Liquid-phase hybrid capture kit based on double-stranded probe, washing kit and application of kits

A technology of hybrid capture and kit, which is applied in the field of molecular biology, can solve the problems of high synthesis cost of molecular probes, insufficient detection sensitivity, and difficult region amplification, etc., so as to improve target enrichment efficiency, reduce costs, and achieve high efficiency Effect

Active Publication Date: 2017-11-17
DALIAN SHUANGDI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR method has the advantages of high sensitivity, high specificity, and good repeatability. However, this method cannot easily expand the target area. It is suitable for capturing some small areas, and some areas are difficult to amplify. There is no good solution to the problem of insufficient sensitivity of somatic cell mutation detection
The molecular inverted probe method has the advantages of simple sample pre-treatment and small sample demand. However, the molecular probe synthesis cost is high and the uniformity is poor, and it is difficult to capture some large genome sequences.

Method used

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  • Liquid-phase hybrid capture kit based on double-stranded probe, washing kit and application of kits
  • Liquid-phase hybrid capture kit based on double-stranded probe, washing kit and application of kits
  • Liquid-phase hybrid capture kit based on double-stranded probe, washing kit and application of kits

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 1

[0039] Preparation of Example 1 Capture Solution Preparation

[0040] The solution consists of sodium dihydrogen phosphate (NaH 2 PO 4 ) buffer, sodium citrate buffer (SSC), EDTA

[0041] (EDTA), deionized formamide, Denhardt (Denhardt) solution, Carrier RNA and dextran sulfate

[0042] made. First prepare a 2×concentration solution (without deionized formamide, deionized formamide liquid is added separately to capture

[0043] solution), it can be stored for a long time at -20°C, and the specific formula is as follows:

[0044] 100mM NaH 2 PO 4

[0045]4×SSC

[0046] 2mM EDTA

[0047] 2 x Denhard's solution

[0048] 2mg / ml Carrier RNA

[0049] 20w / w% dextran sulfate

[0050] Solution pH 7.0

[0051] The capture solution includes 2×concentration solution and 100% formamide liquid, which are mixed to form a hybridization capture solution working solution, and the volume mixing ratio is shown in Table 1 below.

[0052] Table 1

[0053]

preparation Embodiment 2

[0054] Preparation Example 2 Preparation of Washing Solution Kit

[0055] The washing solution kit contains 5 washing buffer components, which can be stored for a long time at -20°C. The specific formula is as follows:

[0056] Stringent wash solution: 2 x SSC solution, 30 v / v % deionized formamide.

[0057] Wash solution I: 2 x SSC solution, 0.1% sodium dodecyl sulfate (SDS).

[0058] Wash solution II: 2x SSC solution.

[0059] Wash solution III: 0.2x SSC solution.

[0060] Biotin affinity beads washing solution: 10mM Tris-HCl, pH 7.5; 1mM EDTA; 2M NaCl.

Embodiment 1

[0061] Example 1 Application of capture solution and washing solution kit in hybrid capture enrichment

[0062] Specifically, the entire experimental process includes: firstly, the extracted sample DNA is ultrasonically broken into DNA fragments of 200-250bp (but not limited to the ultrasonic breaking method), and the DNA The fragments are connected to the index adapter; then the constructed capture library is used to hybridize with the DNA fragments containing the target gene region to be captured, the DNA fragments in the target region are captured, the enrichment effect of the DNA fragments in the target region is evaluated, and these DNA fragments are sequenced. The sample detection results were obtained after bioinformatics data analysis.

[0063] 1. Sample genomic DNA extraction and library preparation

[0064] The extraction method of the genomic DNA of the samples refers to the QIAamp DNA Blood Mini Kit kit of Qiagen Company, and the human whole blood genomic samples ...

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Abstract

The invention discloses a genome sequencing library hybrid capture kit, a washing kit and applications of the kits in liquid-phase hybrid capture. The capture kit consists of a 2*concentration solution and 100% de-ionized formamide, wherein the 2*concentration solution comprises 100-150mM of sodium dihydrogen phosphate, a 4*sodium citrate buffer solution, 1-3mM of ethylene diamine tetraacetic acid, a 2*Denhardt solution, 2-5mg/ml of Carrier RNA and 15-20w/w% of dextran sulfate, wherein the 4*sodium citrate solution comprises 0.6M of sodium chloride and 0.06M of sodium citrate and pH value of the 4*sodium citrate solution is 7.0; and the 2*Denhardt solution comprises 0.04% of polysucrose 400, 0.04% of polyvinylpyrrolidone and 0.04% of bovine serum albumin.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a genome sequencing library hybridization capture kit, a washing kit and their application in liquid phase hybridization capture. Background technique [0002] At the beginning of the 20th century, the next generation sequencing technology (Next Generation Sequencing, NGS) was born, and the emergence of this technology has brought rapid progress in the field of DNA sequencing technology. Compared with the first-generation sequencing technology, the next-generation sequencing technology has the technical characteristics of high throughput and high accuracy, and can simultaneously sequence hundreds of millions of DNA fragments. Compared with traditional Sanger sequencing, the cost of NGS sequencing is greatly reduced. However, the cost of whole-genome sequencing is still high, and many research applications do not require whole-genome sequencing, and the demand for sequencing the ta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6874C12Q2527/125C12Q2535/122
Inventor 赵金银李宏志姜旭刘琦许立志于闯李杰
Owner DALIAN SHUANGDI TECH
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