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

Selective enrichment of population of DNA in mixed DNA sample through targeted suppression of DNA amplification

A population and sample technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as determining the resistance of complex polymorphisms

Pending Publication Date: 2020-08-21
零日诊断有限公司
View PDF1 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR-based diagnostics can be rapid, but their reliance on target probes limits them to a limited number of targets, which prevents them from comprehensively characterizing known pathogen species (of which there are thousands) and known antimicrobial resistance genes (of which there are at least 1,600 PCR-based diagnostics are also not always accurate in determining complex, polymorphism-based resistance)

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
  • Selective enrichment of population of DNA in mixed DNA sample through targeted suppression of DNA amplification
  • Selective enrichment of population of DNA in mixed DNA sample through targeted suppression of DNA amplification
  • Selective enrichment of population of DNA in mixed DNA sample through targeted suppression of DNA amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Design of Assassin Blocker DNA Oligonucleotide Sequence

[0128] In order to inhibit the amplification of a first population of DNA molecules comprising mammalian DNA sequences, Assassin blocker oligonucleotides were designed that are complementary to DNA sequences that are more abundant in mammalian DNA populations than microbial DNA populations appears more frequently in . The selected mammalian sequences are unique to the first population with minimal overlap with the second population of DNA to be amplified.

[0129] The human mitochondrial genome is small (approximately 16.6 kb) and contains sequences distributed on the plus and minus strands that are unique relative to both the human nuclear and bacterial genomes . The sequences of the Assassin blocker oligonucleotides are designed to be complementary to sequences that have a higher frequency per kilobase (kb) in mitochondrial genomes compared to bacterial, fungal, and viral genomes. Some non-limiting example...

Embodiment 2

[0168] Cross-linking of Assassin blockers to complementary sequences

[0169] Amplification of the first population of human mitochondrial DNA can be blocked by cross-linking the Assassin blocker oligonucleotide designed in Example 1 with its complementary sequence in the first population of human mitochondrial DNA. This cross-linking blocks genome-wide amplification of sequences by strand-displacing polymerases such as phi29 ( figure 2 ).

[0170] Lyophilized Assassin blocker oligonucleotides with modified 3' amino groups were designed as described in Example 1 and purchased (Integrated DNA Technologies, Coralville, IA). Lyophilized oligonucleotides were resuspended in phosphate-buffered saline (1×PBS) and mixed with NHS-SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butanol ester, "psoralen") molecules (ThermoFisher, Waltham, MA) were conjugated. The conjugation reaction was quenched with Tris-HCl and excess psoralen was removed with a desalting column (ThermoFisher, Wa...

Embodiment 3

[0172] Demonstration of sequence-specific covalent crosslinking of Assassin blockers to target DNA

[0173] To demonstrate sequence-specific covalent crosslinking of Assassin blocker oligonucleotides to target DNA, different Assassin blocker oligonucleotides were conjugated to the 3' end with psoralen, target oligonucleotide The reaction mixture of acid and off-target oligonucleotides was added at 0.5 μM to a mixture of 40 mM Tris HCl (pH 7.9), 50 mM NaCl and 10 mM MgCl 2 Composition of the annealing buffer. The reaction mixture was heated to 94°C for 5 minutes and cooled to 16°C within 15 minutes. The reaction mixture was then treated with 350 nm ultra-violet (UV) light for 20 minutes. The reaction mixture products were separated on a 10% Tris-borate-EDTA(TBE)-urea gel in 1×TBE buffer. Gels were stained with SYBR-gold (ThermoFisher, Waltham, MA) and visualized on an E-gel Imager (ThermoFisher, Waltham, MA). ( image 3 ).

[0174] A reduction in human mitochondrial DNA...

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 present disclosure provides method and products for the selective enrichment of one population of DNA in a mixed sample comprising multiple populations of DNA. In some embodiments, the mixed sample comprises one or more populations of microbial DNA and the mammalian host DNA, particularly including pathogenic microbial DNA mixed with mammalian host DNA in a clinical sample from an infected individual.

Description

[0001] technical background [0002] Pathogenic microbial infection in mammalian tissues is a major healthcare concern. Bacterial infections of the blood (called bacteremia) are usually treated with antibiotics, but sometimes lead to sepsis, a life-threatening condition in which the entire body becomes inflamed if left untreated, leading to multiple Organ failure and eventual death. With increasing antimicrobial resistance, the treatment of bacterial infections has become more challenging. In the United States alone, approximately 2,000,000 patients suffer from bacterial infections with antimicrobial resistant organisms each year and approximately 100,000 of these patients die from the infection. Nearly half of the infections currently diagnosed in hospitals are resistant to at least one common antibacterial drug, and the risk of death can double in the case of drug-resistant infections. Rapid and correct identification of pathogens involved in infection and their patterns of...

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): C07H19/00C07H21/02C07H21/04C12M1/00C12M1/34C12N5/02
CPCC12Q1/6806C12Q2525/186C12Q2537/159C12N15/11C12P19/34C12Q1/689C12Q2523/101
Inventor 奇亚豪·徐梅利斯·N·阿纳赫塔尔杜格尔·麦克劳林米丽娅姆·H·亨特利杰斯里·D·布鲁斯特
Owner 零日诊断有限公司
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