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Method for detecting sequence of double chain DNA based on procedure of DNA automaton

A technology of DNA sequence and automaton, applied in the field of DNA sequence determination

Inactive Publication Date: 2005-08-31
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It can effectively avoid the sequencing difficulties caused by the advanced structure of single-stranded DNA molecules, overcome the problem that the secondary structure of the single-stranded template affects sequencing in the existing dideoxy sequencing technology, and reduce costs on this basis. The present invention not only It can replace the existing technology, and overcome the defects of the existing technology, and has great application prospects

Method used

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  • Method for detecting sequence of double chain DNA based on procedure of DNA automaton
  • Method for detecting sequence of double chain DNA based on procedure of DNA automaton
  • Method for detecting sequence of double chain DNA based on procedure of DNA automaton

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Effect test

Embodiment 1

[0027] Determine the DNA sequence information of the unknown sequence that has been inserted into the multiple cloning site of the plasmid. Using DNA recombination technology to insert the sequence to be tested into the plasmid, the multiple cloning site should contain a recognition site for a restriction endonuclease such as FokI for the DNA calculation reaction; the known sequence of the multiple cloning site is the start of the determination sequence. The state transition molecule is designed so that the cut distance of each DNA automaton is 1 base, and the sequence of the DNA input molecule can be read directly. If FokI is used to run the automaton, the exposed cohesive ends are 4 bases, so the number of all state transition molecules that need to be considered is 4 4 = 256 pieces. It is only necessary to know the first 3 bases of the determined sequence (the known sequence of the multiple cloning site) to infer all subsequent sequences according to the fluorescent color...

Embodiment 2

[0052] Determine the sequence of the double-stranded DNA of the PCR product. It is necessary to design a FokI site in the PCR primer, so that one end of the double-stranded DNA molecule that is amplified by PCR (the specific method of the PCR reaction is not limited) contains a FokI site, which is far away from the end of the double-stranded DNA molecule. There is a distance of 6-10 bases at the end to ensure that this site can be effectively cleaved by FokI. In this way, the sequence determination of PCR products can be realized by using the DNA calculation reaction system corresponding to FokI. Before the reaction, the double-stranded DNA sequence amplified by PCR was cut with FokI to determine whether the sequence contained another FokI recognition site. If there is no other FokI recognition site, then directly perform the sequencing reaction, that is, the entire PCR product double-stranded DNA is added to the DNA calculation reaction system as an input molecule for DNA ca...

Embodiment 3

[0054] For unknown sequences that already contain a FokI recognition site, the following strategy can be used. The sequence was cleaved with FokI, and the cleaved fragments were recovered and directly subjected to a sequencing reaction. There are many ways to recover the fragments. The products of the FokI cleavage reaction can be separated by agarose gel according to the size of the fragments, and each fragment can be purified by tapping the gel separately. These recovery and purification processes utilize standard DNA recombination techniques, and any kits on the market can be selected to carry out. The purified DNA fragments can be directly subjected to the DNA calculation reaction to determine the sequence, that is, the purified DNA double-stranded fragment is added to the DNA calculation reaction system as the input molecule of the DNA calculation. Finally, the sequences of these fragments are spliced ​​together.

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Abstract

A method based on DNA robot process for testing the double-strained DNA sequence includes such steps as using the double-strained DNA sequence as input molecular, severing it by restriction endonuclease to expose adhesive end, linking with state transition molecular via base, cyclic severing and linking to gradually shorten the sequence, and reading the variation of length to reflet the information about DNA sequence.

Description

technical field [0001] The invention relates to a method for DNA sequence determination in the field of biotechnology, in particular to a method for double-stranded DNA sequence determination based on a DNA automaton process. Background technique [0002] Since the publication of the human genome sequence, the world has entered the post-genome era. Thousands of biological species require genome sequence determination. Among the current DNA sequencing solutions, Sanger's dideoxy method is the most popular. At present, DNA sequencing has a number of international patent technologies [such as US patents, European patents and Japanese patents], except for a few methods that are not very practical. , most of which are patents implemented on a series of technical issues surrounding the dideoxy method. The sequence of the human genome was determined using the dideoxy method. In recent years, DNA computing and DNA computer have aroused people's widespread interest. DNA computing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张治洲胡钧贺林
Owner SHANGHAI JIAO TONG UNIV
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