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

Technology for identifying HBV (hepatitis B virus) gene integration sites and recurrently targeted genes in host genome

A technology of integration sites and genomes, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problem that the sequencing throughput cannot identify integration sites.

Active Publication Date: 2014-04-16
HANGZHOU PROPRIUM BIOTECH
View PDF2 Cites 17 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have technical drawbacks (i.e., only isolate integration sites that are restricted to certain parts of the genome) and are limited by limited sequencing throughput to identify more integration sites

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
  • Technology for identifying HBV (hepatitis B virus) gene integration sites and recurrently targeted genes in host genome
  • Technology for identifying HBV (hepatitis B virus) gene integration sites and recurrently targeted genes in host genome
  • Technology for identifying HBV (hepatitis B virus) gene integration sites and recurrently targeted genes in host genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] 1. Experimental samples

[0141] Tissue samples used in this application were agreed to by each patient and the Ethics Committee of the First Affiliated Hospital of Zhejiang University, which approved this study.

[0142] HBV-positive HCC tissue samples were obtained from clinical tissue sections of the First Affiliated Hospital of Zhejiang University School of Medicine. Each case was divided into cancer tissue and paracancerous tissue, a total of 40 pairs of tissue sections. Tissue samples were stored in a -80°C freezer.

[0143] 2. Reagents and equipment

[0144] 1) Reagent

[0145] Kits: DNA extraction kit (DNeasy Blood & Tissue Kit, QIAGEN), DNA general purification kit (QIAquick PCR Purification Kit, QIAGEN), DNA tapping gel purification kit (QIAquick Gel Extraction Kit, QIAGEN).

[0146]Enzymes: T4DNA Polymerase (T4DNA Polymerase, NEB), T4DNA Ligase (T4DNA Ligase, NEB), T4 Polynucleotide Kinase (T4Polynucleotide Kinase, NEB), Klenow DNA Polymerase (DNA Polymer...

Embodiment 2

[0222] In this example, the integration sites found in Example 1 were randomly selected for verification.

[0223] 1. PCR verification and Sanger sequencing verification

[0224] 1) Primer design

[0225] In order to be consistent with the amplification reaction when isolating the integration site, the verification reaction also used nested PCR. On the human gene sequence of the integrant, two primers (for the first round of PCR primers and nested primers in Table 5) were designed towards the integration site to correspond to the nested primers of the HBX gene. The final PCR product was controlled within 1kb. The integration sites and their validation primers are listed in Table 5, and the integration sites used in the validation experiments are represented by the codes N9+chr1-1 to N13-chrX-1 in the "Information" column, a total of 28.

[0226] Table 5. Primers and results of integration site nested PCR validation

[0227]

[0228]

[0229] 2) Nested PCR validation ...

Embodiment 3

[0242] This embodiment further examines the reliability and relative quantitative possibility of the MAPS identification results of Example 1.

[0243] 1. Semi-quantitative PCR

[0244] 1) Genomic DNA template dilution

[0245]Take 10 μL of HCC genomic DNA (60ng / μL) and add it to tube 1 of the PCR eight-tube strip. Add 15 μL of double distilled water to tubes 2-8 respectively. Take 5 μL of DNA from tube 1 to tube 2; mix well and pipette 5 μL of the mixture from tube 2 to tube 3; mix well and then pipette into the next tube to dilute, and then dilute to tube 7. Finally, the concentration of DNA in the eight tubes was approximately: 60ng, 15ng, 3.75ng, 0.94ng, 0.23ng, 0.06ng and 0ng (pure water, blank control).

[0246] 2) Nested PCR

[0247] For the 28 integration sites in Table 5, according to the method of "2) nested PCR verification" in the step "9. PCR verification and Sanger sequencing verification" of Example 1, basically the same PCR reaction system and cycle conditi...

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
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses a novel MAPS (massive anchored parallel sequencing) technology which can be used for separating and sequencing integration sequences so as to identify HBV gene integration sites and recurrently targeted genes in a host genome. The invention further discloses a kit used for implementing the technology as well as six genes recurrently integrated by the HBV and related kits.

Description

technical field [0001] The present invention relates to a novel large-scale anchored parallel sequencing technology (massive anchored parallel sequencing, MAPS), which can be used to isolate and measure HBV gene integration sequences (ie, integrons, integrants), thereby identifying HBV genes in the host genome Integration sites and integrated target genes (targeted genes); the invention also relates to kits for implementing the technique; the invention also relates to genes recurrently integrated by HBV determined using the technique, and uses of these genes . Background technique [0002] Chronic hepatitis B virus (HBV) infection can cause a series of liver diseases, including chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). About 2 billion people worldwide are infected with HBV, 350 million of whom are chronically infected. HBV infection causes 500,000-1.2 million deaths per year, 320,000 of which are caused by HCC (Parkin 2006). Many epidemiological stu...

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
CPCC12Q1/6869C12Q2535/122C12Q2525/191C12Q2537/1373
Inventor 林标扬丁东
Owner HANGZHOU PROPRIUM BIOTECH
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