One-step Wash Magnetic Bead Method Saliva DNA Extraction Kit

A kit and washing solution technology, which is applied in the field of magnetic bead method saliva DNA extraction kit, can solve the problems of insufficient concentration of extraction effect, easy pollution, time-consuming and other problems

Active Publication Date: 2022-07-26
NINGBO AJCORE BIOSCIENCES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently known phenol chloroform method, salting-out method, adsorption column method, immunoaffinity method, magnetic bead method and other existing DNA extraction methods that can be used for saliva DNA extraction mainly include adsorption column method and magnetic bead method. There are obvious deficiencies in the extraction effect and the concentration of the extracted DNA, and the magnetic bead method is easy to operate (such as the magnetic bead method of CN201611240409, CN201810169271, CN201610848178, CN201110105238, etc.) in the extraction of DNA by the magnetic bead method. , not only consumes time, but also requires more delicate operation when absorbing the supernatant after washing, which is prone to pollution / not suitable for on-site operation outside the laboratory) and the extraction effect also has a lot to be improved. It is necessary to develop a method that is easier to operate Advanced magnetic bead DNA extraction method to improve extraction efficiency / meet the needs of on-site rapid sample processing in screening, forensic investigation, etc.

Method used

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  • One-step Wash Magnetic Bead Method Saliva DNA Extraction Kit
  • One-step Wash Magnetic Bead Method Saliva DNA Extraction Kit
  • One-step Wash Magnetic Bead Method Saliva DNA Extraction Kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Reagent preparation and basic extraction process

[0039] Prepare Extraction Reagent 1 according to the following recipe:

[0040]Lysate: Tris 55mM, EDTA 15mM, NaCl 0.3M, NaDC 0.4%, Dodecyl-N-Betaine 0.2%, NLS 0.5%, GuHCl 75%, TritonX100 2%, NH 4 Cl 0.15%, mercaptoethanol 0.5%, pH 7.0;

[0041] Binding solution: isopropanol 65%, PEG 8000 10%, Brij 58 3%, pH 7.0;

[0042] Proteinase K solution: proteinase K 20mg / ml;

[0043] Washing solution: GuHCl 7M, Tris 10mM, ethanol 40%, sodium bicarbonate 40mM, pH 9.0;

[0044] Elution buffer: Tris 20 mM, EDTA 0.2 mM, pH 8.5.

[0045] Control Reagent 2 was prepared according to the recipe of Reagent 1 (replace sodium bicarbonate with sodium acetate 10mM)

[0046] Control reagent 3 was prepared according to the formula of reagent 1 (dodecyl-N-betaine-free, NaDC 0.6%)

[0047] Follow the steps below for DNA extraction:

[0048] (1) Take 200ul of fresh saliva into a 1.5ml centrifuge tube, then add a total of 400ul of ...

Embodiment 2

[0053] Example 2 Effect verification of the kit formulation of this application

[0054] 1 The role of sodium bicarbonate and DPTA

[0055] Use the reagents 1-3 and DNA extraction methods in Example 1 to extract the same saliva sample, use the Qubit® (fluorescent dye-based method) detector and the matching kit to detect the concentration of the extracted DNA and detect OD A260 / 280, the results as follows:

[0056]

[0057] Repeated results across multiple samples were similar. We speculate that the acid-base / charged conditions in the washing solution have a great influence on the washing effect of mucin hydrolysates. Among the various formulations in the experiment, only slightly more alkaline sodium bicarbonate can effectively remove proteins without affecting DNA stability. effect; the addition of zwitterionic surfactants is beneficial to fully lyse cytolytic proteins and make the proteins easier to remove during washing.

[0058] Comparison of the one-step washing kit...

Embodiment 3

[0063] Example 3 Use the kit of this application for actual detection

[0064] Use the kit of this application to extract 4 pairs of 8 subjects' saliva (collected simultaneously with blood samples) DNA for STR locus typing in paternity testing (Promega Power Plex16, 16 loci, ABI9700 PCR instrument), and Compared with the genotyping of DNA extracted from blood, the genotyping of all 128 loci of the 8 subjects was exactly the same. It is preliminarily proved that the samples extracted by the kit of the present application can be used for subsequent molecular biology detection.

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Abstract

The invention belongs to the technical field of molecular biology detection. Specifically, the application provides a magnetic bead method saliva DNA extraction kit that only needs one-step washing, which includes a lysis solution, a binding solution, a proteinase K solution, a washing solution and an elution solution. buffer. A saliva DNA extraction kit includes lysing solution, binding solution, proteinase K solution, washing solution and elution buffer. The lysate contains Tris, EDTA, NaCl, NaDC, dodecyl-N-betaine, NLS, GuHCl, TritonX100, NH 4 Cl, mercaptoethanol; binding solution including isopropanol, PEG 8000, Brij 58; washing solution including GuHCl, Tris, ethanol, sodium bicarbonate; elution buffer including Tris, EDTA. In the present application, by preparing a washing solution containing a relatively high concentration of sodium bicarbonate (slightly stronger than sodium acetate and sodium chloride), the ion balance and the effect of providing alkalinity are taken into account at the same time, and a zwitterionic detergent is used in combination. Alkyl-N-betaine, a magnetic bead-based saliva DNA extraction kit was constructed that only required one-step washing.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection. Specifically, the application provides a magnetic bead method saliva DNA extraction kit that only needs one-step washing, which includes a lysis solution, a binding solution, a proteinase K solution, a washing solution and an elution solution. buffer. Background technique [0002] Nucleic acid extraction is the basis of various molecular biology detection methods, and efficient and complete DNA extraction is the basis for PCR amplification, library construction, and sequencing. In addition to tissue and organ samples, the most commonly used in vitro samples in molecular biology testing are blood samples, but blood collection requires professionals to use sterile blood collection equipment, which is costly and complicated. Immediate pain and fear are rejected by many people; for these reasons, molecular biology testing with blood samples is inconvenient in many situations, esp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6806
Inventor 周杰锋
Owner NINGBO AJCORE BIOSCIENCES INC
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