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Method for using DNA tetrahedron as scaffold on nano-particle surface and initiating rolling circle amplification reaction

A rolling circle amplification reaction and nanoparticle technology, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms, etc. problem, to achieve the effect of improving hybridization efficiency

Inactive Publication Date: 2015-03-04
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, improper control of DNA density on the surface of nanomaterials not only leads to inefficient amplification, but also results in long single-stranded DNA products entangled with each other and leads to aggregation, making receptor recognition difficult

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Equivalent amount of chain A ( 5’- GTG AAAAA AAAAA GCA -ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3'), B strand (5'- TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C -3'), C chain (5'- TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C -3'), D chain (5'- TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T -3') with TM buffer (20mM, Tris, 50mM MgCl 2 , pH 8.0), after mixing thoroughly, heat at 95°C for 2min, then rapidly cool down to 4°C within 30s. The prepared DNA tetrahedron was recovered and verified by PAGE electrophoresis and AFM.

Embodiment 2

[0019] Equivalent amount of chain A ( 5’- GTG AAAAA AAAAA GCA -ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3'), B strand (5'-HS-C6- TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C -3'), C strand (5'-HS-C6- TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C -3'), D strand (5 '-HS-C6- TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T -3') with TM buffer (20mM, Tris, 50mM MgCl 2 , pH 8.0), after mixing thoroughly, heat at 95°C for 2min, then rapidly cool down to 4°C within 30s. The prepared DNA tetrahedron was recovered and characterized by PAGE electrophoresis and AFM.

[0020] Add 20 μL (10 μM) DNA tetrahedron structure to 1mL 15nm gold colloid solution, shake at room temperature overnight, add 100mM PB buffer (pH7.4), the final concentration is 10mM, add aging solution (10 mM PB, 2M NaCl, pH 7.4) The final concentration of salt in the solution was 0.15M. After overnight a...

Embodiment 3

[0022] Equivalent amount of chain A ( 5’- GTG AAAAA AAAAA GCA -ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3'), B strand (5'-NH 2 -C6- TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C -3'), C strand (5'-NH 2 -C6- TCA ACT GCC TGG TGA TAA AAC GAC ACT ACGTGG GAA TCT ACT ATG GCG GCT CTT C -3'), D strand (5'-NH 2 -C6- TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T -3') with TM buffer (20mM, Tris, 50mM MgCl 2 , pH 8.0), after mixing thoroughly, heat at 95°C for 2min, then rapidly cool down to 4°C within 30s. The prepared DNA tetrahedron was recovered and characterized by PAGE and AFM. 20 μL (10 μM) DNA tetrahedron structure in 10 μL magnetic bead solution (1 mM), shake overnight at 37°C and place on a magnetic rack, magnetic suspension, discard the supernatant (unconnected DNA tetrahedron), add 100 μL pH7.4 PB (10 mM, 0.15 M NaCl) three times. Add 20 μL PB buffer solution to the precipitate to re...

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PUM

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Abstract

The invention relates to a method for using a DNA tetrahedron as a scaffold on a nano-particle surface and initiating rolling circle amplification reaction; after the prepared DNA tetrahedron is assembled on the nano-particle surface, a section of single-stranded DNA extending on the DNA tetrahedron can be used as primer DNA; circular DNA complementary with a special aptamer sequence is used as an amplification template; the circular DNA is hybridized with the primer DNA while the rolling circle amplification reaction is initiated; and the prepared product is multiple long single-stranded DNA composed of multiple apatmer structures on the nano-particle surface. The method disclosed by the invention is capable of effectively regulating and controlling the DNA distribution density on the nano-particle surface, so that the hybridization efficiency of the primer DNA and the circular DNA is increased; by means of long single-stranded DNA composed of the multiple aptamer structures prepared through the rolling circle amplification technology, the CTC (Carbon Tetra Choride) capture efficiency is greatly increased; and thus, the method disclosed by the invention has potential application value in the research fields, such as cell detection, cell capture, cell imaging and the like.

Description

technical field [0001] The invention belongs to the field of functionalization of nanomaterials and application of DNA nanotechnology, and relates to a DNA self-assembled DNA tetrahedron used as a scaffold to assemble on the surface of nanomaterials, and a section of single-stranded DNA extended from the DNA tetrahedron triggers rolling circle expansion. The resulting product is a long single-stranded DNA loaded with multiple specific recognition properties on the surface of nanoparticles. This structure can be used for cell detection, capture, and cell imaging research. Background technique [0002] Malignant tumors have become one of the major diseases with the highest mortality rate in the world. With the increasing incidence of tumors, people's understanding and research on malignant tumors are also deepening: early, efficient and specific CTC Capture is not only a powerful means for early diagnosis of tumors, but also facilitates the risk assessment and treatment plan f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6811C12Q2525/191C12Q2531/125C12Q2563/155C12Q2565/537
Inventor 何丹农颜娟胡冲娅
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH
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