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Method for testing sequence of nucleic acid single molecule

A single-molecule sequencing and nucleic acid technology, which is used in biochemical equipment and methods, material excitation analysis, and microbial determination/inspection. The effect of high quality and low measurement cost

Inactive Publication Date: 2010-02-24
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current methods are difficult to meet the needs of various sequencing
The current method has many defects: slow speed, high cost, poor accuracy and short one-time sequencing length, so new sequencing technology should be developed to replace the current sequencing method
Since this method requires the use of expensive fluorescently-labeled dNMPs, the cost of sequencing is the main limiting factor for the widespread use of this method.
In addition, the accuracy rate is also far lower than the 99.99% proposed by the sequencing community.

Method used

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  • Method for testing sequence of nucleic acid single molecule
  • Method for testing sequence of nucleic acid single molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] DNA fragment sequencing

[0021] Step 1, detection of dNMPs handwriting

[0022] Add water or aqueous solution to the flow chamber 1 connected by nanochannels embedded with zero-mode waveguides, and only add one ultra-diluted nucleotide each time, turn on the vacuum pumping and Raman spectroscopy detection system to record when a single nucleotide passes through the ZMW In this way, the Raman spectrum signals of various nucleotides are detected, and finally the characteristic spectra of their specific phonon vibrations are determined, a standard curve is established, and software is written.

[0023] Step 2, immobilization of single-molecule DNA fragments

[0024] A magnetic bead carrying a target nucleic acid molecule is fixed to the magnet of the flow chamber 1 of the sequencing unit, so that one end of a target nucleic acid is fixed. The sequencing unit is made into an array, and finally each sequencing unit of the entire array has a target nucleic acid.

[0025] ...

Embodiment 2

[0030] Complete Chromosome Sequencing

[0031] Step 1, single-molecule operation: with the aid of an optical microscope, flow cytometer or other methods, a single chromosome is extracted, histones are removed, and the chromosome is immobilized on magnetic beads according to the method in Example 1.

[0032] Step 2, Chromosome fixation: fix the magnetic beads with 1 complete chromosome on the magnet installed in the flow chamber 1 of a sequencing unit to make an array, and finally make each sequencing unit of the entire array have a chromosome.

[0033] Step 3, sequence detection: add exonuclease and buffer in flow chamber 1, after a single enzyme molecule binds to DNA, add magnesium ions, immediately turn on the vacuum device, collect Raman spectrum signals, and use software to read the DNA sequence .

[0034] The implementation effect of this embodiment: the whole chromosome is used as the substrate for sequencing, although the effect of the array cannot be fully exerted as ...

Embodiment 3

[0036] RNA sequencing

[0037] Step 1, the detection of rNMPs handwriting is the same as in Example 1, and a standard curve is established according to the specific Raman spectrum signals generated by them.

[0038] Step 2, according to the method of Example 1, fix the RNA single molecule to the flow chamber 1, use RNA exonuclease to degrade the nucleotides one by one, when the nucleotide passes through the ZWM, record the Raman spectrum data, and convert the Raman spectrum according to the standard curve The signal is converted into sequence information.

[0039]In step 3, RNA can also be used as a template to synthesize cDNA (complementary DNA) by reverse transcriptase, and then follow the steps in Example 1 to obtain the RNA sequence.

[0040] Implementation effect of this embodiment: suitable for the RNA / DNA exonuclease activity of this embodiment is 1000nts / second, such as detection of mRNA sequence, according to the average length of mRNA of 1000nts, the array of 100 * ...

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Abstract

The invention relates to a method for testing a sequence of a nucleic acid single molecule in the technical field of biology, which comprises the following steps: respectively detecting Raman spectrumsignals of deoxyadenosine 5'-phosphoric acid, deoxyguanosine 5'-phosphoric acid, deoxycytidine 5'-phosphoric acid, deoxythymidine 5'-phosphoric acid, methyldeoxycytidine 5'-phosphoric acid, adenosine5'-phosphoric acid, vernine 5'-phosphoric acid, cytidine 5'-phosphoric acid and uridine 5'-phosphoric acid and establishing a standard curve; cutting a nucleic acid molecule to be detected by exonclease and detecting the Raman spectrum signal transmitted by a cut product when passing through a zero mode waveguide by the trigger of a laser; converting the Raman spectrum signal obtained in the step2 into concrete ribotide according to the standard curve obtained in the step 1 and obtaining the concrete sequence of the nucleic acid molecule to be detected by combining the cutting direction of the exonclease. The method can directly detect a natural nucleic acid sequence without a mark and has high detecting speed and detecting quality and low detecting cost.

Description

technical field [0001] The invention relates to a sequencing method in the field of biotechnology, in particular to a nucleic acid single-molecule sequencing method. Background technique [0002] With the advent of the post-genomic generation, the demand for sequence determination of biological macromolecules such as DNA and RNA has increased substantially. However, the current sequencing methods have problems such as slow speed, high cost, gaps, short sequencing length or low accuracy, which restrict the development of related disciplines. According to the results released by NCBI on June 23, 2009, 22 eukaryotic genomes have been sequenced, 174 have been assembled, and 172 are in progress. In fact, this is just the prelude to large-scale genome sequencing, because there are many needs for genome sequencing, such as personalized sequencing, individual sequencing in animal and plant genetic improvement, monitoring of pathogenic microorganisms, and so on. However, the curren...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/65
Inventor 王志民詹黎程秀兰
Owner SHANGHAI JIAO TONG UNIV
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