CN102181441B - Method for coupling single long-chain DNA (deoxyribonucleic acid) molecules through single quantum dots - Google Patents
Method for coupling single long-chain DNA (deoxyribonucleic acid) molecules through single quantum dots Download PDFInfo
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- CN102181441B CN102181441B CN201110095077A CN201110095077A CN102181441B CN 102181441 B CN102181441 B CN 102181441B CN 201110095077 A CN201110095077 A CN 201110095077A CN 201110095077 A CN201110095077 A CN 201110095077A CN 102181441 B CN102181441 B CN 102181441B
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Abstract
The invention relates to a method for coupling single long-chain DNA (deoxyribonucleic acid) molecules through single quantum dots. An oligonucleotide primer coupled with the quantum dots is used for replacing a primer in polymerase chain reaction, long-chain DNA (which is greater than 100 base pairs) of a target fragment is obtained through the polymerase chain reaction, and a single long-chain DNA molecular compound coupled by the single quantum dots is further obtained through the electrophoretic separation method. By adopting the method, the problems that the long-chain DNA molecules can not be coupled on the surfaces of the quantum dots and the number of the coupled molecules can not be controlled in the traditional coupling method can be solved, and the method can be widely applied in probe preparation, nano-structure assembly, nano-scale biochemical reaction and other related fields.
Description
Technical field
The present invention relates to the method for the single long-chain dna molecular of a kind of single quantum point coupling, belong to field of nanometer material technology and biology field.
Technical background
Nanometer material science is the focus of studying at present.Nano material typically refers to the material particle diameter, and wherein the unidimensional yardstick is between 1～100nm arbitrarily, and the dimensional structure owing to unique makes them have special physics and chemical property.(quantum dots QD) typically refers to the semiconductor fluorescence nano material to quantum dot, because it has unique photoluminescent property, has been widely used in bio-science and the Materials science.Thymus nucleic acid (DNA) is because the double-spiral structure of its distinctive nanoscale; And have many different conformations; The complementary pairing of base, end carry out functional modification (like amino, carboxyl etc.) easily, on sequence, have designability; And can be very suitable for the nano material of constructing function property through the amplification of enzyme extension, elongation.Yet in present research; Main difficulty is can not effectively control the quantity of quantum point coupling DNA (Chembiochem 2009; 10,1781), so often need utilize the method for gel electrophoresis to separate quantum dot surface coupling different quantities dna fragmentation (J. Am. Chem. Soc. 2004; 126,10832).On the other hand, because influence sterically hindered and electrostatic repulsion, traditional coupling method can only be at the dna molecular (less than 100 bases, Nano lett 2001,1,32) of quantum dot surface coupling short segments.
(polymerase chain reaction is one of method the most frequently used in the modern life science research PCR), can synthesize the long-chain dna fragmentation, and in a short period of time, produce a large amount of target dna fragments in the polymerase chain reaction.Retrieve through prior art; Find the existing report of one Chinese patent application 200410018098.X disclosed " based on the PCR of nanoparticle "; Mainly utilize nanometer gold and magnetic nano-particle to be material; And the enzyme extension (J. Am. Chem. Soc. 2002,124,7314) based on nanometer gold also has report.These research proofs are feasible based on the pcr amplification and the enzyme extension of nano material, but report the mixture that obtains the single long-chain dna molecular of single quantum point coupling.The single long-chain dna molecular of single quantum point coupling will make quantitative analysis and the unit molecule based on quantum dot become possibility, and will prepare at probe, the association areas such as biochemical reaction of nanostructure assembling, nanoscale play a significant role.
Summary of the invention
Technical problem to be solved by this invention is to provide the method for the single long-chain dna molecular of a kind of single quantum point coupling.
The method of the single long-chain dna molecular of single quantum point coupling provided by the invention comprises the steps:
1) according to the long-chain dna fragmentation that will obtain, design and synthesize a pair of Oligonucleolide primers, and 5 ' end of one of them primer carried out chemically modified, make Oligonucleolide primers be coupled to the quantum dot surface through chemical bond;
2) with coupling the Oligonucleolide primers and the another one primer of quantum dot, and contain the segmental dna profiling of purpose and add together in the system of polymerase chain reaction, obtain the long-chain dna molecular through the polymerase chain reaction;
3) mixture that obtains of separation of polymeric PCR obtains the mixture of the single long-chain dna molecular of single quantum point coupling.
Further optimized technical scheme is:
Sterically hindered when reducing Oligonucleolide primers and quantum point coupling before the Oligonucleolide primers with quantum point coupling is carried out chemically modified, connects (CH at 5 ' end
2)
6-TTTTTT.
The coupling method of Oligonucleolide primers and quantum dot can be the chemical reaction between amino and carboxyl, sulfydryl and maleimide or sulfydryl and the metallic element.
The single long-chain dna molecular of single quantum point coupling mixture obtains through the agarose gel electrophoresis separation.
Said quantum dot is water miscible, and particle diameter is in 1-100 nm scope.
Template as the polymerase chain reaction can be genomic dna (comprising plant, animal, mikrobe etc.), also can be DNA.
Said PCR system is example referring to various commercial polysaccharase specification sheetss with FERMENTAS company's T aq enzyme, the consisting of of PCR system:
| The Taq enzyme | 1 U |
| 10 * PCR damping fluid | 2.5 uL |
| dNTP（2 mM） | 2.5 uL |
| Mg 2+（25 mM） | 1.5 uL |
| Oligonucleolide primers and quantum point coupling thing (0.4 uM) | 1 uL |
| Another primer (0.4 uM) | 1 uL |
| Template DNA | 50 ng |
| ddH 2O | Supplying TV is 25uL |
The mixture that will obtain through electrophoretic separation detects through AFM, and confirmation is the mixture of the single long-chain dna molecular of single quantum point coupling.
What the present invention obtained is the mixture of the single quantum dot of dna molecular coupling of single long-chain; And can obtain containing the segmental long-chain dna molecular of purpose (100bp-2000bp) arbitrarily; This conjugate detects through electrophoresis detection, AFM detection, fluorescence spectrum; The characteristic of its quantum dot and DNA does not all have to change (like Fig. 1-shown in Figure 3), can be used for follow-up operation.This method is simple to operate, and is applied widely.Can be applied to the association areas such as biochemical reaction of probe preparation, nanostructure assembling, nanometer meso-scale.
Description of drawings
Fig. 1 is the quantum dot that obtains after embodiment 1 polymerase chain reaction and the electrophorogram of long-chain DNA conjugate, has reacted the rate of migration of its conjugate.
Fig. 2 separates the mixture that obtains to embodiment 1 to carry out the observation of AFM, and embodiment is the mixture of the dna molecular of the single long-chain of single quantum point coupling.
Fig. 3 carries out fluorescent spectroscopy to embodiment 1 polymerase chain reaction product, and the photoluminescent property that embodies its quantum dot does not change.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these embodiment only to be used to the present invention is described and be not used in the scope of restriction requirement of the present invention protection.
Embodiment 1:
1) synthesize 5 ' end carry out amido modified Oligonucleolide primers, sequence is 5 '-NH
2-(CH
2)
6-TTTTTT-GGCCTTCAGAGACGAGGTTG-3 ';
2) Oligonucleolide primers is coupled to carboxyl water-soluble CdSe/ZnS quantum dot surface (product type: W4-605, an ancient woman's ornament source, Wuhan quantum dot development technique ltd);
3) with synthetic Oligonucleolide primers and carboxyl water-soluble CdSe/ZnS quantum point coupling thing as primer 1; Primer 2 is 5 '-GCTGTGCATCAGGAATAATTTG-3 '; Corn gene group DNA is as template; Carry out with the polymerase chain reaction that the carboxyl water-soluble CdSe/the ZnS quantum dot is the basis amplification
Fatty aldehyde dehydrogenase 1Gene obtains the long dna molecular of 480 bp;
Said PCR system is example referring to various commercial polysaccharase specification sheetss with FERMENTAS company's T aq enzyme, the consisting of of PCR system:
| The Taq enzyme | 1 U |
| 10 * PCR damping fluid | 2.5 uL |
| dNTP（2 mM） | 2.5 uL |
| Mg 2+（25 mM） | 1.5 uL |
| Primer 1 (0.4 uM) | 1 uL |
| Primer 2 (0.4 uM) | 1 uL |
| Template DNA | 50 ng |
| ddH 2O | Supplying TV is 25uL |
4) agarose gel electrophoresis separates the mixture that obtains the long dna molecular of the single 480bp of single carboxyl water-soluble CdSe/ZnS quantum point coupling, and is as shown in Figure 1, before (a) EB dyes, after (b) EB dyes.M road: DNA marker (DL 2000); 1 road: normal PCR product; 2 roads: quantum dot is the PCR product on basis; 3 roads: the conjugate of quantum dot and Oligonucleolide primers; 4 roads: carboxyl water-soluble CdSe/ZnS quantum dot.Because the optical property of quantum dot can be seen band in 4 roads 2,3 before EB dyes, and mobility 23 < 4, prove that PCR extends successfully, quantum dot and Oligonucleolide primers coupling success.And we see that the band in 2 roads wants narrower, and guess is in the PCR process, because sterically hindered influence has only one of them to be connected the surperficial Oligonucleolide primers of quantum dot and extends successfully.After EB dyes, in 1 road, can see the band of normal PCR product, and in 2 roads, see a band the same with 1 road mobility, our guess maybe be in the PCR process part quantum dot and Oligonucleolide primers disengaging, carried out the process of normal PCR.
The mixture that 5) will obtain through electrophoretic separation carries out AFM again and detects, and confirmation is the long dna molecular mixtures of single 480 bp of single carboxyl water-soluble CdSe/ZnS quantum point coupling, and is as shown in Figure 2.
6) will carry out fluorescent spectroscopy with the polymerase chain reaction product that carboxyl water-soluble CdSe/ZnS quantum dot is the basis, as shown in Figure 3, its fluorescence intensity just reduces slightly, and photoluminescent property does not change.
Embodiment 2:
1) synthesize 5 ' end carry out amido modified Oligonucleolide primers, sequence is 5 '-NH
2-(CH
2)
6-TTTTTT-TCTGAGTACGCACGGCCGGT-3 ';
2) Oligonucleolide primers is coupled to the CdSe/ZnS quantum dot surface (product type: W1-605, an ancient woman's ornament source, Wuhan quantum dot development technique ltd) that Polyethylene Glycol (PEG) Lipids modifies;
3) the CdSe/ZnS quantum point coupling thing of synthetic Oligonucleolide primers and PEG Lipids being modified is as primer 1; Primer 2 is 5 '-CATCGCCGGTCGGCATCGTT-3 '; The human gene group DNA is as template; Carry out the polymerase chain reaction that is basis with the CdSe/ZnS quantum dot that PEG Lipids modifies, increase 1
8S ribosomal 1Gene obtains the long dna molecular of 1046 bp;
Said PCR system is example referring to various commercial polysaccharase specification sheetss with FERMENTAS company's T aq enzyme, the consisting of of PCR system:
| The Taq enzyme | 1 U |
| 10 * PCR damping fluid | 2.5 uL |
| dNTP（2 mM） | 2.5 uL |
| Mg 2+（25 mM） | 1.5 uL |
| Primer 1 (0.4 uM) | 1 uL |
| Primer 2 (0.4 uM) | 1 uL |
| Template DNA | 50 ng |
| ddH 2O | Supplying TV is 25uL |
4) agarose gel electrophoresis separates the long dna molecular mixture of single 1046 bp of CdSe/ZnS quantum point coupling that obtains single PEG Lipids modification.
The mixture that 5) will obtain through electrophoretic separation carries out AFM again and detects, and confirmation is the mixture of the long dna molecular of single 1046 bp of CdSe/ZnS quantum point coupling that modify of single PEG Lipids.
6) the CdSe/ZnS quantum dot of PEG Lipids being modified carries out fluorescent spectroscopy for basic polymerase chain reaction product.
Claims (3)
1. the method for the single long-chain dna molecular of single quantum point coupling is characterized in that comprising the steps:
1) according to the long-chain dna fragmentation that will obtain, design and synthesize a pair of Oligonucleolide primers, and 5 ' end of one of them primer connected (CH
2)
6-TTTTTT also carries out chemically modified, makes Oligonucleolide primers be coupled to the quantum dot surface through the chemical reaction between amino and carboxyl, sulfydryl and maleimide or sulfydryl and the metallic element;
2) with coupling the Oligonucleolide primers and the another one primer of quantum dot, and contain the segmental dna profiling of purpose and add together in the system of polymerase chain reaction, obtain the long-chain dna molecular through the polymerase chain reaction;
3) mixture that obtains through agarose gel electrophoresis separation of polymeric PCR obtains the mixture of the single long-chain dna molecular of single quantum point coupling.
2. the method for claim 1, it is characterized in that: said quantum dot is water miscible, particle diameter is in 1-100 nm scope.
3. according to claim 1 or claim 2 method, it is characterized in that: the template as the polymerase chain reaction is genomic dna or DNA.
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| CN104395471B (en) * | 2012-02-19 | 2018-01-16 | 纳维基因股份有限公司 | Purposes of the IDED nanostructureds in nucleic acid |
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| CN101603085B (en) * | 2009-07-07 | 2011-11-09 | 天津工业大学 | Magnetic DNA fluorescent probe and preparation method thereof |
| CN101698889A (en) * | 2009-10-21 | 2010-04-28 | 江南大学 | Method for detecting DNA base number based on quantum dot and PCR technology |
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