Sequencing method based on gene capture technology

A gene capture and sequencing technology, applied in the biological field, can solve the problems of poor stability of sgRNA and dCas9, difficult sgRNA design, hindering capture efficiency and specificity, etc., and achieve the effects of low synthesis cost, low cost and short time-consuming.

Inactive Publication Date: 2021-11-26
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
View PDF6 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the traditional probe hybridization capture technology, this capture method has the advantages of low cost, short time-consuming (2h), high capture efficiency, simple operation, and no need for heat denaturation of DNA, but the non-specific binding of dCas9 itself to DNA and Off-target effects of sgRNAs severely hamper capture efficiency an

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
  • Sequencing method based on gene capture technology
  • Sequencing method based on gene capture technology
  • Sequencing method based on gene capture technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Comparison of the application effects of biotin-modified probes and polyA-modified probes in RATE capture technology.

specific Embodiment approach

[0047] In this example, we compared the capture effect of biotin-modified probes and polyA-modified probes on RATE capture technology (see figure 1 with figure 2 ), the target DNA site we picked was double-stranded DNA derived from 5.8S rRNA. The specific implementation is as follows:

[0048] 1) RNA library construction: 1 ug of 293F RNA was constructed using the Hieff NGS® Ultima Dual-modeRNA Library Prep Kit for Illumina (Cat#12252) of Yisheng Biology, and then captured using the RATE-seq process.

[0049] 2) Biotin-modified probe capture

[0050] Table 2

[0051] components Dosage Biotin-5.8S probe (SEQ ID NO: 6) 10-100nM UvsX recombinase 20-200nM UvsY recombinase accessory protein 20-200nM 10× reconstitution buffer 3 µL total capacity 20 µL

[0052] 10× reconstitution buffer including 100-1000 mM tris, 100-3000 mM potassium acetate, 30-500 mM magnesium acetate, 3-30 mM dithiothreitol, 10-200 mM adenosine triphosphate, ...

Embodiment 2

[0068] Example 2: Comparison of the application effects of different 3' end modified probes in the RATE capture technology.

[0069] In this example, we compared the dideoxyribonucleotide modified ddN (SEQ ID NO: 3) and the amino modified NH2C6 (SEQ ID NO: 2) two 3' end modified probes SEQ ID NO: capture at RATE For the technical capture effect, the selected target DNA site is double-stranded DNA derived from 5.8S rRNA. The specific implementation is the same as the biotin-modified probe capture process in Example 1. The result is as Figure 5 As shown, both ddN modification and NH2C6 modification at the 3' end of the probe have good RATE capture efficiency, and the ddN modified probe has better capture efficiency.

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 invention provides a sequencing method based on a gene capture technology. The sequencing method has the principle that a recombinase compound is used for assisting a single-stranded DNA probe with a closed 3' end to efficiently and specifically hybridize into a double-stranded target DNA molecule under the condition of 37 DEG C, and then an affinity label on the single-stranded DNA probe is used for carrying out specific enrichment on the target DNA. The method not only has the advantages of relatively strong long probe pairing, low probe design requirement and the like of a probe hybridization capture technology, but also has the advantages of simplicity in operation, short time consumption, no need of thermal denaturation of DNA, high capture efficiency, low cost and the like of a CRISPR/dCas9 system, and the probe does not participate in subsequent amplification, so that the identification accuracy is improved. Therefore, compared with other capture technologies, the RATE-seq has the obvious advantages of simplicity in operation, high capture efficiency, extremely short consumed time (30 minutes), good stability, low cost and the like, and is very suitable for gene capture and automatic application. In addition, the RATE-seq can effectively separate and remove human host DNA in a pathological sample, and the detection rate of pathogenic microorganisms is increased.

Description

technical field [0001] The patent of the present invention relates to a sequencing method based on gene capture technology, which belongs to the field of biotechnology. Background technique [0002] The second-generation and third-generation sequencing technologies have become the core technologies in the field of gene diagnosis and treatment due to their advantages of large throughput, complete information, and easy operation. Gene trapping has been a major challenge for DNA and RNA high-throughput sequencing technologies. Due to the huge amount of human genome information, which contains about 3 billion base pairs, the real coding gene region accounts for less than 4%, and the exon region and transcription regulatory element region account for less than 1%. The vast majority of pathogenic mutations are enriched in exon regions and transcriptional regulatory element regions, so the genetic information in these regions plays a key role in the diagnosis and treatment of dise...

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
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2565/519C12Q2523/308C12Q2521/507C12Q2522/101C12Q2535/122
Inventor 刘倩江翱马海玲陈晶晶孙睿王嫚曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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