CN101220356B - Low copy biochemical reaction method - Google Patents
Low copy biochemical reaction method Download PDFInfo
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- CN101220356B CN101220356B CN200710036291XA CN200710036291A CN101220356B CN 101220356 B CN101220356 B CN 101220356B CN 200710036291X A CN200710036291X A CN 200710036291XA CN 200710036291 A CN200710036291 A CN 200710036291A CN 101220356 B CN101220356 B CN 101220356B
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Abstract
The invention discloses a low-copy biochemical reaction method, which includes that a low-copy reactant is introduced onto the tip of a nano-probe of a scanning probe microscope for carrying out reaction. The invention takes the nano-tip as a carrier, a surrounding water film as a support and creates a novel nano-reaction space. The molecules of the low-copy reactant are arranged in the small limited space of the limited tip surface area, which can greatly increase the probability of the collisions between the molecules and further improve the efficiency of the reaction. The invention is combined with the development of nano-manipulation technology which is based on the scanning probe microscope, the method of the invention can be used for the research of single-molecule biochemical reactions and single-molecule detection and other aspects, thereby having broad application prospect.
Description
Technical field
The present invention relates to a kind of method of low copy biochemical reaction.
Background technology
(low copy number LCN) is defined as the dna profiling amount [Gill P, Whitaker J.Forensic Sci.int., 2001,112:17-40.] that is lower than 100pg by Gill etc. to so-called low copy template at first.Therefore, now usually will be by the few reactant of at least a molecule number, the biochemical reaction that is less than 100 DNA participation as the molecule number is low copy biochemical reaction, but from present bibliographical information, unit molecule and low copy biochemical reaction are difficult for realizing.Because in essence, chemical reaction or biochemical reaction always occur in the particular environment, reaction molecular carries out effective collision in regular hour and space, when the reaction molecular number after a little while, if utilize conventional reaction method, the probability that effective collision takes place is just very little.Therefore, to nano-reactor, how people improve on the reaction efficiency problem quickly is being made great efforts always from the chemical reactor of routine.In fact, nature provides the reactor of ready-made specific region, from the simple relatively system of nano-scale, such as enzyme system, to the system of the relative complex of micron-scale, such as cell.For a synthetic chemistry man, the generation of specified time and spatial reaction is exactly need be in a limited space, the needed molecule that here responds, and conversion is finished in their effective collisions in the space of limitation.Therefore, how making up an artificial high-level efficiency reaction member is human ultimate illusion, and at present, the self-assembly system of micella, vesica even some molecules also becomes the carrier space of chemical reaction or biochemical reaction gradually.But these nanometer spaces all are to exist in a large number in the solution body, for concrete each nanometer or micron unit, and imperfectly understand, and can not obtain self-existent nano unit reaction lattice truly.And the technique means that generally relies in traditional unit molecule research is the doubling dilution in the solution, yet dilution itself is just inhomogeneous.Also there is bibliographical information to adopt the micro emulsion drop to improve efficient [the Anna Musyanovych of reaction as the nano-reactor of individual molecule, Volker Mailander, Katharina Landfester, Biomacromolecules 2005,6,1824-1828], still, these methods all have certain limitation, and they all are to have constituted whole reaction effect with each nanometer space of dispersive.
Summary of the invention
Therefore, the technical problem to be solved in the present invention provides a kind of method of low copy biochemical reaction, focuses on to bring up a really nano-reactor independently, can finish required biochemical reaction in the space of such limitation.
The inventor is surprised to find: because the nano-probe needle point of Scanning Probe Microscopy is under certain humidity, the probe tip surface can form water membrane by nature, thereby under the support of this moisture film, create one and be similar to the unitary independent nanometer reaction compartment of cell, reaction molecular is included the localized area arround needle point, increase the probability of collision of reactant molecule effectively, and then improve the efficient of reaction greatly.
Therefore, the present invention solves above-mentioned technical problem by following technical proposal.A kind of method of low copy biochemical reaction, it comprises and reacting after introducing the reactant of low copy on the nano-probe needle point of Scanning Probe Microscopy.
According to the present invention, all reactions all are the surfaces that is carried on probe tip, though the scan probe microscopic probe tip portion can not with the naked eye observe directly, probe base of Cun Zaiing and cantilever still are macroscopic observable as a whole.Thereby the temperature and humidity of nanometer pinpoint reactor can macro adjustments and controls, as long as can form water membrane by nature on the probe tip surface, so easy and simple to handle.The natural condition of the preferred normal temperature and pressure of the present invention, as under room temperature (18-25 ℃), controlling moisture reacts in the 50%-90% scope.
For ease of operation, the present invention is preferably under automatic inserting needle program, the reactant of low copy is introduced on the nano-probe needle point of Scanning Probe Microscopy, specifically, allow the automatic contact of nano-probe needle point of Scanning Probe Microscopy place the suprabasil first kind of reactant solution of microscope, first kind of reactant of low copy introduced on the nano-probe needle point, treated to introduce the remaining reaction thing with the same manner again after the needle point drying.After treating all to be incorporated into all reaction moleculars on the needle point, the various molecules of modifying needle surface all can converge to the tip portion of needle point under the effect of the moisture film that needle surface forms naturally, form a nano-area, finish reaction.Under automatic inserting needle program, probe can progressively play pin, behind the solution below the nanometer pinpoint contact, can stop automatically, just as dipping in the pen action, and probe tip is of a size of nano level, as calculated, the molecule number less (being less than 100 molecules) that mode like this is modified up belongs to the biochemical reaction to copy.
Because the ligation of DNA is convenient to observe and detect, the present invention as reactant, is that example illustrate the of the present invention biochemical reaction that support copy with the DNA ligation with dna molecular.Wherein, the composition of DNA ligation system is similar with the popular response system.
According to the present invention,, under certain humidity, under the moisture film that needle surface forms automatically supports, can bring up a nanometer reaction compartment as long as the probe tip size that adopts is in Nano grade.Because any scan probe microscopic probe all possesses these characteristics, so all applicable the present invention of all kinds probe of any Scanning Probe Microscopy.The present invention selects for use atomic force microscope (AFM) to illustrate for example.
Scanning Probe Microscopy all is to come " groping " world with small probe, not only can be on atomic level the detecting material pattern, and unit molecule nano-manipulation technology has based on this also grown up.The present invention with the probe tip of the Scanning Probe Microscopy reacting environment as low copy biochemical reaction, in conjunction with the advantage of Scanning Probe Microscopy, has realized the biochemical reaction of low copy dexterously.For example, can realize effectively that the DNA of low copy connects, and the biochemical reaction that conventional dependence dilution process carries out is not easy effective collision owing to the scope of activity that a few molecules has than large space, and then reaction efficiency is lower, reaction failure easily usually.And adopt method of the present invention, and dna molecular can be confined to nano-area, increased the collision of molecules probability effectively, solved the low problem of joint efficiency in the low copy DNA connection, and good reproducibility, simple to operate.Present method also can be used for the generation of other biochemical reactions effectively, and the combining nano manipulation technology can also carry out the research of faces such as unit molecule biochemical reaction and Single Molecule Detection.The inventive method has broad application prospects undoubtedly.
Description of drawings
Fig. 1 is a DNA linked system I synoptic diagram of the present invention.
Fig. 2 is the electrophorogram after the DNA linked system I of the present invention ligation; Wherein, 1: blank negative control, 2-7: the DNA after the ligation of the present invention (wherein 3 is embodiment 1), 8: the positive control of linked system, M:DL2000.
Fig. 3 DNA linked system of the present invention II synoptic diagram.
Fig. 4 is the electrophorogram after the DNA linked system II of the present invention ligation; Wherein, 1: blank negative control, 2-5: the DNA after the ligation of the present invention (wherein 3 is embodiment 5), 6: the positive control of linked system, M:DL2000.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.
Scanning Probe Microscopy of the present invention is selected atomic force microscope for use in the following example, and the probe tip model is NSC35.Wherein, embodiment 1-4 is DNA linked system I of the present invention, its ligation synoptic diagram as shown in Figure 1, embodiment 5-10 is DNA linked system II of the present invention, its ligation synoptic diagram as shown in Figure 3.
Embodiment 1
With 1 μ L15ng/ μ L, the DNA of 209bp (nucleotide sequence is shown in SEQ ID No.1 in the sequence table) solution is placed on the mica surface that 3-aminopropyltriethoxywerene werene (AP) is modified, under the automatic inserting needle program of atomic force microscope, progressively play pin, after needle point contact solution surface, automatically stop inserting needle, carry out the modification of needle surface, after drying, modify the DNA (nucleotide sequence is shown in SEQ ID No.2 in the sequence table) of 50bp of the 10ng/ μ L of the 1 μ L be complementary with it once more, the T4DNA ligase enzyme of 1 μ L, the mixed system of the 10 μ L that 1 μ L connection damping fluid and 7 μ L water are formed, the modification mode is the same, then, remained on room temperature and humidity and be 50% condition following two hours, take off needle point at last, with the dna molecular after connecting is that template is carried out pcr amplification, primer designs respectively at (the primer nucleotide sequence is respectively shown in SEQ ID No.3 in the sequence table and SEQ ID No.4) above two segment DNAs, electrophoresis result as shown in Figure 2, target stripe (175bp) has appearred, successful connection.By calculating as can be known, the molecule number of so modifying the DNA of the 209bp that gets on is less than 60, and the DNA that belongs to low copy connects.
The dna solution of 1 μ L5ng/ μ L, 209bp is placed on the mica surface that 3-aminopropyltriethoxywerene werene (AP) is modified, under the automatic inserting needle program of atomic force microscope, progressively play pin, after the contact solution surface, automatically stop inserting needle, carry out the modification of needle surface, after drying, T4DNA ligase enzyme, the 1 μ L that modifies 1ng/ μ L50bpDNA, the 1 μ L of the 1 μ L be complementary with it once more connects the mixed system of the 10 μ L that damping fluid and 7 μ L water are formed, and the modification mode is the same.Then, remained on room temperature and humidity and be 50% condition following two hours, take off needle point at last, verification method and result are with embodiment 1, successful connection.By estimation, the molecule number of so modifying the 209bp DNA that gets on is less than 20, and the DNA that belongs to low copy connects.
Kept being reflected at room temperature and humidity and be 60% condition following two hours, surplus with embodiment 1, take off needle point at last, verification method and result are with embodiment 1, successful connection.
Kept being reflected at room temperature and humidity and be 60% condition following two hours, surplus with embodiment 2, take off needle point at last, verification method and result are with embodiment 1, successful connection.
The dna solution of 1 μ L20ng/ μ L, 341bp is placed on the mica surface that 3-aminopropyltriethoxywerene werene (AP) is modified, under the automatic inserting needle program of atomic force microscope, progressively play pin, after the contact solution surface, automatically stop inserting needle, carry out the modification of needle surface, after drying, T4DNA ligase enzyme, the 1 μ L that modifies 10ng/ μ L50bpDNA, the 1 μ L of the 1 μ L be complementary with it once more connects the mixed system that damping fluid and 7 μ L water are formed, and the modification mode is the same.Then, remained on room temperature and humidity and be 70% condition following two hours, take off needle point at last, with the dna molecular (nucleotide sequence is shown in SEQ ID No.5 in the sequence table) after connecting is that template is carried out pcr amplification, primer designs respectively at (the primer nucleotide sequence is respectively shown in SEQ ID No.6 in the sequence table and SEQ IDNo.7) above two segment DNAs, electrophoresis result target stripe (391bp) occurs as shown in Figure 4, shows successful connection.By estimation, the molecule number of so modifying the 341bp DNA that gets on is less than 60, and the DNA that belongs to low copy connects.
Kept being reflected at room temperature and humidity and be 80% condition following two hours, surplus with embodiment 5, take off needle point at last, verification method and result show successful connection with embodiment 5.
Embodiment 7
The dna solution of 1 μ L5ng/ μ L, 341bp is placed on the mica surface that 3-aminopropyltriethoxywerene werene (AP) is modified, under the automatic inserting needle program of atomic force microscope, progressively play pin, after the contact solution surface, automatically stop inserting needle, carry out the modification of needle surface, after drying, T4DNA ligase enzyme, the 1 μ L that modifies 2.5ng/ μ L50bp DNA, the 1 μ L of the 1 μ L be complementary with it once more connects the mixed system that damping fluid and 7 μ L water are formed, and the modification mode is the same.Then, remained on room temperature and humidity and be 90% condition following two hours, take off needle point at last, verification method and result show successful connection with embodiment 5.By estimation, the molecule number of so modifying the 341bp DNA that gets on is less than 15, and the DNA that belongs to low copy connects.
Embodiment 8
The dna solution of 1 μ L5ng/ μ L, 341bp is placed on the mica surface that 3-aminopropyltriethoxywerene werene (AP) is modified, progressively play pin under the automatic inserting needle program under atomic force microscope, after the contact solution surface, automatically stop inserting needle, carry out the modification of needle surface, after drying, T4DNA ligase enzyme, the 1 μ L of DNA, 1 μ L that modifies the 1ng/ μ L50bp of the 1 μ L be complementary with it once more connects the mixed system that damping fluid and 7 μ L water are formed, and the modification mode is the same.Then, remained on room temperature and humidity and be 80% condition following two hours, take off needle point at last, verification method and result are with embodiment 5, and the result shows successful connection.
Embodiment 9
Kept being reflected at room temperature and humidity and be 60% condition following two hours, surplus with embodiment 8, take off needle point at last, verification method and result show successful connection with embodiment 5.
Embodiment 10
Kept being reflected at room temperature and humidity and be 70% condition following two hours, surplus with embodiment 8, take off needle point at last, verification method and result show successful connection with embodiment 5.
Sequence table
＜110〉Shanghai Inst. of Applied Physics Chinese Academy of Sciences
＜120〉a kind of method of low copy biochemical reaction
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＜213〉Artificial (artificial sequence)
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<220>
＜223〉primer
<400>6
<210>7
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＜213〉Artificial (artificial sequence)
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＜223〉primer
<400>7
Claims (1)
1. the method for a low copy biochemical reaction, it comprises and reacting after introducing the reactant of low copy on the nano-probe needle point of Scanning Probe Microscopy; The described reactant that will hang down copy is introduced and is comprised the following steps: on the nano-probe needle point of Scanning Probe Microscopy under inserting needle program automatically, allow the nano-probe needle point contact of Scanning Probe Microscopy place the suprabasil first kind of reactant solution of microscope, first kind of reactant of low copy introduced on the nano-probe needle point, treated to introduce the remaining reaction thing with the same manner again after the needle point drying; Carry out under the described humidity that is reflected at room temperature and 50%-90%; Described reactant is a dna molecular, and described reaction is the DNA ligation; Described Scanning Probe Microscopy is an atomic force microscope.
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| CN200710036291XA CN101220356B (en) | 2007-01-09 | 2007-01-09 | Low copy biochemical reaction method |
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| CN200710036291XA CN101220356B (en) | 2007-01-09 | 2007-01-09 | Low copy biochemical reaction method |
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| CN101220356B true CN101220356B (en) | 2011-06-15 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1232813C (en) * | 2003-03-13 | 2005-12-21 | 东南大学 | Method for preparing probe tip of nano tube |
| CN1285737C (en) * | 2004-08-03 | 2006-11-22 | 湖南大学 | Process for testing SARS virus genome segment by silicon shell nano particle |
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- 2007-01-09 CN CN200710036291XA patent/CN101220356B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1232813C (en) * | 2003-03-13 | 2005-12-21 | 东南大学 | Method for preparing probe tip of nano tube |
| CN1285737C (en) * | 2004-08-03 | 2006-11-22 | 湖南大学 | Process for testing SARS virus genome segment by silicon shell nano particle |
Non-Patent Citations (1)
| Title |
|---|
| 郭万林，台国安，姜燕.针尖的化学物理力学研究.《力学进展》.2005,第35卷(第4期),585-599. * |
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