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

DNA sequence measurement based on primer extension

A DNA sequence and determination method technology, applied in the field of DNA sequence determination, can solve problems such as background interference, interference with fluorescence intensity measurement accuracy, complex cleavage groups, etc., and achieve the effect of high accuracy

Inactive Publication Date: 2007-04-04
SOUTHEAST UNIV
View PDF0 Cites 22 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of these routes are the need to synthesize complex cleavage groups; the second is that the fluorophore may not be completely cut off and removed, and background interference will occur when it is extended to a certain length
The specificity of DNA polymerase makes this method have high sensitivity and resolution, but its disadvantage is that a multi-color fluorescence system and corresponding detection system must be used, and the excitation light and emission fluorescence spectra of various dyes often have different Most of the overlap, which interferes with the measurement accuracy of the fluorescence intensity

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 sequence measurement based on primer extension
  • DNA sequence measurement based on primer extension
  • DNA sequence measurement based on primer extension

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A nucleic acid sequence determination method for determining an unknown nucleic acid sequence:

[0027] A) Introduce a known universal DNA sequence into the DNA template to be tested, and fix it on a solid-phase substrate; B) Design 1-15 oligonucleotide DNA primer combinations, so that it contains a section and the above-mentioned addition The sequence that is complementary to the common DNA sequence, and make the 3' ends of the 1-15 oligonucleotide DNA primers contain 0, 1, 2, ..., 14 degenerate nucleotides respectively; C) according to the The position of the nucleotide site, select a primer from the above primer combination, and hybridize it with the fixed DNA template described in A, so that the nucleotide site to be tested is next to the 3' end of the primer; D) use DNA polymerase and dideoxynucleotide substrates labeled with four distinguishable markers or four deoxynucleotides or dideoxynucleotides labeled with one or more markers respectively, hybridized to the ...

Embodiment 2

[0045] Example 2: Sequencing the artificially synthesized DNA template

[0046] The following DNA sequences were synthesized by Invitrogen:

[0047] Seq1: 5'NH2-(T)25 TT CT C AGG TAG AGT TGG AGC TCA GGA CAT GGC AT

[0048] Seq2: 5'NH2-(T)25 TT AC G TGC GTC AGT TGG AGC TCA GGA CTG GGC TG

[0049] Seq3: 5'NH2-(T)25 TT GT C AGA CGA AGT TGG AGC TCA GGA ATA CTT AT

[0050] Seq4: 5'NH2-(T)25T T AGT CCT AGT TGG AGC TCA GGA TCC TTG GA

[0051] Seq5: 5'-CTGAGCTCCAACT 3'

[0052] Seq6: 5'-TGAGCTCCAACTN 3'

[0053] Seq7: 5'-GAGCTCCAACTNN 3'

[0054] Seq8: 5'-GAGCTCCAACTNNN3'

[0055] Seq9: 5'-AGCTCCAACTNNNN3'

[0056] Seq10: 5'-GCTCCAACTNNNNN 3'

[0057] Sequ11: 5'-CTCCAACTNNNNNN 3'

[0058]Seq1-seq4 are used as DNA templates, and seq5-seq11 are sequencing primers. The oblique and bold part of the template sequence is the primer binding region, the underlined part is the sequence to be tested, and NH2 represents amino modification. N is a degenerate nucleotide, and ...

Embodiment 3

[0079] Example 3: Sequencing of T7 phage DNA library clones

[0080] Materials: T7 bacteriophage DNA was purchased from Shanghai Bioengineering Company; Tsp509 I was purchased from NEB Company; pUC118 vector, competent bacteria and transformation kit, T4 DNA ligase were purchased from Dalian Bao Biological Company; klenow DNA polymerase was purchased from Fermentas Company; 序列订购自invitrogen公司:M13前引物:NH-CAGGAAACAGCTATGAC(seq12);M13后引物:GTAAAACGACGGCCAGT(seq13);测序引物:GCATGCCTGCAGGTCAATT(seq14);CATGCCTGCAGGTCAATTN(seq15);ATGCCTGCAGGTCAATTNN(seq16);ATGCCTGCAGGTCAATTNNN(seq17);TGCCTGCAGGTCAATTNNNN (seq18);

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

A method for measuring DNA sequence based on primer extension is carried out by inducing a section of DNA sequence on DNA template, fixing it onto solid-phase substrate, designing 1-15 oligonucleotide DNA primer combination to make it contain a section of DNA sequence, whose 3'end of 1-15 oligonucleotide DNA primer contains 0,1,2......14 degeneracy nucleic acid, selecting one primer from the primer combination by site position to be measured, hybridizing it with fixed DNA template, adhering site to primer 3'end, nucleic acid extending for primer by dideoxynucleic acid substrate or deoxyribonucleotide or dideoxyribonucleotide and inspecting extended nucleic acid type. The base type on DNA template is complementary with extended nucleic acid type.

Description

technical field [0001] The present invention relates to a DNA sequence determination method, in particular to a DNA sequencing method based on primer extension. Background technique [0002] The two most commonly used DNA sequencing methods are Sanger's dideoxy chain termination method and Maxam and Gilbert's chemical cleavage sequencing method, but both rely on electrophoresis to separate DNA fragments of different sizes. Sanger sequencing employs dideoxynucleotide random termination of polymerization, which produces extended strands of various lengths that are separated by electrophoresis. The sequence can be read because the nucleotides at the stop can be identified, and there is a stop everywhere. Although the application of electrophoresis technology, especially capillary electrophoresis, has greatly improved the automation of sequencing, its throughput is still limited to a certain extent. Therefore, for large-scale genome projects or diagnostic sequencing that requir...

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 施小龙唐超陆祖宏
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