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

DNA sequencing method by using thiooligonucleotide probe

A technology of oligonucleotide probes and thiooligonucleotides, which is applied in the field of DNA sequencing in biotechnology, can solve the problems of PCR amplification bias, easy introduction of replication errors, and short read length of synthetic sequencing methods, achieving Simple synthesis, easy implementation, accurate and reliable effect determination

Inactive Publication Date: 2008-08-13
SOUTHEAST UNIV
View PDF0 Cites 16 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the sequencing-by-synthesis method has made important progress, the read length of the sequencing-by-synthesis method is relatively short and the preparation of the sequencing DNA template array relies on PCR amplification, which not only easily introduces replication errors during the amplification copy process of the sequencing template, Moreover, there is obvious bias in PCR amplification, which forms an important technical bottleneck for effectively obtaining whole-genome sequencing template arrays.

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
  • DNA sequencing method by using thiooligonucleotide probe
  • DNA sequencing method by using thiooligonucleotide probe
  • DNA sequencing method by using thiooligonucleotide probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Utilize the DNA sequencing method of thiooligonucleotide probe

[0022] Using the DNA sequencing method of thiooligonucleotide probes, the sequencing steps are: construction of thiooligonucleotide probes: the 5'-3' end of the thiooligonucleotide probe sequence is the anchor region in turn , the recognition region and the splicing region, the anchor region contains n nucleotides or base analogs, where 0<n≤10; the recognition region contains m nucleotides or base analogs, where 0<m≤10 , and between the first nucleotide or base analog at the 3' end of the recognition region and the adjacent nucleotide or base analog in the 5' direction is a thio-modified phosphate bond; the cleavage region contains k nuclei Nucleic acid or base analogs, wherein 0<k≤10; the sequence of the shear region is provided with a marker corresponding to the recognition region; the sequencing cycle: a). Using sequencing positioning primers to hybridize with the single-stranded DNA templ...

Embodiment 2

[0023] Example 2: Determination of the whole human genome by single-base hybridization-ligation sequencing

[0024] Digest the human genome with enzymes (or sonicate) into fragments with a size of 50-200 bases, use T4 polymerase, kelnow polymerase, Tag polymerase and T4 phosphokinase to repair the ends of the fragments and form a phosphate group at the 5' end Under the action of T4 ligase, connect these fragmented nucleic acid sequences with a pair of universal linkers (5'-p-CAG TCA GTC AGT CAG TCA G T-3' and 3'-T GTC AGT CAGTCA GTC AGT C-p-5', where p represents a phosphate group) for connection, one oligonucleotide sequence of the universal linker is completely complementary to the sequence of the amplification primer, and the other oligonucleotide sequence of the universal linker is aligned with the sequencing Primers are the same.

[0025] The fragmented nucleic acid sequences connected by these linkers are subjected to emulsion parallel PCR reaction with microbeads immob...

Embodiment 3

[0027] Example 3: Determination of the whole genome of rice by two-base hybridization-ligation sequencing

[0028] Cut the rice genome with enzymes (or sonicate) into fragments with a size of 50-200 bases, use T4 polymerase, kelnow polymerase, Tag polymerase and T4 phosphokinase to repair the ends of the fragments and form a phosphate group at the 5' end Under the action of T4 ligase, connect these fragmented nucleic acid sequences with a pair of universal linkers (5'-p-CAG TCA GTC AGT CAG TCA G T-3' and 3'-T GTC AGT CAGTCA GTC AGT C-p-5', where p represents a phosphate group) for connection, one oligonucleotide sequence of the universal linker is completely complementary to the sequence of the amplification primer, and the other oligonucleotide sequence of the universal linker is aligned with the sequencing Primers are the same.

[0029] The fragmented nucleic acid sequences connected by these linkers are subjected to emulsion parallel PCR reaction with microbeads immobilizi...

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 DNA sequencing method using a sulfooligonucleotide probe further lower the sequencing cost, lengthen the sequencing length of each sequence, and shorten the reading time of each base. By a sulfonucleotide-contained sequencing primer, employing a hybrid- enzyme connection - enzyme cutting high throughput DNA sequence technology, the invention provides a new method for DNA sequence analysis, establishing a rapid, precise and low-cost high throughput DNA sequence technology. The most advantage of the invention is realizing simple synthesis of maker hybrid sequence tested by DNA sequence and convenient cutting of maker. Since the hybrid high throughput primer is synthesized and purified by a very mature solid-phase DNA method, the method has no accumulative effect of erroneous extension and is capable of keeping the quantity of DNA template and sequencing primer, and the sequencing is reliable and correct.

Description

technical field [0001] The invention belongs to the field of DNA sequencing in biotechnology, in particular to a DNA sequencing method using thiooligonucleotide probes. Background technique [0002] Existing technology: The development and completion of the Human Genome Project and various model organism genome projects have had a huge impact on contemporary biological and medical research. People can understand the differences of life phenomena, the law of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. As far as gene sequence analysis is concerned, the focus of the post-genome era has shifted from the determination of the whole genome sequence of a single species to the comparison of individual genetic differences and inter-species genetic differences of a certain species at the level of genome DNA sequence. In terms of basic research, it studies the genetic laws of disease genes and clones disease-causing g...

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
IPC IPC(8): C12Q1/68C12N15/11
Inventor 陆祖宏罗俊峰肖鹏峰孙蓓丽贾超
Owner SOUTHEAST UNIV
Features
  • Generate Ideas
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com