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Method for detecting influence of mechanical force on interaction of DNA (deoxyribose nucleic acid) and DNA polymerase

A polymerase, mechanical force technology, applied in the nano field, that can solve problems such as lack of methods for the interaction of DNA and DNA polymerases

Inactive Publication Date: 2016-06-08
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of methods to detect the effect of mechanical forces other than the direction of the DNA double helix on the interaction between DNA and DNA polymerase.

Method used

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  • Method for detecting influence of mechanical force on interaction of DNA (deoxyribose nucleic acid) and DNA polymerase
  • Method for detecting influence of mechanical force on interaction of DNA (deoxyribose nucleic acid) and DNA polymerase
  • Method for detecting influence of mechanical force on interaction of DNA (deoxyribose nucleic acid) and DNA polymerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of Equilateral Triangular Hollow DNA Origami

[0035] The hollow DNA origami designed in this example utilizes M13mp18 DNA and staple single-stranded self-assembly to form an equilateral triangle pattern. The preparation method of the equilateral triangle hollow DNA origami is as follows: the single-stranded DNA M13mp18 of the circular long scaffold and the two strands of A17 and C33 are respectively The staple strand DNA collection ABCL of the sequence shown in SEQIDNO.1 and SEQIDNO.2 is replaced by the sequence table and mixed in TAE-Mg 2+ Buffer system (40mM Tris-acetic acid, 1mM EDTA, 12.5mM MgCl 2 , pH8.0), and then placed in a PCR instrument to anneal from 95°C to 20°C at an annealing rate of 0.1°C / 10s, where the molar concentration ratio of M13mpl8 to staple chains was 1:10, and TAE-Mg 2+ The buffer was ultrafiltered to prepare the assembled hollow DNA origami.

[0036] The side length of equilateral triangle hollow DNA origami in the pres...

Embodiment 2

[0037] Example 2 Preparation of single-stranded DNA template-DNA origami

[0038] Hybridize the 114nt DNA template whose sequence is shown in the sequence table SEQ ID NO.3 to the hollow DNA origami prepared in Example 1, so that it is located in the center of the hollow DNA origami, and its two ends are fixed on the hollow DNA origami by hybridization to form a single strand DNA template-hollow DNA origami, the steps are as follows: mix excess 114nt DNA template with hollow DNA origami, the molar ratio of 114ntDNA template and hollow DNA origami is 50:1, then place in a PCR instrument, anneal from 50°C to 15°C, the annealing rate 0.5°C / min, with TAE-Mg 2+ Single-stranded DNA template-hollow DNA origami was prepared after buffer ultrafiltration. It was determined that the molar concentration of the prepared single-stranded DNA template-hollow DNA origami was 0.15nM.

Embodiment 3

[0039] Example 3 AFM imaging and analysis of interaction between DNA and DNA polymerase Klenow fragment

[0040] Mix 3.0 μL of the single-stranded DNA template-hollow DNA origami prepared in Example 2 with 1.0 μL of Klenow fragment (KF) at a concentration of 85 U / mL in a test tube, incubate at 25°C for 10 min, and add 1.0 μL of Klenow fragment (KF) at a concentration of 62.5 After μM of dNTPs, immediately take 3 μL of the above sample and drop it on the newly dissociated mica sheet, and inject TAE-Mg into the liquid pool 2+ After 30 μL of buffer solution was scanned and imaged. With "J" scanning head, "tapping" mode. Adjust the scanning parameters in the small force area (F<200pN), and use the real-time dynamic scanning method of AFM in situ to collect AFM images of samples at different stages, including DNA origami, single-stranded DNA template-hollow DNA origami, and during replication. After copying the hollow DNA origami, the images were processed and analyzed with NanoS...

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Abstract

The invention discloses a method for detecting influence of mechanical force on interaction of DNA (deoxyribose nucleic acid) and DNA polymerase. The method comprises steps as follows: a single-stranded DNA template containing dNTP (deoxyribonucleotide triphosphate) and a buffering system of a DNA polymerase Klenow fragment combined with the single-stranded DNA template are scanned by means of an atomic force microscope, two ends of the single-stranded DNA template are fixed on two different sites on inner edges of hollow DNA origami, the two different sites are not located on the edge of the same straight line, the buffering system is arranged on a newly dissociated mica substrate, and synthesis of double-stranded DNA is observed through imaging of the atomic force microscope. The information about the effect of mechanical force in the non-double-helix direction on the structure and the function of DNA and DNA polymerase is acquired with the detection method, then influence of the mechanical force on DNA synthesis is obtained, accurate positioning on the nano scale can be realized, and a condition is provided for detection of the interaction relation of DNA and DNA polymerase under a certain tension condition is provided.

Description

technical field [0001] The invention relates to the field of nanotechnology, in particular to a method for detecting the influence of mechanical force on the interaction between DNA and DNA polymerase. Background technique [0002] The interaction between DNA and DNA polymerase is closely related to the occurrence, development and evolution of cells and tissues, and is also widely used in modern biotechnology, including DNA sequencing, forensic testing, nanobiological sensing and DNA amplification. technology etc. The interaction between DNA and DNA polymerase is regulated and influenced by multiple factors. For example, in vivo, it is closely related to molecular functional states such as single-chain binding proteins, helicases, and topoisomerases; in vitro, it is also affected by various environmental factors such as temperature, ion concentration, and pH value. [0003] The final product of the interaction between DNA and DNA polymerase is double-stranded DNA (dsDNA), ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2565/601C12Q2521/101
Inventor 张萍李宾张峰周星飞胡钧
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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