Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Molecular stamp sequencing device and sequencing method

A sequencing and detector technology, applied in the field of microarray molecular stamp sequencing devices, can solve the problems of large consumption of drugs, high-throughput detection limitation, and large reaction system, and achieves reduction of raw material consumption, sequencing cost, and dosage. Effect

Inactive Publication Date: 2007-04-11
SOUTHEAST UNIV
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One is the pyrosequencing method that generally uses a single-tube reaction, that is, the sequence determination of one sample at a time, which undoubtedly limits high-throughput detection in this case
The other is a foreign parallel pyrosequencing method based on a 96-well plate, which places 96 different samples in 96 wells and then sequences these samples, although the analysis throughput of this method has increased , but sample preparation is cumbersome and consumes a lot of medicine
Although the above two pyrosequencing devices can realize the determination of the target DNA sequence, due to their large reaction systems and troublesome sample preparation, the sequencing cost is greatly increased.
More importantly, since the pyrosequencing reaction system is a liquid phase system, each sequencing reaction requires an independent reaction chamber, which greatly limits the throughput of DNA sequencing

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Molecular stamp sequencing device and sequencing method
  • Molecular stamp sequencing device and sequencing method
  • Molecular stamp sequencing device and sequencing method

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0021] Example 1. Sequence determination of 50 DNA fragments using rolling circle amplification products.

[0022] 1. Digest the 50 DNA fragments with corresponding restriction endonucleases, and connect one strand of the digested fragments with a universal adapter (containing a 36-base DNA sequence) under the action of the target DNA to form a circular DNA.

[0023] 2. Use an 18-base universal amplification reverse primer (with acrylamide modification at the 5' end and complementary to the 18 bases at the 3' end of the adapter) and another 18-base universal amplification forward primer (with the adapter 5 ' end 18bps complementary) to the above-mentioned circular DNA by double-primer rolling circle amplification; then the rolling circle amplification products were mixed with acrylamide gel solution, and 50 treated samples were spotted on glass slides with a chip spotter , and aggregated into a gel lattice, and prepared into a microarray chip for sequencing, as shown in Figur...

example 2

[0030] Example 2. Using PCR products to detect 100 SNP genotypes.

[0031] 1. First, extract human genomic DNA, and obtain 100 fragments of PCR amplification products containing different SNP sites through PCR technology. The 5' end of the reverse primer of the above PCR product is modified with biotin, and the first 18 base sequences of the reverse primer are identical, and the remaining bases are the specific sequence part of each fragment to be amplified.

[0032] 2. The 100 PCR products were respectively fixed with streptavidin-modified magnetic beads, then denatured with NaOH and washed with buffer to obtain single-stranded DNA.

[0033] 3. Fix the above-mentioned single-stranded DNA into a 10×10 array with a magnetic DNA extension substrate.

[0034] 4. Hybridize the microarray DNA on the extension substrate with universal sequencing primers, and wash away excess primers.

[0035] 5. Spread DNA polymerase evenly on the microarray extension substrate of the DNA extensio...

example 3

[0041] Example 3. Re-determination of the whole genome DNA sequence of Escherichia coli using rolling circle amplification products.

[0042] 1. Genomic DNA of Escherichia coli was extracted and fragmented by ultrasonic method. By combining one end of the DNA fragment with a universal 5' terminal phosphate adapter (containing a 36-base DNA sequence) under the action of the target DNA, one strand of the DNA fragment is ligated into a circular DNA, and suitable Dilute the template solution for whole genome sequencing of E. coli to form a single circular DNA.

[0043] 2. Use a 18-base universal amplification reverse primer (with acrylamide modification at the 5' end and complementary to the 18 bases at the 3' end of the adapter) to perform double-primed rolling circle amplification on the above circular DNA; The circle amplification product is mixed with acrylamide gel solution, evenly spread on the glass slide, and polymerized into an ultra-thin gel layer to prepare a rolling c...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The molecular stamp sequencing device and sequencing method is based on pyrophosphoric acid sequencing principle. The device includes one DNA extending part, one chemiluminescence part and one mechanical connecting part. The sequencing method includes fixing rolling circle amplified single strand DNA template or PCR amplified single strand template and DNA polymerase on one side of the DNA extending substrate; fixing the chemiluminescence related enzyme and auxiliary matter on one side of the transparent luminescence chip; and fixing the light signal detector on the other side of the chip. When the substrate and the chip are contacted and impressed, the pyrophosphoric acid will be transferred to the chip to release chemical reaction light signal in certain strength, and the light signal may be real-time detected. Four types of dNTP mononucleotides may be added successively into the system for the analysis and sequencing of each DNA sequence.

Description

technical field [0001] The invention relates to a molecular seal sequencing device and a sequencing method based on the principle of pyrosequencing, in particular to a low-cost, high-throughput microarray molecular seal sequencing device and a sequencing method. Background technique [0002] The completion of the Human Genome Project is largely due to the development of sequencing technology and the reduction of sequencing costs. The cost of DNA sequencing has dropped from about ten dollars per base more than a decade ago to about ten cents per base now. However, with the in-depth study of the human genome, people have become more and more aware that people should really study the function of genes, decipher the genome, a molecular program produced by natural evolution, and then carry out clinical diagnosis and disease diagnosis at the genetic level. For prevention and susceptibility assessment, only a large amount of personal genome information can be tested, and by compar...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陆祖宏潘志强肖鹏峰
Owner SOUTHEAST UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com