, and generates phosphate simultaneously.Act on this with containing the active enzyme of ligase enzyme, then two oligonucleotide connect.
First kind of oligonucleotide and second kind of situation that oligonucleotide is had an effect of wild-type in (2) expression polymorphism nucleic acid of Fig. 1.In this case, therefore 3 ' end of first kind of oligonucleotide of wild-type does not hybridize owing to not complementary with the polymorphism position.And the G of 5 ' end of second kind of oligonucleotide is in the state that does not take place 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 second kind of oligonucleotide.Therefore contain the effect of the active enzyme unable to get up of ligase enzyme, 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 first kind of oligonucleotide of polymorphism is not connected with second kind of oligonucleotide, and first kind of oligonucleotide of wild-type is connected with second kind of oligonucleotide.
Under situation shown in Figure 1,, can have to the first kind of oligonucleotide of polymorphism and the connector of second kind of oligonucleotide when purpose nucleic acid during only with polymorphism nucleic acid homology; When purpose nucleic acid during, then have to the first kind of oligonucleotide of wild-type and the connector of second kind of oligonucleotide only with the wild-type nucleic acid homology; When purpose nucleic acid is allogenic polymorphism nucleic acid and wild-type nucleic acid, then obtain the mixture of ratio near 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.Promptly, if it is different that mark is used for the fluorescent mark with the first kind of oligonucleotide that is used for polymorphism of wild-type at least, to be used to identify that first kind of oligonucleotide of multiple nucleotide polymorphisms mixes with second kind of oligonucleotide, in same reactive system, make the enzyme that contains nuclease and contain the active enzyme of ligase enzyme to act on them.Like this, carry out relevant multiple nucleotide polymorphisms when detecting, can detect as follows at same reactive system.
Detect multiple nucleotide polymorphisms, be used to detect the multiple detection probe of these nucleotide polymorphisms, can adopt every kind of probe fixed solid phase and the method that do not interfere with each other separately.To act on the resultant of reaction that multiple nucleotide polymorphisms produces in order being determined to use the enzyme that contains nuclease in few extremely a kind of reactive system simultaneously and contain the active enzyme of ligase enzyme, can to adopt and fix the dna microarray of above-mentioned detection with oligonucleotide.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 based compositions of successive.Can contain the partial sequence complementary sequence with second kind of oligonucleotide, or contain simultaneously and the partial sequence at the nucleotide polymorphisms position of containing first kind of oligonucleotide and the partial sequence complementary sequence of second kind of 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 on MONO-Q post or anti-phase post on 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, mean include can both the complementary base with wild-type or polymorphism base.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:, T and G can be synthesized when synthetic with phosphoramidite method etc.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.
When using employed in the present invention detection to prepare dna microarray, can use existing method with probe.The preparation method of dna microarray, known have at the direct synthesising probing needle of surface of solid phase carriers method (on-chip method) and the method for the probe stationary for preparing in advance on the solid-phase matrix surface, dna microarray of the present invention is then preferably with back a kind of method preparation.When the probe stationary for preparing in advance in solid-phase matrix when surface, then synthesize the probe that has imported functional group, again with probe points on the surface treated solid-phase matrix surface, make it that covalent attachment (Lamture for example take place, 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 the polyacrylamide gel proper alignment that makes small on glass surface is arranged, 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 poly-L-Methionin or with probe.In addition, also know the array that preparation microelectrode on siliceous microarray is arranged, 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, probe being dropped in when carrying out point sample on the solid-phase matrix surface, can carry out, also can adopt the ink-jetting style of in TOHKEMY 2001-116750 communique or TOHKEMY 2001-186881 communique, delivering by pin mode (pin mode).Behind the point sample,, adding moisture (under the humidity about 80%, keeping certain hour) on the drop, dryouting the dry immobilization processing etc. of carrying out, each point is fixed on the solid substrate, can finish the preparation of dna microarray by cooling.In addition, the solid-phase matrix of dna microarray except that the glass (slide glass) that uses usually, can also use plastics, silica gel and pottery etc. on dna microarray.
Dna microarray provided by the invention with first kind that is connected by ligase enzyme and second kind of oligonucleotide 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 kind of oligonucleotide that is used for wild-type of the fluorescent substance mark that different wavelength of fluorescence are arranged and be used for first kind of oligonucleotide of polymorphism and second kind of 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 the advantage of the occurrence probability of non-specific responding in addition.Non-specific responding usually becomes the obstacle of industrialization, and the present invention has made bigger 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 first kind of oligonucleotide more than 2 kinds of the sequence part of nucleotide polymorphisms;
(ii) contain with first kind of oligonucleotide and be connected, and second kind of oligonucleotide more than 2 kinds of the sequence of not hybridizing with this nucleotide polymorphisms sequence part;
At least a enzyme that (iii) contains nuclease;
(iv) contain the active at least a enzyme of ligase enzyme;
(v) two or more with a part of complementary of second kind of oligonucleotide at least detection probes, and/or two or more with some complementary of second kind of oligonucleotide and first kind of oligonucleotide at least detection probes.
In addition, among the present invention, test kit also can comprise:
(i) first of two or more polymorphisms kind of oligonucleotide, they contain the sequence with the base complementrity that shows polymorphism in this nucleotide polymorphisms sequence part of inferring;
(ii) second of two or more wild-types kind of oligonucleotide, they contain the sequence with the base complementrity that shows wild-type in this nucleotide polymorphisms sequence part of inferring;
(iii) two or more second kind of oligonucleotide, first kind of oligonucleotide of they and polymorphism and/or wild-type sequence adjacent and that have the sequence part of this nucleotide polymorphisms not hybridize with supposition;
At least a enzyme that (iv) contains nuclease;
(v) contain a kind of active at least a enzyme of ligase enzyme that contains;
(vi) two or more with a part of complementary of second kind of oligonucleotide at least detection probes, and/or two or more with a part of complementary of second kind of oligonucleotide and first kind of oligonucleotide at least detection probes
Detection has following feature with the detection probes on the dna microarray: with nuclease remove those when the oligonucleotide hybridization after nucleotide polymorphisms sequence part does not form the base sequence of base pair, detect first kind of oligonucleotide and the second kind of oligonucleotide conjugates that connects into the ligase enzyme active function again.Simultaneously, first kind and second kind of 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) the PCR primer used of gene amplification and probe is synthetic
Gene and SNP (single nucleotide polymophism, the single nucleotide polymorphism) position measured are illustrated in the table 1.In order to resolve this 5 kinds of single nucleotide polymorphism simultaneously, 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 ヮ デ ィ one, GENSET KK, AmershamPharmacia, Biotech) of DNA.
Table 1
| 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 |
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 | First kind of oligonucleotide Cy3 mark of SNP No.1 |
| Few 12 | CAGAAGGAAGAGTTC TGGGGGA | First kind of oligonucleotide Cy5 mark of SNP No.1 |
| Few 13 | GTCATCTGGGGCCTG CAGCAG | Second kind of oligonucleotide of SNP No.1 |
| Few 14 | TGCTGTCCACACTGGC TCCCG | First kind of oligonucleotide Cy3 mark of SNP No.2 |
| Few 15 | GTGCTGTCCACACTG GCTCCCA | First kind of oligonucleotide Cy5 mark of SNP No.2 |
| Few 16 | CTCAGGGAGCAGCCA GTCTTCCA | Second kind of oligonucleotide of SNP No.2 |
| Few 17 | AGCACTTCACTACCA AATGAGCC | First kind of oligonucleotide Cy3 mark of SNP No.3 |
| Few 18 | CAGCACTTCACTACC AAATGAGCA | First kind of oligonucleotide Cy5 mark of SNP No.3 |
| Few 19 | GTTAGCTACTTTTCAG AATTGAAGGAG | Second kind of oligonucleotide of SNP No.3 |
| Few 20 | GGTCACAGCGAGGTG AGCCCG | First kind of oligonucleotide Cy3 mark of SNP No.4 |
| Few 21 | AGGTCACAGCGAGGT GAGCCCA | First kind of oligonucleotide Cy5 mark of SNP No.4 |
| Few 22 | CGAGGCAGGGCCTGT AAGTCAGG | Second kind of oligonucleotide of SNP No.4 |
| Few 23 | AGCAGCAGAGCCCTC ATGGCG | First kind of oligonucleotide Cy3 mark of SNP No.5 |
| Few 24 | GAGCAGCAGAGCCCT CATGGCA | First kind of oligonucleotide Cy5 mark of SNP No.5 |
| Few 25 | CTCCGTCCGTTGGTCC AGCTG | Second kind of 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
Add following reagent with benzene phenol-chloroform method extractive dna solution from people's white cell as sample, carry 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 ℃-30 seconds, 60 ℃-30 seconds, 72 ℃-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
* 10bTaq damping fluid 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 ℃-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), thorough mixing adds cover glass on the dna microarray of step (a) preparation, 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 whizzer (2000rpm, 2 minutes) centrifugal drying at last.
(d) measure
After the ァ レ ィ ス キ ャ Na FLA8000 mensuration with Fuji Photo Film Co., Ltd.'s product.Analyze with ァ レ ィ ゲ ィ ジ software (Fuji Photo Film Co., Ltd.'s product), measure the fluorescent value of spot.By table 3 as seen, can resolve 5 kinds of SNP simultaneously, and judge its nucleotide polymorphisms.
Table 3
| SNP No. | The gene name | The SNP position | dbs SNP No. |
| 1 | Nitricoxide synthase 3 | Glu298Asp | rs1799983 |
| 2 | Proangiotensin | T704C(M235T) | Jrs699 |
| 3 | Angiotensin II 1 receptor | A1166C | rs5186 |
| 4 | Glycoprotein I IIa | C1565T | rs5918 |
| 5 | Factor VII | Arg 353Gln | rs1801020 |
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 oligonucleotides and specific nucleic acid hybridization, after active with ligase in nuclease act in turn or simultaneously, whether there are the first of connection and the method that the second oligonucleotides is judged with combining the array detection that detects with probe. Owing to by the activity of nuclease, 5 ' terminal phosphate of the second oligonucleotides is exposed, when the preparation oligonucleotides, can omit the reaction that makes the phosphate combination. Simultaneously, therefore the second oligonucleotides generation coupled reaction that only goes out phosphate in 5 ' ends exposed owing to the effect of nuclease has reduced non-specific coupled reaction. The present invention also provides a method of using simultaneously nuclease and the multiple nucleotide polymorphisms of ligase active function at least a reaction system. More since the first and the second oligonucleotides that ligase connects have at least about 26 to 70 bases, so have the reactive good of detector probe, 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 circle.
Claims (33)
1. the authentication method of a plurality of nucleotide polymorphisms, be to identify the method that is contained in the multiple nucleotide polymorphisms in the sample simultaneously, it is characterized in that, sample with multiple nucleotide polymorphisms to be measured, hybridize with multiple first kind of oligonucleotide and multiple second kind of oligonucleotide, with the enzyme that contains nuclease and contain the active enzyme of ligase enzyme first kind of oligonucleotide is connected with second kind of 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 the described a plurality of nucleotide polymorphisms of claim 1 is characterized in that, contain with first kind of oligonucleotide in nucleotide polymorphisms sequence part complementary sequence.
3. the authentication method of the described a plurality of nucleotide polymorphisms of claim 1 is characterized in that, contain with second kind of oligonucleotide in infer the sequence part complementary sequence not that nucleotide polymorphisms is arranged.
4. the authentication method of each described a plurality of nucleotide polymorphisms in the claim 1～3 is characterized in that, contains with 5 ' of second kind of oligonucleotide terminal to infer the sequence part complementary sequence not that nucleotide polymorphisms is arranged.
5. the authentication method of each described a plurality of nucleotide polymorphisms in the claim 1～3, it is characterized in that, with first kind of oligonucleotide and second kind of oligonucleotide when containing the purpose nucleic acid of inferring the sequence part that nucleotide polymorphisms is arranged and contact, have in the sequence part 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 the claim 1～3 is characterized in that, first kind of oligonucleotide is labeled in advance.
7. the authentication method of the described a plurality of nucleotide polymorphisms of claim 6, it is characterized in that mark is any selected method from a group material that comprises enzyme, vitamin H, fluorescent substance, heparin, antigen, antibody, radioactive substance, luminophore and specific nucleotide sequence.
8. the authentication method of claim 6 or 7 described a plurality of nucleotide polymorphisms, it is characterized in that the first kind of oligonucleotide that is used to detect wild-type carries out mark with the fluorescent substance with different wavelength of fluorescence respectively with the first kind of oligonucleotide that is used to detect nucleotide polymorphisms.
9. the authentication method of each described a plurality of nucleotide polymorphisms in the claim 1～3 is characterized in that, is hybridization with the method that detects the oligonucleotide that connects with probe in detecting.
10. the authentication method of each described a plurality of nucleotide polymorphisms in the claim 1～3 is characterized in that, detects with probe to contain at least a part of complementary sequence with second kind of oligonucleotide.
11. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in the claim 1～3, detects with probe to contain at least a part of complementary sequence with first kind of oligonucleotide and second kind of oligonucleotide.
12. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in the claim 1～3, detects a part that contains polymorphic sequence with probe.
13. the authentication method of the described a plurality of nucleotide polymorphisms of claim 12 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 the claim 1～3, more than one detection is fixed on the solid phase with probe.
15. the authentication method of the described a plurality of nucleotide polymorphisms of claim 14 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 the claim 1～3, detects make the oligonucleotide amplification of connection by amplified reaction after.
17. the authentication method of the described a plurality of nucleotide polymorphisms of claim 16 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 a plurality of nucleotide polymorphisms is characterized in that in the claim 1～3, sample is the nucleic acid that increases in advance.
19. the authentication method of the described a plurality of nucleotide polymorphisms of claim 18 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 the claim 1～3, adopts the enzyme that contains nuclease in turn and/or simultaneously and contains the active enzyme of ligase enzyme.
21. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in the claim 1～3, containing the enzyme of nuclease and containing the active enzyme of ligase enzyme is the same enzyme.
22. the authentication method of each described a plurality of nucleotide polymorphisms is characterized in that in the claim 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 the claim 1～3, contains the active enzyme of ligase enzyme and 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 the claim 1～3, containing the enzyme of nuclease and containing the active enzyme of ligase enzyme is the thermotolerance enzyme.
25. the authentication method of the described a plurality of nucleotide polymorphisms of claim 24 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 the claim 1～3, the enzyme that contains nuclease is an archaeal dna polymerase.
27. the authentication method of claim 26 described a plurality of nucleotide polymorphisms, it is characterized in that, archaeal dna polymerase for from comprise Taq DNA polymerase from Tth, from the Taq DNA polymerase of Taq, from the Taq DNA polymerase of KOD with from the selected archaeal dna polymerase more than a kind or 2 kinds a group enzyme of the Taq DNA polymerase of Pfu.
28. the authentication method of each described a plurality of nucleotide polymorphisms in the claim 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 the method that is contained in the multiple nucleotide polymorphisms in the sample simultaneously, its feature is as follows:
(1) preparation contains multiple first kind of oligonucleotide of this nucleotide polymorphisms sequence part, and preparation contains multiple second kind of oligonucleotide of sequence adjacent with this first kind of oligonucleotide and that do not hybridize with this nucleotide polymorphisms sequence part;
(2) this first kind of oligonucleotide and this second kind of oligonucleotide are hybridized with the karyomit(e) or the nucleic acid fragment that are contained in the sample;
(3) when hybridization has taken place in various oligonucleotide, remove the nucleotide polymorphisms sequence does not partly form base pair on this second kind of oligonucleotide base sequence with the enzyme that contains nuclease, then with containing the active enzyme effect of ligase enzyme, this oligonucleotide is connected, forms the oligonucleotide that connects;
(4) adopt to contain to detect with the multiple base of a part of complementary of this second kind of oligonucleotide at least and use probe, detect this oligonucleotide that be connected with the multiple base of a part of complementary of this second kind of oligonucleotide and first kind of oligonucleotide with probe in detecting or/and contain at least.
30. a test kit is to identify the test kit that is contained in the multiple nucleotide polymorphisms in the sample simultaneously, it is characterized in that comprising at least following (i)～(v):
(i) the two or more first kind of oligonucleotide that comprises nucleotide polymorphisms sequence part;
The (ii) two or more second kind of oligonucleotide that comprises sequence adjacent with first kind of oligonucleotide and that do not hybridize with this nucleotide polymorphisms sequence part;
The (iii) at least a enzyme that contains nuclease;
The (iv) at least a active enzyme of ligase enzyme that contains;
(v) detect with a part of complementary of second kind of oligonucleotide at least and use probe, and/or at least with a part of complementary detection probe of second kind of oligonucleotide and first kind of oligonucleotide.
31. a test kit is to identify the test kit that is contained in the multiple nucleotide polymorphisms in the sample simultaneously, it is characterized in that comprising at least
Below (i)～(vi):
(i) have in the sequence part of nucleotide polymorphisms in supposition, contain first kind of oligonucleotide of two or more polymorphisms with the sequence of the base complementrity that shows polymorphism;
(ii) have in the sequence part of nucleotide polymorphisms, contain second kind of oligonucleotide of two or more wild-types with the sequence of the base complementrity that shows wild-type in supposition;
(iii) contain with polymorphism and/or first kind of oligonucleotide of wild-type is adjacent and second kind of two or more oligonucleotide of the sequence that nucleotide polymorphisms sequence part do not hybridize arranged in this supposition;
The (iv) at least a enzyme that contains nuclease;
(the v) at least a active enzyme of ligase enzyme that contains;
(vi) detect with a part of complementary of second kind of oligonucleotide at least and use probe, and/or at least with a part of complementary detection probe of second kind of oligonucleotide and first kind of oligonucleotide.
32. claim 30 or 31 described test kits, containing predetermined fixed has the solid phase that detects with probe.
33. claim 30 or 31 described test kits is characterized in that solid phase is a dna microarray.
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| CN103328956A (en) * | 2011-01-26 | 2013-09-25 | 奥林巴斯株式会社 | Method for identifying polymorphism of nucleic acid molecule |
| CN106755402A (en) * | 2016-12-22 | 2017-05-31 | 远见生物科技（上海）有限公司 | A kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII |
| CN109371115A (en) * | 2018-08-24 | 2019-02-22 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs5918 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4294732B2 (en) * | 1996-02-09 | 2009-07-15 | コーネル・リサーチ・ファンデーション・インコーポレイテッド | Detection of nucleic acid sequence differences using ligase detection reactions in addressable arrays |
| US6506594B1 (en) * | 1999-03-19 | 2003-01-14 | Cornell Res Foundation Inc | Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays |
| JP4310599B2 (en) * | 2000-07-05 | 2009-08-12 | 東洋紡績株式会社 | Method for detecting nucleotide polymorphisms |
| CN1197976C (en) * | 2003-01-31 | 2005-04-20 | 中国人民解放军南京军区联勤部军事医学研究所 | Method for detecting polymorphism of genes |
-
2005
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- 2005-12-12 CN CN200580040391.9A patent/CN101065483B/en not_active Expired - Fee Related
- 2005-12-12 JP JP2006520590A patent/JPWO2006064745A1/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103328956A (en) * | 2011-01-26 | 2013-09-25 | 奥林巴斯株式会社 | Method for identifying polymorphism of nucleic acid molecule |
| CN103328956B (en) * | 2011-01-26 | 2015-08-12 | 奥林巴斯株式会社 | Differentiate the method for polymorphic nucleic acid molecule |
| CN106755402A (en) * | 2016-12-22 | 2017-05-31 | 远见生物科技（上海）有限公司 | A kind of method for detecting single nucleotide polymorphism based on heat stable nuclease HII |
| CN109371115A (en) * | 2018-08-24 | 2019-02-22 | 山东德诺生物科技有限公司 | For detecting the primed probe group and its application of rs5918 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2006064745A1 (en) | 2006-06-22 |
| CN101065483B (en) | 2013-02-06 |
| JPWO2006064745A1 (en) | 2008-06-12 |
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