Preparation method of sequence and length customized circular single-stranded DNA (Deoxyribonucleic Acid) and application of sequence and length customized circular single-stranded DNA to DNA origami

A circular and sequence technology, applied in the field of DNA nanotechnology and biological applications, can solve the problems of lack of circular single-stranded DNA, low efficiency of single-stranded DNA, and inability to obtain single-stranded DNA, etc., to achieve efficient preparation, abundant sources, The effect of rich diversity

Pending Publication Date: 2018-10-26
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

The former method is inefficient in obtaining single-stranded DNA because it involves enzyme digestion and column separation; and due to the limitation of PCR amplification ability, it is impossible to obtain larger lengths of single-stranded DNA
Although the latter method is more capable of obtaining larger lengths of single-stranded DNA, it can only obtain specific DNA based on the hybrid sequence of M13 and lambda phage
[0005] At present, there is a lack of a new method for preparing circular single-stranded DNA with customized sequence and length to break through its use as the backbone chain of DNA origami to construct larger-scale and more complex DNA origami structures

Method used

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  • Preparation method of sequence and length customized circular single-stranded DNA (Deoxyribonucleic Acid) and application of sequence and length customized circular single-stranded DNA to DNA origami
  • Preparation method of sequence and length customized circular single-stranded DNA (Deoxyribonucleic Acid) and application of sequence and length customized circular single-stranded DNA to DNA origami
  • Preparation method of sequence and length customized circular single-stranded DNA (Deoxyribonucleic Acid) and application of sequence and length customized circular single-stranded DNA to DNA origami

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Embodiment 1

[0030] A method for preparing circular single-stranded DNA with customized sequence and length according to the present invention comprises the following steps:

[0031] (1) Design and synthesize circular double-stranded recombinant phagemids; design the length and sequence of circular DNA according to different application requirements; use different plasmids or different biological genomes as templates for PCR amplification to obtain DNA fragments, Or directly chemically synthesize DNA fragments; the length of DNA fragments can be designed to vary from 100 base pairs to 10,000 base pairs. By adding short overlapping sequences of about 15-30bp at both ends of the fragment, the amplified DNA fragment can be seamlessly spliced ​​with the replication origin site (M13ori) of M13mp18 phage and the replication origin (pBR322ori) of plasmid pBR322. Mix each fragment with Gibson Assembly Master Mix and incubate at 50°C for 15 minutes to 1 hour to complete the reaction; up to 6 DNA fr...

Embodiment 2

[0036] Regarding the preparation of circular DNA single strands of four different sequences and lengths in the present invention, the specific operations are as follows:

[0037] Design single-strand DNA of different lengths and sequences according to different needs. The sequence should contain pBR322ori and M13ori.

[0038] Using PET-28a and M13mp18RF DNA plasmids as templates, PCR primers were designed to amplify pBR322ori and M13ori respectively, and two short DNA fragments were obtained. Using the same method, use different plasmids or biological genomes as templates for PCR amplification, or directly through chemical synthesis, to obtain DNA fragments ranging in length from 100 base pairs to 10,000 base pairs. A short overlapping sequence of about 15-30 bp should be added to the two ends of each DNA fragment according to the connection sequence for seamless connection.

[0039] The different DNA fragments obtained above were purified by agarose gel electrophoresis and ...

Embodiment 3

[0048] Regarding the assembly and characterization of DNA origami structures of 4 different sizes and shapes in the present invention, the specific operations are:

[0049] (1) Mix the prepared circular single-stranded DNA with its corresponding staple strand at a molar ratio of 1:10, wherein the concentration of the circular single-stranded DNA is in the range of 3-6 nM. Add 10×TAE / Mg to the system 2+ Buffer (Mg 2+ Concentration is 12.5mM), mix well.

[0050] (2) Place the mixture in step (1) in a PCR machine, and anneal from 85°C to 25°C. The annealing process is about 12 hours.

[0051] (3) After the annealing is completed, use a 100kDa ultrafiltration tube to centrifuge to remove excess staple chains, or use agarose gel electrophoresis to cut and recover for purification. After electrophoresis, the bands assembled to form the DNA origami structure were excised and collected in Freeze'N Squeeze DNA Gel Extraction Spin Columns. Freeze the spin column at -20°C for 5 minu...

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Abstract

The invention discloses a preparation method of sequence and length customized circular single-stranded DNA (Deoxyribonucleic Acid) and application of the sequence and length customized circular single-stranded DNA (ssDNA) to DNA origami. The preparation method of the sequence and length customized circular single-stranded DNA, disclosed by the invention, comprises the following steps: (1) designing and synthesizing circular double-stranded recombinant phagemid; (2) transforming escherichia coli through the recombinant phagemid and infecting through helper phage to prepare the circular single-stranded DNA with a corresponding form; (3) assembling different origami structures by taking prepared the ssDNA as a framework chain respectively, and carrying out characterization through atomic force microscope imaging. According to the method disclosed by the invention, aiming at the certain specific DNA, various types of single-stranded DNAs with different base sequences and lengths can be designed according to application requirements, so that customization is realized.

Description

technical field [0001] The invention belongs to the field of DNA nanotechnology and biological application, and specifically relates to the preparation of single-stranded DNA (single-stranded DNA, ssDNA) of different sequences and lengths, and using it as the skeleton chain of DNA origami to construct DNA origami structures, making a breakthrough Size limitations imposed by traditional methods. Background technique [0002] As a natural biological macromolecule, DNA has unique advantages in constructing functional nanostructures due to its unique chemical structure and unique intermolecular interactions. Using complementary base pairing, DNA molecules can self-assemble into ordered structures with nanoscale precision. This DNA nanostructure can also modify the DNA strand at a specific position, making it a scaffold to guide other molecules or nanomaterials to self-assemble in a controlled manner. [0003] In 2006, Rothemund proposed DNA origami, a brand-new DNA self-assemb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1027
Inventor 李喆陈小星汪倩于涵洋
Owner NANJING UNIV
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