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DNA quantitative method based on rolling circle amplification

A technology of rolling circle amplification and rolling circle amplification reaction, which is applied in the field of DNA quantification, can solve the problems of limited application, decline, and inability to distinguish free nucleotides of double-stranded DNA, and achieves wide concentration range, low error, and high sensitivity. high effect

Inactive Publication Date: 2016-06-22
GENEWIZ INC SZ
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Spectrophotometry is convenient and widely used, but has some drawbacks: it cannot distinguish double-stranded DNA, single-stranded DNA and free nucleotides, and proteins, polysaccharides, phenols and other organic and inorganic substances can also affect the absorbance at 260nm , the most important thing is that the sensitivity will drop when the DNA concentration reaches 5ng / μl (5μg / ml), and the lowest measurement limit is 2ng / μl
However, fluorescence quantitative method requires special instruments, such as microplate reader and Qubit, not all laboratories have such conditions, which limits the application of this method

Method used

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  • DNA quantitative method based on rolling circle amplification
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  • DNA quantitative method based on rolling circle amplification

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 RCA quantification of pGEX4T-1 (4969bp)

[0033] Prepare 150μL sample buffer and 150μL reaction buffer; sample buffer includes random primer, Tris-HCl (400mM, pH8.0), KCl (160mM); reaction buffer includes Tris-HCl (125mM, pH7.5), MgCl 2 (25mM), (NH 4 ) 2 SO 4 (25 mM), DTT (10 mM), dNTPs (used at a final concentration of 0.2 mM).

[0034]The sample buffer was added to 35 wells, 4 μL of sample buffer in each well; then pGEX4T- 2 μL of 1’s double-distilled aqueous solution, five groups of each were added to 4 μL of sample buffer, and five groups of 300 pg / μL of pGEX4T-1 double-distilled aqueous solution (called U1) 2 μL were added to 4 μL of sample buffer at the same time. Amplify, react at 95°C for 3 minutes, and centrifuge instantly; then add the reaction buffer to 35 wells, 4 μL of reaction buffer per well, and simultaneously react at 30°C for 3 hours; and finally react at 65°C for 10 minutes to obtain three. Fifteen groups of rolling circle amplification...

Embodiment 2

[0038] Example 2 RCA quantification of pUC19 (2686bp)

[0039] Prepare 160μL sample buffer and 160μL reaction buffer; sample buffer includes random primer, Tris-HCl (400mM, pH8.0), KCl (160mM); reaction buffer includes Tris-HCl (125mM, pH7.5), MgCl 2 (25mM), (NH 4 ) 2 SO 4 (25 mM), DTT (10 mM), dNTPs (used at a final concentration of 0.2 mM).

[0040] The sample buffer was added to 32 wells, 4 μL of sample buffer in each well; then the mass concentrations were 0pg / μL, 3pg / μL, 10pg / μL, 30pg / μL, 100pg / μL, 1000pg / μL, 3000pg / μL, respectively. 2 μL of double-distilled aqueous solution of pUC19, four groups were added to 4 μL of sample buffer, and 2 μL of four groups of 320 pg / μL double-distilled aqueous solution of pUC19 (referred to as U2) were added to 4 μL of sample buffer, and rolled at the same time. Loop amplification, react at 95°C for 3 minutes, and centrifuge instantly; then add the reaction buffer to 32 wells, 4 μL of reaction buffer per well, and react at 30°C for 3 ...

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Abstract

The invention discloses a DNA quantitative method based on rolling circle amplification. The method comprises the following steps: performing rolling circle amplification reaction, restriction enzyme digestion reaction and electrophoresis on DNS standard solutions with different concentrations, and quantitatively analyzing the brightness of an objective strap to obtain a brightness value of the objective strap, thereby acquiring a standard curve between the concentration of the DNA standard solution and the brightness value of the objective strap; and then substituting the brightness value of the objective strap corresponding to the to-be-quantified DNA solution into a standard curve formula, thereby finishing the quantification of the to be quantified DNA. Through the adoption of the method disclosed by the invention, the sensitivity is close to the PicoGreen fluorescent dye quantitative method, an expensive device is unnecessary, and only a water bath pot, an electrophoresis apparatus and a plastic blooming instrument are required. The method disclosed by the invention can be used for quantitatively testing chain-typed DNA in a pg level.

Description

technical field [0001] The invention relates to a quantitative method for DNA, in particular to a quantitative method for DNA based on rolling circle amplification. Background technique [0002] DNA quantification is important for biological research, food and pharmaceutical industries. A number of different methods are used to quantify DNA: [0003] The most classic and commonly used method is UV spectrophotometry. The principle of this method is that the absorbance of the sample at 260nm corresponds to the concentration of nucleic acid in the sample. The latest spectrophotometer only needs 1 microliter of DNA solution, and Test without the use of cuvettes. Spectrophotometry is convenient and widely used, but has some drawbacks: it cannot distinguish double-stranded DNA, single-stranded DNA and free nucleotides, and proteins, polysaccharides, phenols and other organic and inorganic substances can also affect the absorbance at 260nm , most importantly, the sensitivity wil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2563/107C12Q2531/125C12Q2565/125
Inventor 洪媛媛李世红孙中平陈维之
Owner GENEWIZ INC SZ
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