Methods of amplifying and sequencing nucleic acids

一种核酸序列、测序的技术，应用在化学仪器和方法、生物化学设备和方法、通过光学手段进行材料分析等方向，能够解决昂贵、DNA测序仪慢、测序仪慢等问题

Inactive Publication Date: 2008-10-01
454 LIFE SCIENCES CORP
View PDF22 Cites 15 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DNA sequencers are still slower and more expensive than needed
In pure research situations, may be acceptable if the sequencer is slow and expensive
But when a DNA sequencer is required for clinical diagnostic situations, said inefficient sequencing method is prohibitive even for well-financed institutions

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
  • Methods of amplifying and sequencing nucleic acids
  • Methods of amplifying and sequencing nucleic acids
  • Methods of amplifying and sequencing nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0135] The attachment of the anchor primer to the solid or removable solid substrate can occur before, during, or after extension of the annealed anchor primer. Therefore, in one embodiment, one or more anchor primers are attached to a solid or removable fixed substrate, after which the anchor primers anneal to the target nucleic acid and are extended in the presence of a polymerase. Alternatively, in the second embodiment, the anchor primer is first annealed to the target nucleic acid, and the 3'OH end of the annealed anchor primer is extended with a polymerase. The extended anchor primer is then attached to a solid or removable solid substrate. By changing the sequence of the anchor primer, the unique target nucleic acid present in the nucleic acid population can be specifically amplified.

[0136]The following summarizes preferred embodiments for preparing template nucleic acids for amplification and sequencing reactions. The present invention includes a method for preparing sa...

Embodiment 1

[0389] Example 1: Sample preparation

[0390] DNA sample:

[0391] DNA should be of high quality and free of contaminants such as proteins, nucleic acids, lipids, and other chemicals (such as residual EDTA from preparations) and salts. The preferred genomic DNA should have a 260 / 280 ratio of 1.8 or higher. If it is necessary to sequence the genome of only one organism, the DNA should be quality checked to ensure that it does not contain contaminating DNA. For example, a preparation of human DNA can be tested by PCR to ensure that it is not contaminated by bacterial DNA molecules. Another way to test for contamination is to use a suitable probe known to be specific to a known organism (e.g. human or mouse) and a second probe known to be specific to a possible contaminating organism (e.g. Escherichia coli). Enzyme digestion mode, especially Southern blot after restriction enzyme digestion. If necessary, the DNA should be from a single clone of the organism (for example, if it is fro...

Embodiment 2

[0499] Example 2: Primer design

[0500] As discussed above, the universal adaptor is designed to include: 1) a set of unique PCR primer regions, whose length is typically 20 bp (adjacent to (2)); 2) a set of unique sequencing primer regions, whose length is typically 20 bp; And 3) optionally followed by a unique distinguishing key sequence, which consists of at least one of the four deoxyribonucleotides (ie A, C, G, T). The possible cross-hybridization between the primer and the unexpected region of the genome of interest increases as the size of the genome increases and the length of the complete match with the primer decreases. However, for the reasons explained below, this potential interaction with the cross-hybridization region (CHR) is not expected to cause problems.

[0501] In a preferred embodiment of the present invention, the single-stranded DNA library is used for PCR amplification and subsequent sequencing. The sequencing method requires random digestion of a given g...

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

An apparatus and method for performing rapid DNA sequencing, such as genomic sequencing, is provided herein. The method includes the steps of preparing a sample DNA for genomic sequencing, amplifying the prepared DNA in a representative manner, and performing multiple sequencing reaction on the amplified DNA with only one primer hybridization step.

Description

Invention field [0001] The present invention relates to a method and device for determining the base sequence of DNA. More specifically, the present invention relates to a method and device that can be used for automated or semi-automated amplification and determination of the base sequence of the genome. Background of the invention [0002] The development of rapid and sensitive nucleic acid sequencing methods using automated DNA sequencers has revolutionized modern molecular biology. Through the collaborative efforts of a series of machines and a team of technicians, the complete genomes of plants, fungi, animals, bacteria and viruses can now be analyzed. However, the goal of rapid and automated or semi-automated sequencing of the genome in a short period of time has not yet been achieved. There are still technical problems of accurate sample preparation, amplification and sequencing. [0003] One technical problem that hinders genome sequence analysis is that researchers canno...

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): G06F19/00B01L3/00C07H21/00C12N15/10C12P19/34C12Q1/68G01N21/25G01N21/64G16B30/10G16B30/20
CPCG01N21/6428B01L2300/0636G01N21/6452C12N15/1075B01L2300/0819C07H21/00B01L3/502761B01L3/5085G01N21/6458G01N21/253B01L2300/0877B01L3/5027G06F19/22G16B30/00G16B30/10G16B30/20
Inventor K·E·麦达德J·M·费罗J·R·奈特J·乍鲁米林J·H·利蒙E·W·迈尔斯J·W·辛普森G·A·福尔克默
Owner 454 LIFE SCIENCES CORP
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap