One-step washing magnetic bead method blood dna extraction kit

The technology of a kit and washing solution is applied in the field of magnetic bead method blood DNA extraction kit, which can solve the problems such as easy pollution

Active Publication Date: 2020-10-02
NINGBO AJCORE BIOSCIENCES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The magnetic bead method is easy to operate (such as CN201611240409, CN201810169271, CN201610848178, CN201110105238, etc.) in the magnetic bead method to extract DNA, it will go through 2-4 times of washing, which not only consumes time, but also needs to be more delicate when absorbing the supernatant after washing. It is easy to cause 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 for magnetic bead DNA extraction that is easier to operate to improve extraction efficiency / meet screening, The need for rapid on-site sample processing in forensic investigations, etc.

Method used

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  • One-step washing magnetic bead method blood dna extraction kit
  • One-step washing magnetic bead method blood dna extraction kit
  • One-step washing magnetic bead method blood dna extraction kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 Reagent preparation and basic extraction process

[0049] Prepare extraction reagent 1 according to the following recipe:

[0050] Lysis buffer: Tris 60mM, EDTA 15mM, NaCl 0.6M, SDS 0.3%, CHAPS 0.2%, NLS 7%, GuHCl72%, sodium laurate 3%, NH 4 Cl 0.2%, mercaptoethanol 1.0%, pH 7.1;

[0051] Binding solution: isopropanol 70%, PEG 8000 8%, Brij 58 3%, pH 7.0;

[0052] Proteinase K solution: proteinase K 20mg / ml;

[0053] Washing solution: GuHCl 7M, Tris 15mM, ethanol 50%, sodium bicarbonate 40mM, pH 9.0;

[0054] Elution buffer: Tris 20mM, EDTA 0.2mM, pH 8.5.

[0055] Control reagent 2 was prepared according to the formula of reagent 1 (replace sodium bicarbonate with sodium acetate 10mM)

[0056] Control reagent 3 was prepared according to the formula of reagent 1 (without CHAPS, with NaDC 0.6%)

[0057] Follow the steps below for DNA extraction:

[0058] (1) Take 200ul of fresh blood into a 1.5ml centrifuge tube, then add a total of 400ul of a 1:1 mixt...

Embodiment 2

[0063] Example 2 Validation of the effect of the kit formula of the present application

[0064] 1 The role of sodium bicarbonate and DPTA

[0065] Using reagents 1-3 and DNA extraction method in Example 1 to extract the same blood sample, use Qubit® (based on fluorescent dye method) detector and matching kit to detect the concentration of extracted DNA and detect ODA260 / 280, the results as follows:

[0066]

[0067] Repeat results in multiple samples were similar. We speculate that the acid-base / charge conditions in the washing solution have a great influence on the washing effect of mucin hydrolyzate. Among the various formulas in the experiment, only slightly alkaline sodium bicarbonate can effectively remove protein without affecting DNA stability. The effect; the addition of zwitterionic surfactant is conducive to fully lysing the lysed protein and making it easier to remove the protein during washing.

[0068] Comparison between the one-step washing kit of this app...

Embodiment 3

[0074] Example 3 Use the kit of this application for actual detection

[0075] Use the kit of this application to extract 4 pairs of DNA from the blood of 8 subjects (collected synchronously with blood samples) for STR locus typing in paternity testing (Promega Power Plex16, 16 sites, ABI9700 PCR instrument), and Compared with the typing done by the DNA extracted from the blood, the typing situation of all 128 loci of the 8 test subjects is exactly the same. It is preliminarily proved that the sample 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 biological detection, and particularly provides a blood DNA extraction kit only needing one-step washing by using a magnetic bead method. Theblood DNA extraction kit comprises a lysis solution, a binding solution, a proteinase K solution, a washing solution and an elution buffer solution. The method is characterized in that the lysis solution comprises Tris, EDTA, NaCl, NaDC, dodecyl-N-betaine, NLS, GuHCl, Triton X100, NH4Cl and mercaptoethanol; the binding solution comprises isopropanol, PEG 8000 and Brij 58; the washing solution comprises GuHCl, Tris, ethanol and sodium bicarbonate; and the elution buffer solution comprises Tris and EDTA. According to the invention, the blood DNA extraction kit only needing one-step washing by using the magnetic bead method is constructed by preparing the washing solution containing high-concentration sodium bicarbonate (the alkalinity is slightly stronger than that of sodium acetate and sodium chloride), simultaneously giving consideration to ion balance and providing alkalinity, and cooperatively using a zwitterionic detergent CHAPS.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and specifically, the application provides a magnetic bead method blood DNA extraction kit which only needs one-step washing. Background technique [0002] Nucleic acid extraction is the basis of various molecular biology detection methods, and efficient and complete extraction of DNA is the basis for PCR amplification, library construction, and sequencing. In addition to tissue and organ samples, the most commonly used isolated samples in molecular biology testing are blood samples. Among the currently known DNA extraction methods such as phenol-chloroform method, salting-out method, adsorption column method, immunoaffinity method, and magnetic bead method, the methods that can be used for blood DNA extraction mainly include the adsorption column method and the magnetic bead method. The extraction effect of the method and the concentration of the extracted DNA are obviously ...

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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