CN101280338B - Nucleic acid amplification method for detecting polymorphism of nucleic acid - Google Patents
Nucleic acid amplification method for detecting polymorphism of nucleic acid Download PDFInfo
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Abstract
The invention provided a nucleic acid amplification method for detecting nucleic acid polymorphyism. The method is to utilize the probe the basic structure of which is shown in the figure to be interbred with a target molecule, and a base interstice exists in the formed probe-target molecule crossbred, then the interstice in the probe-target molecule crossbred is filled through polymerization-connection reaction, in order to ensure the two monomers to connected into a single chain to be the template used for the next polymerase chain reaction amplification, finally, the analyzing on nucleotide polymorphyism of the target molecule is performed through detecting the polymerase chain reaction amplification product. The method has the advantages that the template used for the next polymerase chain reaction amplification can be obtained only through polymerization-connection reaction, therefore the steps, such as restriction enzyme or extension, are omitted, and the detecting efficiency is improved.
Description
Technical field
The present invention relates to a kind of nucleic acid amplification method, in particular for the nucleic acid amplification method of polymorphic nucleic acid detection.
Background technology
Single nucleotide polymorphism (single nucleotide polymorphisms, SNP) and the detection of polymorphic nucleic acid such as transgenation normally utilize multiplex PCR (polymerase chain reaction) technology, the principle of this technology is the multiple target molecule that increases simultaneously in same reaction tubes, analysis throughput is improved greatly, also can save the nucleic acid template consumption of sample to be checked, 5-10 the target molecule fragment but most at present multiple PCR methods all is confined to increase simultaneously, its major cause is that the heavy PCR of n-must add n to different primers, be 2n primer, this makes the condition of pcr amplification reaction be difficult to control, in addition, the interaction between the primer is easy to produce primer two aggregates and non-specific amplification.Therefore, the bottleneck of multiple PCR technique is how to reduce the usage quantity of primer when improving its amplification multiplicity, thereby makes multiplex PCR become real high-throughput nucleic acid analysis technology platform.
Universal primer is one of a kind of important method that improves the multiplex PCR strategy.In the design of multiplex PCR, the design of universal primer adopts certain mode to make it to be present in the nucleic acid template or elementary pcr amplification product to be amplified usually, for example based on the multiple PCR technique of GoldenGate, MIP and SNPlex, thereby can adopt universal primer that the target molecule sequence is carried out high-pass expanding, greatly improve the multiplicity of multiplex PCR.
MIP technology (Hardenbol P, Baner J, Jain M et al.Multiplexed genotypingwith sequence-tagged molecular inversion probes.Nat Biotechnol.2003; 21 (6): 673-678) with SNPlex technology (Tobler AR, Short S, Andersen MR et al.TheSNPlex genotyping system:a flexible and scalable platform for SNPgenotyping.J Biomol Tech.2005; 16 (4): though probe structure 398-406.) has than big-difference, method is basic identical, and its main process is: 1, hybridize with target gene complementary sequence and target gene in the probe; 2, under the effect of archaeal dna polymerase, mend in the flat probe and the corresponding base in SNP site, and two sections sequences are connected by ligase enzyme; 3, shear the restriction enzyme site that is arranged on primer sequence part in the probe with restriction enzyme, make the probe linearizing; 4, the probe after cutting with enzyme adds primer as template and carries out pcr amplification; 5, detect amplified production, mend the sequence that the base of going into can be judged target gene SNP site according to step 2.These two kinds of technology all need to utilize enzyme cut with target gene hybridization after the probe linearizing could make operation become complicated loaded down with trivial details as the template of follow-up pcr amplification, reduced the efficient that detects, even might influence the tolerance range of detection.
GoldenGate technology (Shen R, Fan JB, Campbell D et al.High-throughputSNP genotyping on universal bead arrays.Mutat Res.2005; 573 (1-2): characteristics 70-82) are that probe is made up of two oligonucleotide chains, every chain has all comprised and primer sequence and two parts of target-gene sequence complementary, wherein a chain also contains sequence label, the end of another chain and target-gene sequence complementary part and the SNP site of target gene are corresponding, after these two probes and target gene hybridization, if in the probe wherein with the end of target-gene sequence complementary part and the SNP site complementation of target gene, under the effect of archaeal dna polymerase, be that template extends to another probe junction then with the target gene, and two fragments are connected by ligase enzyme, add primer then and carry out pcr amplification as template with the sequence after connecting, detect amplified production at last, can be according to judging the SNP site sequence with the corresponding terminal bases in SNP site of target gene in the probe.Though this method does not need to make the pcr template linearizing with the enzyme blanking method, need use by the extension of a probe wherein and realize the connection of two probes, thereby obtain the template of follow-up PCR, therefore, this technology does not still solve the defective that above-mentioned technology exists.
Summary of the invention
The technical problem to be solved in the present invention is to improve the detection efficiency of the nucleic acid amplification method that is used for the polymorphic nucleic acid detection.
The technical scheme that the present invention solves the problems of the technologies described above is:
A kind of nucleic acid amplification method that detects nucleotide polymorphisms, this method may further comprise the steps:
(1) design and preparation LDPP probe, described LDPP probe comprises upstream oligonucleotide monomer and downstream oligonucleotide monomer; Wherein, upstream oligonucleotide is monomeric 5 '-and 3 '-end be respectively universal sequence district and complementary district, the downstream oligonucleotide is monomeric 5 '-and 3 '-end then be respectively complementary district and universal sequence district; The corresponding sequence complementation of mode that upstream and downstream oligonucleotide monomer adjoins with head-tail and same nucleic acid strand of target molecule, and, in LDPP probe-target molecule crossbred, (structure of LDPP probe of the present invention-target molecule crossbred as shown in Figure 1 only the gap of a base between the monomeric head-tail of upstream and downstream oligonucleotide, P1, P2 is the universal sequence district of probe, C1, C2 is the complementation district of probe, sequence among the figure only is on illustrating in the crossbred, the monomeric position of downstream oligonucleotide relation is not the definite sequence of probe of the present invention or target gene);
(2) establish 1～4 reaction system, each reaction system all adds target gene and at least a LDPP probe, carry out polymerization-ligation then under the condition that triphosphate deoxyribose nucleotide, archaeal dna polymerase and dna ligase exist, the triphosphate deoxyribose nucleotide described in each reaction system is respectively a kind of among dATP, dTTP, dCTP and the dGTP;
(3) with the reaction product of step (2) gained as reaction template, add with the corresponding universal primer of LDPP probe and carry out pcr amplification; Described universal primer, wherein a universal primer is identical with the sequence in the monomeric universal sequence of an oligonucleotide district in the probe, another universal primer then with probe in the sequence complementation in the monomeric universal sequence of another oligonucleotide district;
(4) amplified production of detection step (3) gained carries out qualitative and/or quantitative analysis according to whether the exist amplified production and the abundance thereof of each reaction system.
The described LDPP probe of the inventive method can also comprise upstream complementary oligonucleotide monomer and downstream complementary oligonucleotide monomer, part or all of other nucleic acid array complementation outside the monomeric complementary district of described upstream complementary oligonucleotide monomer and upstream oligonucleotide, part or all of other nucleic acid array complementation outside downstream complementary oligonucleotide monomer is then distinguished with the monomeric complementation of downstream oligonucleotide, thereby improve complementary validity of hybridizing of LDPP probe and target molecule and specificity (as shown in Figure 2, LDPP-1 and LDPP-2 are respectively upstream oligonucleotide monomer and downstream oligonucleotide monomer, and LDPP-3 and LDPP-4 are respectively upstream complementary oligonucleotide monomer and downstream complementary oligonucleotide monomer; Sequence among the figure only is for the monomeric position of upstream and downstream oligonucleotide relation in the crossbred is described, is not the definite sequence of probe of the present invention or target gene).
The upstream complementary oligonucleotide monomer of the described LDPP probe of the inventive method and/or downstream complementary oligonucleotide monomer can be designed to have the structure of following one or more features:
(1) the universal sequence district in upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer directly links to each other with complementary district, as shown in Figure 3A: P1 and P2 are respectively upstream oligonucleotide monomer and the monomeric universal sequence of downstream oligonucleotide district, and C1 and C2 are respectively upstream oligonucleotide monomer and the monomeric complementary district of downstream oligonucleotide; Perhaps
(2) there is the intervening sequence district between universal sequence district in upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer and the complementary district, described intervening sequence district has versatility, the minimum length of its sequence is a base, maximum length is unrestricted, intervening sequence each other can be same or similar, but and there is not cross hybridization between the universal primer, and, the species gene group nucleotide sequence of described intervening sequence and thing to be detected does not have homology or homology is lower, shown in Fig. 3 C, the S in the probe is the intervening sequence district; Perhaps
(3) there is the sequence label district between universal sequence district in upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer and the complementary district, described sequence label district has autospecific, the length of sequence label is the oligonucleotide of 10-40 aggressiveness, each other and and universal primer between do not have cross hybridization, and, the species gene group nucleotide sequence of described sequence label and thing to be detected does not have homology or homology is lower, and shown in Fig. 3 B, the T in the probe is the sequence label district; Perhaps
(4) have restriction enzyme site between universal sequence district in upstream oligonucleotide monomer and/or the downstream oligonucleotide monomer and the complementary district, shown in Fig. 3 B, the X in the probe is a restriction enzyme site.
The inventive method can also become the LDPP probe design of the different target molecules of target the fragment of different lengths, can directly come qualitative and/or detection by quantitative target molecule like this, to realize detecting simultaneously the purpose of a plurality of different target molecules by the difference of pcr amplification product length and/or abundance.When target molecule was RNA or mRNA, the complementation district of LDPP probe of the present invention can also be Oligo d (T), and the length of Oligo d (T) is the 8-40 aggressiveness.
The part or all of base of LDPP probe of the present invention can be base analogue or base modification thing, can also introduce artificial base mismatch in the complementary district of two target molecules in the LDPP probe, described artificial base mismatch is natural four kinds of bases of A, T, C, G or its analogue.
Detect amplified production for convenience, one or more that can be in upstream oligonucleotide monomer, downstream oligonucleotide monomer, upstream complementary oligonucleotide monomer and the downstream complementary oligonucleotide monomer of LDPP probe of the present invention are monomeric 5 '-and/or 3 '-end carry out the modification or the mark of phosphate group, oh group or other similar group; Also can be in one or more mark fluorescent molecules, luminophore, rare elements or non-fluorescence chromophoric group in four kinds of triphosphate deoxyribose nucleotides that step (2) adds, vitamin H, digoxin molecule, isotropic substance, other similar light emitting molecule or the tagged molecule that is fit to separation or differentiates.
Polymerization-the ligation of the inventive method step (2) comprises following four processes: sex change, renaturation, polymerization and be connected.Wherein,
1. sex change: target molecule is sex change in advance, or sex change after having added a series of reagent, makes the target molecule single stranded; Normally 92～99 ℃ of denaturation temperatures, the time length is 1sec～10min, preferred temperature is 94～96 ℃, sex change time 2～5min.
2. renaturation: whole polymerization-ligation system is placed certain temperature, the complementation district that makes the LDPP probe respectively with the complementary hybridization of target molecule, form LDPP probe-target molecule crossbred; Normally 25～72 ℃ of this temperature, time length 30sec～30min; 45～65 ℃ of preferred temperature, time length 5～15min.
3. polymerization-connection: under proper temperature, when single base (as: base T) coupling of a kind of triphosphate deoxyribose nucleotide (as: dATP) of unique existence in the reaction system and LDPP probe-target molecule crossbred middle and upper reaches and downstream oligonucleotide monomer head-tail gap location or when complementary, then under the effect of archaeal dna polymerase, archaeal dna polymerase extends LDPP probe-monomeric 3 '-not terminal sequence of target molecule crossbred middle and upper reaches oligonucleotide, is padded to monomeric 5 '-not end of downstream oligonucleotide according to 5 ' → 3 ' direction.Archaeal dna polymerase described here does not possess the strand displacement activity, therefore, chain extension to the downstream oligonucleotide monomeric 5 '-not the end after, archaeal dna polymerase promptly comes off from nucleic acid-templated, at this moment, dna ligase sealing LDPP probe upstream and downstream oligonucleotide is monomeric 3 '-and 5 '-not breach between holding, make the LDPP probe be transformed into a complete oligonucleotide single chain molecule.In this stage, extend and be connected coexistence, have in the formed single strand dna and universal primer bonded universal sequence, utilize the universal primer corresponding with universal sequence, can carry out pcr amplification to single strand dna.When the LDPP probe shown in use Fig. 3 B, just contain the sequence label district in the single strand dna that polymerization-ligation forms; When the LDPP probe shown in use Fig. 3 C, just contain the intervening sequence district in the single strand dna that polymerization-ligation forms.
Archaeal dna polymerase in polymerization-ligation of the present invention and dna ligase (the perhaps nucleic acid polymerase of other type and nucleic acid ligase, other enzyme that perhaps has ligase enzyme and polymerase activity simultaneously) can be low temperature or normal temperature, also can be heat-stable.In the reaction system except adding a kind of dna ligase (perhaps nucleic acid ligase of other type, other enzyme that perhaps has ligase enzyme and polymerase activity simultaneously) outside, also can add dna ligase more than 2 kinds or 2 kinds (the perhaps nucleic acid ligase of other type perhaps has other enzyme of ligase enzyme and polymerase activity simultaneously) mixture.In the reaction system except adding a kind of archaeal dna polymerase (the perhaps nucleic acid polymerase of other type, other enzyme that perhaps has ligase enzyme and polymerase activity simultaneously), also can add archaeal dna polymerase more than 2 kinds or 2 kinds (the perhaps nucleic acid polymerase of other type perhaps has other enzyme of ligase enzyme and polymerase activity simultaneously) mixture.
Polymerization-the ligation of the inventive method step (2) can repeatedly circulate, and promptly passes through the reaction of repetitious sex change, renaturation, extension and four steps such as being connected, to improve the efficient of polymerization-ligation.The polymerization of step (2)-ligation product can also adopt one or more exonucleases to handle, and only makes the LDPP probe that has connected into an integral body keep its integrity, reduces the complicacy of polymerization-ligation product.
Target molecule described in the inventive method step (2) or thing to be detected are meant various nucleic acid molecule, comprise the oligonucleotide analogs, polymerase chain reaction (PCR) of polymerized nucleoside acid-like substance, RNA or the DNA of DNA, RNA, RNA or DNA or the preparation of other technology nucleic acid product, mRNA, cDNA, comprise nucleic acid that bisulfite modifies the whole bag of tricks of (bisulfite-modification) and modify, comprise the responsive restriction enzyme that methylates (methylation-sensitive restriction endonuclease, MSRE; For example HpaII and HhaI) nucleic acid product, other similar nucleic acid fragment of handling at interior various restriction enzymes or other nucleic acid enzyme.
One or more of four kinds of triphosphate deoxyribose nucleotides (dATP, dCTP, dGTP, dTTP) that the inventive method step (2) is used are marked with fluorescence molecule, luminophore, rare elements or non-fluorescence chromophoric group, vitamin H, digoxin molecule, isotropic substance, or other similar light emitting molecule or suitable mark or the decorating molecule that separates or differentiate pcr amplification product.
Polymerization-the ligation of the inventive method step (2) can be carried out respectively in 1,2,3 or 4 reaction tubess, but, only use a kind of of four kinds of triphosphate deoxyribose nucleotides (dATP, dCTP, dGTP, dTTP) in each reaction tubes, other reacted constituent of each reaction tubes is then identical with condition, for example, when the polymerization ligation when 4 reaction tubess carry out respectively, 4 reaction tubess use dATP, dCTP, dGTP and dTTP respectively, and other reacted constituent of 4 reaction tubess is then identical with condition.
The inventive method step (3) is to be template with step (2) products therefrom, carries out pcr amplification with universal primer, wherein,
The processing of 1. polymerization-ligation product: the upstream and downstream oligonucleotide monomer in the LDPP probe in described LDPP probe-target molecule crossbred is after polymerization-ligation forms a complete oligonucleotide single strand dna, adopt one or more exonucleases or nuclease or other enzyme to handle polymerization-ligation product, thereby the complete single strand dna that LDPP probe is only formed still keep its integrity; Perhaps use and catch elution system collection polymerization-ligation product; The perhaps not treated pcr amplification that directly carries out.
2. pcr amplification: polymerization-ligation product behind above-mentioned treatment step as the pcr amplification template, perhaps not treated directly as the pcr amplification template.In the pcr amplification system, use a pair of universal primer corresponding that polymerization-ligation product is carried out pcr amplification with the universal sequence of LDPP probe.The formation of PCR reaction system mainly comprises: polymerization-ligation product, a pair of universal primer, four kinds of triphosphate deoxyribose nucleotides (dATP, dCTP, dGTP, dTTP), hot resistant DNA polymerase and suitable buffer system thereof.Utilize universal primer, guaranteed that each polymerization-ligation product all can be by the equivalence amplification.
The pcr amplification of the inventive method step (3) can carry out in 1～4 reaction system, and its reaction template is respectively the corresponding product of step (2) gained; Can also carry out in 1 reaction system, its reaction template is the mixture of each reaction system products therefrom of step (2).
The inventive method step (3) used universal primer can be marked with fluorescence molecule, luminophore, rare elements or non-fluorescence chromophoric group, vitamin H, digoxin molecule, isotropic substance, or other similar light emitting molecule or be fit to separate or differentiate the mark or the decorating molecule of pcr amplification product.The all or part of base of universal primer can be base analogue or base modification thing.
One or more of four kinds of triphosphate deoxyribose nucleotides (dATP, dCTP, dGTP, dTTP) that the inventive method step (3) is used are marked with fluorescence molecule, luminophore, rare elements or non-fluorescence chromophoric group, vitamin H, digoxin molecule, isotropic substance, or other similar light emitting molecule or suitable mark or the decorating molecule that separates or differentiate pcr amplification product; Perhaps applying marking has the dUTP of above-mentioned tagged molecule partly or entirely to substitute dTTP.
Can also use universal primer to carry out for the second time or the pcr amplification product of the inventive method step (3) gained that repeatedly increases, this reaction can be carried out in 1～4 reaction system, and its reaction template is respectively the pcr amplification product of step (3) gained; Can also carry out in 1 reaction system, its reaction template is the mixture of each reaction system products therefrom of step (3).
The method of the described detection pcr amplification product of the inventive method step (4) can realize by following several modes: 1. directly detect amplified production with agarose gel electrophoresis, polyacrylamide gel electrophoresis, hair cell electrophoresis tube or other similar electrophoretic technique.
2. the detection system of PCR-based amplified production length: when the length of the LDPP probe of employed each target molecule of target in the reaction system is different, the length of the pcr amplification product of each target molecule correspondence is different, can use the quantitative and/or qualitative detection target molecule of detection system of PCR-based product length.The detection system of PCR-based amplified production length comprises agarose gel electrophoresis, polyacrylamide gel electrophoresis, hair cell electrophoresis tube and other similar electrophoretic technique.
3. the detection system of PCR-based amplified production sequence label: there is not the difference of length in the LDPP probe of the different target molecules of employed target in reaction system, perhaps the difference of length is very little, but when all having the sequence label of autospecific, can directly come quantitatively and/or the qualitative detection target molecule by the sequence label that detects in the PCR product.The detection system of the sequence label in the PCR-based amplified production is included in its solid-phase matrix surface and is fixed with identical with above-mentioned sequence label or complementary general-purpose chip, other similar array or microballon technology etc., the single check point that it is characterized in that general-purpose chip, other similar array or microballon technology etc. all be fixed with every kind of target molecule pcr amplification product in the identical or complementary oligonucleotide fragment of sequence label, after pcr amplification product and its hybridization, can having or not and the next quantitative and/or qualitative detection target molecule of intensity level by the hybridization point.
4. based on the detection system of real-time fluorescence PCR or real-time fluorescence quantitative PCR: adopt identical or complementary sequence is the target sequence with the LDPP probe sequence, perhaps adopt identical or complementary sequence is the target sequence, adopt based on TaqMan probe, molecular beacon or other similar real-time fluorescence PCR or real-time fluorescence quantitative PCR technology and come quantitative and/or qualitative detection target molecule with target molecule row in polymerization-ligation product or the pcr amplification product.
After described LDPP probe of the inventive method and target molecule form LDPP probe-target molecule crossbred, the upstream and downstream oligonucleotide monomer of LDPP probe adjoins the corresponding sequence complementation of form and same nucleic acid strand of target molecule with head-tail, this moment, upstream and downstream oligonucleotide monomer was only to exist the gap of a base between the head-tail of head-tail, then by the archaeal dna polymerase of high-efficiency polymerization ability mend the upstream and downstream oligonucleotide of LDPP probe in flat LDPP probe-target molecule crossbred monomeric 5 '-and 3 '-between the end behind the gap of 1 base, use the dna ligase closing gap again, so just formed the required template of next step universal primer PCR amplification, and do not need to obtain through steps such as restriction enzyme processing or further extensions, not only improve the efficient that detects, also improved the tolerance range that detects.When in polymerization-ligation, in 4 reaction tubess, carrying out respectively, and when only using a kind of triphosphate deoxyribose nucleotide in each reaction tubes, just can whether exist the male pcr amplified fragment to analyze the target molecule base corresponding with above-mentioned triphosphate deoxyribose nucleotide, thereby reach high throughput testing and analysis polymorphic nucleic acids such as SNP and transgenations by detecting its corresponding polymerization-ligation product in above-mentioned gap.
In the present invention, except that other special instruction, following word/term has following implication:
" nucleic acid " of the present invention is meant Yeast Nucleic Acid (RNA), thymus nucleic acid (DNA), the polymerized nucleoside acid-like substance of RNA or DNA, the oligonucleotide analogs of RNA or DNA, the nucleic acid product of polymerase chain reaction (PCR) or the preparation of other technology, mRNA, cDNA, the nucleic acid that process is modified (for example: the genomic dna that bisulfite is modified), the nucleic acid product that various restriction enzymes or other enzymic digestion or enzyme are cut (for example: the responsive restriction enzyme that methylates (methylation-sensitive restriction endonuclease, digestion product MSRE)), other similar nucleic acid fragment.
" oligonucleotide " of the present invention is meant small molecules nucleic acid, connects (polymerization) by nucleotide residue (fragment) by phosphodiester bond and forms, and molecular weight and tends to Nucleotide between nucleic acid and Nucleotide.The present invention there is no strict boundary to the number of nucleotide residue.
" monomer " of the present invention is meant by single stranded oligonucleotide.
" target molecule " of the present invention is meant the material that adopts the method for the invention directly or indirectly to detect, and mainly comprises (but being not limited to) nucleic acid.
" thing to be detected " of the present invention is meant and contains the various samples or the analysans that maybe may contain target molecule.
" qualitative detection " of the present invention is meant that whether direct or indirect detection target molecule exists, and perhaps directly or indirectly detects target molecule and whether is present in thing to be detected.
" detection by quantitative " of the present invention is meant the concentration of direct or indirect detection target molecule, perhaps directly or indirectly detects the concentration of target molecule in the thing to be detected, for example, detects the copy number of target molecule in the thing to be detected.
" complementary district " described herein is meant in the probe and target molecule complementary oligonucleoside sequence area.
" universal sequence district " described herein is meant oligonucleoside sequence area corresponding with universal primer in the probe.
" sequence label district " described herein is meant distinctive one section oligonucleoside sequence area in every kind of LDPP probe, has autospecific, the length of sequence label is the oligonucleotide of 10-40 aggressiveness, each other and and universal primer between do not have cross hybridization, and the species gene group nucleotide sequence of described sequence label and thing to be detected does not have homology or homology is lower.
" intervening sequence district " described herein is meant the same or analogous one section oligonucleoside sequence area that exists at multiple LDPP probe, and, in different LDPP probes, its length can there are differences, and minimum length is a base, and maximum length does not have strict restriction, intervening sequence each other can be same or similar, but and do not have cross hybridization between the universal primer, and the species gene group nucleotide sequence of described intervening sequence and thing to be detected does not have homology or homology is lower.
" restriction enzyme site district " described herein is meant the recognition site of the specific limited restriction endonuclease of artificial design in the LDPP probe.
" LDPP probe-target molecule crossbred " described herein is meant the mode and the complementary hybridization of the corresponding sequence of certain bar single-chain nucleic acid of target molecule that the upstream and downstream oligonucleotide monomer in the LDPP probe adjoins with head-tail, and, the gap of a base is only arranged between the monomeric head-tail of upstream and downstream oligonucleotide of LDPP probe.
" solid-phase matrix " of the present invention comprises sheet glass, silicon chip, ceramic plate, plastics, cellulose nitrate, nylon membrane or the rubber etc. that several different methods is handled.
" fixing " of the present invention is meant the surface that oligonucleotide probe or target molecule is connected in solid-phase matrix by physical adsorption and/or chemical coupling connection mode.
" physical adsorption " of the present invention is meant that oligonucleotide probe or target molecule are by secondary key (for example: ionic linkage) link to each other with the solid-phase matrix surface and fix, or with the non covalent bond effect with oligonucleotide probe or target molecule directly or constant potential be adsorbed onto the surface of solid-phase matrix, perhaps pass through electrostatic interaction and fix by the positively charged decorative layer in phosphate radical negative ion in the meta oligonucleotide probe and solid-phase matrix surface.
" chemical coupling connection " of the present invention is by (for example: amido linkage forming covalent linkage, ester bond, ehter bond etc.) active group on oligonucleotide probe or target molecule and solid-phase matrix surface is interacted, thereby oligonucleotide probe or target molecule are fixed to the surface of solid-phase matrix, for example: at first pre-activated is handled the surface of solid-phase matrix, introduce various required active groups, as amino, carboxyl, sulfydryl, hydroxyl, halogen radical (comprises fluorine, chlorine, bromine, iodine etc.) etc., perhaps derivatized nucleotide, make it with going up suitable functional gene, with the contact of difunctionality reagent or coupling activator oligonucleotide probe or target molecule are fixed to the surface of solid-phase matrix subsequently, double functional group commonly used has glutaraldehyde (GA), p-nitrophenyl chloroformate (NPC), maleimide (MA), diisothio-cyanate etc.
Description of drawings
Fig. 1 is the design and structure principle intention of LDPP probe.
Fig. 2 is the upstream complementary oligonucleotide monomer and the monomeric principle of design synoptic diagram of downstream complementary oligonucleotide of LDPP probe.
Fig. 3 is the principle of design synoptic diagram of multiple structure LDPP probe.
The multiple SNP that Fig. 4 is based on LDPP probe length difference detects principle schematic.
Fig. 5 is based on the principle schematic of the general biological chip high throughput testing SNP of LDPP probe label difference.
Embodiment
To further set forth the inventive method by concrete example below, but the inventive method is not limited to following limited example.Those skilled in the art can make various changes or modifications the present invention according to the content and the actual needs of specification sheets, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment one: based on the multiple SNP detection method of LDPP probe as shown in Figure 4, its concrete implementation step example is as follows:
(1) make up the LDPP probe:
As shown in Figure 4, the LDPP probe by a pair of oligonucleotide monomer (shown in Fig. 4 A, sequence among the figure only is for the monomeric position of upstream and downstream oligonucleotide relation in the crossbred is described, be not the definite sequence of probe of the present invention or target gene) form, wherein, upstream oligonucleotide monomer 5 '-to 3 '-nucleotide sequence of end is universal sequence district, intervening sequence district and complementary district successively, the downstream oligonucleotide is monomeric 5 '-to 3 '-nucleotide sequence of end is complementary district and universal sequence district successively; In LDPP probe-target molecule crossbred, the mode that the upstream and downstream oligonucleotide monomer of LDPP probe adjoins with head-tail and the corresponding sequence complementation of same single-chain nucleic acid of target molecule, only there is the gap of a base in upstream and downstream oligonucleotide monomer between the head-tail in complementation hybridization zone, and this gap is good corresponding to place, SNP site base (shown in Fig. 4 A); There is the above difference (shown in Fig. 4 C) of 5nt in the length of the monomeric intervening sequence of upstream oligonucleotide in the different SNP of target site, therefore, can directly judge the pcr amplification product of different SNP site correspondence by the length scale of PCR product.
(2) polymerization-ligation:
(shown in Fig. 4 B) carried out in polymerization-ligation respectively in 4 reaction tubess, only use a kind of (dXTP among Fig. 4 B) in four kinds of triphosphate deoxyribose nucleotides (dATP, dTTP, dCTP and dGTP) in each reaction tubes respectively, remaining reacted constituent is then identical.Therefore, owing to only use a kind of triphosphate deoxyribose nucleotide in each reaction tubes, therefore, on behalf of the SNP site, the polymerization of each reaction tubes-ligation product whether have corresponding base, simultaneously, because 4 reaction tubess are all taken the base that each SNP site may exist into account, therefore, are suitable for the detection in all SNP sites.
(3) universal primer PCR amplification
Polymerization-ligation product in 4 reaction tubess of step (2) is carried out pcr amplification respectively in 4 reaction tubess, the fluorescence molecule of different colours on upstream primer in the universal primer in each reaction tubes (or downstream primer) mark, remaining reacted constituent is then identical.Owing to only use a kind of triphosphate deoxyribose nucleotide (dXTP among Fig. 4 B) in each polymerization-ligation pipe, and in the amplification in this step, the fluorescence color of universal primer mark is also different in each reaction tubes, therefore, can judge whether the SNP site exists and the corresponding base of triphosphate deoxyribose nucleotide that adds by fluorescence color; In addition, because the upstream oligonucleotide monomer of the LDPP probe in the different SNP of target site is owing to exist the difference of intervening sequence length, thereby finally cause the length of the pcr amplification product in different SNP site also to there are differences, therefore, finally can judge which kind of SNP site of detection by the length of pcr amplification product, and the fluorescence color of the pcr amplification product by its length-specific judges that specifically there is situation in the base in above-mentioned SNP site.
(4) detection of pcr amplification product
Pcr amplification product in 4 reaction tubess in the step (3) is mixed into a reaction tubes, adopt the capillary electrophoresis mode that the pcr amplification product mixed solution is carried out capillary electrophoresis then, at last, judge the kind and the base situation (shown in Fig. 4 D) in SNP site according to clip size and this segmental fluorescence color.
Embodiment two: based on the method for the general biological chip high throughput testing SNP of LDPP probe
As shown in Figure 5, its concrete implementation step example is as follows:
(1) makes up the LDPP probe
The LDPP probe is made up of a pair of oligonucleotide monomer (shown in Fig. 5 A), wherein, upstream oligonucleotide monomer 5 '-to 3 '-nucleotide sequence of end is universal sequence district, sequence label district, restriction enzyme site district and complementary district successively, a specific restriction endonuclease sites (X among Fig. 5 A) is contained in the restriction enzyme site district, and the downstream oligonucleotide is monomeric 5 '-to 3 '-nucleotide sequence of end is complementary district and universal sequence district successively; In LDPP probe-target molecule crossbred, the mode that the upstream and downstream oligonucleotide monomer of LDPP probe adjoins with head-tail and same the single-chain nucleic acid sequence complementation of target molecule, and, only there is the gap of a base in upstream and downstream oligonucleotide monomer between the head-tail in complementation hybridization zone, and this gap is good corresponding to SNP place base (Fig. 5 A); The monomeric sequence label of upstream oligonucleotide in the different SNP of target site has autospecific, therefore, can directly judge the pcr amplification product of different SNP site correspondence by the sequence label of pcr amplification product.
(2) polymerization-ligation:
(shown in Fig. 5 B) carried out in polymerization-ligation respectively in 4 reaction tubess, only use a kind of (dXTP among Fig. 5 B) in four kinds of triphosphate deoxyribose nucleotides (comprising dATP, dTTP, dCTP and dGTP) in each reaction tubes respectively, remaining reacted constituent is then identical.Owing to only use a kind of triphosphate deoxyribose nucleotide in each reaction tubes, therefore, on behalf of the SNP site, the polymerization of each reaction tubes-ligation product whether have corresponding base, simultaneously, because 4 reaction tubess are all taken the base that each SNP site may exist into account, therefore, be suitable for the detection in all SNP sites.
(3) universal primer PCR amplification
Polymerization-ligation product in 4 reaction tubess of step (2) is carried out pcr amplification respectively in 4 reaction tubess, the fluorescence molecule of different colours on upstream primer in the universal primer in each reaction tubes (or downstream primer) mark, remaining reacted constituent then identical (shown in Fig. 5 B).Owing to only use a kind of triphosphate deoxyribose nucleotide (dXTP among Fig. 4 B) in each polymerization-ligation pipe, and in the amplification in this step, the fluorescence color of universal primer mark is also different in each reaction tubes, therefore, can judge whether the SNP site exists and the corresponding base of triphosphate deoxyribose nucleotide that adds by fluorescence color; In addition, because the monomeric sequence label of upstream oligonucleotide of the LDPP probe in the different SNP of target site has autospecific, therefore, finally can judge which kind of SNP site of detection by the sequence label of pcr amplification product, and the fluorescence color of the pcr amplification product by its specific label sequence judges that specifically there is situation in the base in above-mentioned SNP site.(4) detection of pcr amplification product
Pcr amplification product in 4 reaction tubess in the step (3) is mixed into a reaction tubes, adopts the general biological chip to detect then, its concrete steps following (shown in Fig. 5 C):
1. will be fixed in the surface of solid-phase matrix with physical adsorption and/or chemical coupling connection mode with sequence label complementary oligonucleotide in the LDPP probe, it is characterized in that part or all of sequence in the oligonucleotide probe and the sequence label complementation in the LDPP probe.
2. the pcr amplification product in 4 reaction tubess in the step (3) is mixed into a reaction tubes, uses with the corresponding restriction enzyme of the specific limited restriction endonuclease recognition site (X among Fig. 5 A) in LDPP probe upstream oligonucleotide monomer restriction enzyme site district and handle pcr amplification product.
3. step enzyme is 2. cut the chip that 1. product and step prepare and hybridized, then, according to type and the fluorescence color of hybridization point and the type and the base distribution situation that strength of signal is judged SNP of sequence label.
Claims (15)
1. nucleic acid amplification method that detects polymorphic nucleic acid, this method may further comprise the steps:
(1) design and preparation LDPP probe, described LDPP probe comprises upstream oligonucleotide monomer and downstream oligonucleotide monomer; Wherein, upstream oligonucleotide is monomeric 5 '-and 3 '-end be respectively universal sequence district and complementary district, the downstream oligonucleotide is monomeric 5 '-and 3 '-end then be respectively complementary district and universal sequence district; The corresponding sequence complementation of mode that upstream and downstream oligonucleotide monomer adjoins with head-tail and same nucleic acid strand of target molecule, and, in LDPP probe-target molecule crossbred, only there is the gap of a base between the monomeric head-tail of upstream and downstream oligonucleotide;
(2) establish 4 reaction systems, each reaction system all adds target molecule and at least a LDPP probe, carry out polymerization-ligation then under the condition that triphosphate deoxyribose nucleotide, archaeal dna polymerase and dna ligase exist, the triphosphate deoxyribose nucleotide described in each reaction system is respectively a kind of among dATP, dTTP, dCTP and the dGTP;
(3) with the reaction product of step (2) gained as reaction template, add with the corresponding universal primer of LDPP probe and carry out pcr amplification; Described universal primer, wherein a universal primer is identical with the sequence in the monomeric universal sequence of an oligonucleotide district in the probe, another universal primer then with probe in the sequence complementation in the monomeric universal sequence of another oligonucleotide district;
(4) amplified production of detection step (3) gained carries out qualitative and/or quantitative analysis according to whether the exist amplified production and the abundance thereof of each reaction system.
2. method according to claim 1, it is characterized in that described LDPP probe can also comprise upstream complementary oligonucleotide monomer and downstream complementary oligonucleotide monomer, part or all of other nucleic acid array complementation outside part or all of other nucleic acid array complementation outside the monomeric complementary district of described upstream complementary oligonucleotide monomer and upstream oligonucleotide, downstream complementary oligonucleotide monomer are then distinguished with the monomeric complementation of downstream oligonucleotide.
3. method according to claim 1 is characterized in that upstream oligonucleotide monomer and/or the universal sequence district in the oligonucleotide monomer of downstream in the described LDPP probe directly links to each other with complementary district.
4. method according to claim 1, it is characterized in that having the intervening sequence district between upstream oligonucleotide monomer in the described LDPP probe and/or the universal sequence district in the oligonucleotide monomer of downstream and the complementary district, described intervening sequence district has versatility, the minimum length of its sequence is a base, maximum length is unrestricted, intervening sequence each other can be same or similar, but and there is not cross hybridization between the universal primer, and the species gene group nucleotide sequence of described intervening sequence and thing to be detected does not have homology or homology is lower.
5. method according to claim 1, it is characterized in that having the sequence label district between upstream oligonucleotide monomer in the described LDPP probe and/or the universal sequence district in the oligonucleotide monomer of downstream and the complementary district, described sequence label district has autospecific, the length of sequence label is the oligonucleotide of 10～40 aggressiveness, each other and and universal primer between do not have cross hybridization, and the species gene group nucleotide sequence of described sequence label and thing to be detected does not have homology or homology is lower.
6. method according to claim 1 is characterized in that having restriction enzyme site between upstream oligonucleotide monomer in the described LDPP probe and/or the universal sequence district in the oligonucleotide monomer of downstream and the complementary district.
7. method according to claim 1 is characterized in that described LDPP probe design with the different target molecules of target becomes the fragment of different lengths.
8. method according to claim 1 is characterized in that when target molecule is RNA or mRNA, and the complementation district of described LDPP probe can also be Oligo d (T), and the length of Oligo d (T) is 8～40 aggressiveness.
9. method according to claim 1, it is characterized in that in upstream oligonucleotide monomer, downstream oligonucleotide monomer, upstream complementary oligonucleotide monomer and the downstream complementary oligonucleotide monomer of described LDPP probe one or more monomeric 5 '-and/or 3 '-end carry out the modification or the mark of phosphate group, oh group or other similar group.
10. method according to claim 1, the part or all of base that it is characterized in that described LDPP probe can be base analogue or base modification thing.
11. method according to claim 1 is characterized in that the complementary district of two target molecules in the LDPP probe has introduced artificial base mismatch, described artificial base mismatch is natural four kinds of bases of A, T, C, G or its analogue.
12. method according to claim 1 is characterized in that one or more mark fluorescent molecules, luminophore, rare elements or non-fluorescence chromophoric group, vitamin H, digoxin molecule, isotropic substance, other the similar light emitting molecule in four kinds of triphosphate deoxyribose nucleotides that step (2) and/or step (3) add or is fit to the tagged molecule of separating or differentiating.
13. method according to claim 1, the reaction system that it is characterized in that step (2) is 4, and the used triphosphate deoxyribose nucleotide of each reaction system is respectively dATP, dTTP, dCTP and dGTP.
14. method according to claim 1, the upstream primer or the downstream primer that it is characterized in that all general primer of step (3) are marked with fluorescence molecule, luminophore, rare elements or non-fluorescence chromophoric group, vitamin H, digoxin molecule, isotropic substance, or other similar light emitting molecule or the suitable tagged molecule of separating or differentiating pcr amplification product.
15. method according to claim 1 is characterized in that the detection amplified production method of step (4) is:
(1) use agarose gel electrophoresis, polyacrylamide gel electrophoresis, hair cell electrophoresis tube and other similar electrophoretic technique to detect pcr amplification product; Perhaps
(2) use the surface to be fixed with general-purpose chip, other similar array or the microballon technology for detection pcr amplification product of the identical and/or complementary oligonucleotide of sequence label with the LDPP probe; Perhaps
(3) adopt TaqMan probe, molecular beacon or other similar real-time fluorescence PCR or real-time fluorescence quantitative PCR technology to detect polymerization-ligation product; Perhaps
(4) adopt TaqMan probe, molecular beacon or other similar real-time fluorescence PCR or real-time fluorescence quantitative PCR technology to detect pcr amplification product.
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| CN1185181A (en) * | 1995-04-13 | 1998-06-17 | 强生研究有限公司 | Method and amplifying specific nucleic acid squences |
| CN1351671A (en) * | 1999-04-01 | 2002-05-29 | 麦吉尔大学 | Transposon-based genetic marker |
| CN1678753A (en) * | 2002-06-25 | 2005-10-05 | 兰华生物科技公司 | Methods and compositions for monitoring primer extension and polymorphism detection reactions |
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| CN1185181A (en) * | 1995-04-13 | 1998-06-17 | 强生研究有限公司 | Method and amplifying specific nucleic acid squences |
| CN1351671A (en) * | 1999-04-01 | 2002-05-29 | 麦吉尔大学 | Transposon-based genetic marker |
| CN1678753A (en) * | 2002-06-25 | 2005-10-05 | 兰华生物科技公司 | Methods and compositions for monitoring primer extension and polymorphism detection reactions |
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