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

Stabilizing a nucleic acid for nucleic acid sequencing

a nucleic acid and nucleotide technology, applied in specific use bioreactors/fermenters, biomass after-treatment, biochemistry apparatus and processes, etc., can solve the problems of difficult implementation and inconvenient use of bulk sequencing techniques for the identification of subtle or rare nucleotide changes

Inactive Publication Date: 2006-11-09
FLUIDIGM CORP
View PDF99 Cites 119 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides methods and surfaces for nucleic acid sequencing using stabilized primer / target nucleic acid duplexes. The duplex is stabilized by binding pairs on the surface of a substrate. The invention also includes methods for using a stabilizing molecule to increase the stability of the duplex. The invention further describes the use of different polymerases and nucleotides for the sequencing reaction. The technical effects of the invention include improved accuracy and sensitivity of nucleic acid sequencing and the ability to detect single molecules.

Problems solved by technology

Bulk sequencing techniques are not useful for the identification of subtle or rare nucleotide changes due to the many cloning, amplification and electrophoresis steps that complicate the process of gaining useful information regarding individual nucleotides.
While single molecule techniques have several advantages, implementation has been problematic.
However, incomplete binding of the primer to the template, disengagement of the primer from the template and disengagement of the duplex from the substrate are frequent occurrences in such single molecule techniques.

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
  • Stabilizing a nucleic acid for nucleic acid sequencing
  • Stabilizing a nucleic acid for nucleic acid sequencing
  • Stabilizing a nucleic acid for nucleic acid sequencing

Examples

Experimental program
Comparison scheme
Effect test

example 1

Dual Biotinylation

[0066] General methods of the invention were demonstrated using biotin / avidin binding pairs. When a biotin-streptavidin linkage is used to anchor a primer and a target nucleic acid to a substrate, the primer and target nucleic acid are biotinylated, while the surface of the substrate is coated with streptavidin. Because streptavidin is a tetramer, it is possible that both template and primer will bind to the same surface streptavidin. However, the dual biotin labels may bind to adjacent streptavidin molecules as well.

[0067] Two experiments were done to determine the binding stability of the dual biotin constructs. A first experiment was conducted in order to determine the stability of dual biotin duplex on a polyelectrolyte multilayer (PEM) surface. This experiment was done using covalent streptavidin attachment to a PEM surface. The PEM surfaces were prepared as follows. Polyethyleneimine (PEI) and pollyallylamine (PAA, Sigma, St. Louis, Mo.) were dissolved sepa...

example 2

Locked Nucleic Acid

[0076] A primer is designed to be complementary to a known primer attachment site of the target nucleic acid, and locked nucleic acid bases are substituted for certain nucleotides within the selected primer sequence. As many locked nucleic acid bases are selected as desired depending on the temperature and length of primer, up to a primer comprising 100% locked nucleic acids. The more locked nucleic acid substitutions into the primer, the greater the melting point of the primer / target nucleic acid duplex relative a primer of the same length lacking locked nucleic acid residues.

[0077]FIG. 3 shows three primers synthesized with locked nucleic acids. DXS17, 7G7A, and 377 primers were synthesized as complementary strands to known regions of a template, each primer incorporating nine locked nucleic acids. When these primers are annealed to their respective templates, hybridization may be carried out at temperatures between about 80° C. to about 90° 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

PropertyMeasurementUnit
melting temperatureaaaaaaaaaa
pHaaaaaaaaaa
pHaaaaaaaaaa
Login to View More

Abstract

The invention provides methods for sequencing a nucleic acid comprising stabilizing a primer / target nucleic acid duplex on a substrate. Methods of the invention generally contemplate the use of a dual-anchored primer / target nucleic acid duplex, or a stabilizing molecule in a single molecule sequencing reaction.

Description

TECHNICAL FIELD OF THE INVENTION [0001] The invention provides methods for sequencing a nucleic acid comprising stabilizing a primer / target nucleic acid duplex attached to a substrate. Generally, methods of the invention comprise the use of a dual-anchored primer / target nucleic acid duplex or stabilizing molecule. BACKGROUND OF THE INVENTION [0002] Completion of the human genome has paved the way for important insights into biologic structure and function. Knowledge of the human genome has given rise to inquiry into individual differences, as well as differences within an individual, as the basis for differences in biological function and dysfunction. For example, single nucleotide differences between individuals, called single nucleotide polymorphisms (SNPs), are responsible for dramatic phenotypic differences. Those differences can be outward expressions of phenotype or can involve the likelihood that an individual will get a specific disease or how that individual will respond to...

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(United States)
IPC IPC(8): C12Q1/68C12M1/34
CPCC12Q1/6834C12Q1/6869C12Q2563/131C12Q2533/101C12Q2535/101C12Q1/68
Inventor BUZBY, PHILIP
Owner FLUIDIGM CORP
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