Human free DNA (Deoxyribonucleic Acid) extraction method and kit thereof

A kit and human technology, applied in the biological field, can solve the problems of low elution rate, high production cost, cumbersome operation, etc., and achieve the effect of improving extraction purity, saving operating cost, and reducing rinsing times

Inactive Publication Date: 2018-03-09
广州海思医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are some problems in the currently disclosed kits based on the magnetic bead extraction method: first, proteinase K is used to pretreat the samples, resulting in high production costs, cumbersome operations, and high requirements for production, transportation, and storage conditions; , a higher concentration of isopropanol was used in the binding solution, and the use of isopropanol for precipitation is prone to the problem

Method used

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  • Human free DNA (Deoxyribonucleic Acid) extraction method and kit thereof
  • Human free DNA (Deoxyribonucleic Acid) extraction method and kit thereof
  • Human free DNA (Deoxyribonucleic Acid) extraction method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 polysorbate 80 degradation product

[0036]Add polysorbate 80 solution to a 100ml shake flask, inoculate 3wt% Trichoderma viride with a concentration of 1000cfu / mL, set the temperature at 30°C, and rotate at 120r / min, cultivate for 15d, centrifuge, and take the supernatant; Microfiltration, cation exchange and sterilization were performed on the clear water to obtain polysorbate 80 degradation product.

Embodiment 2

[0037] Embodiment 2 A kind of human cell-free DNA extraction kit

[0038] The kit described in embodiment 2 is made up of following reagents:

[0039] ①Cell lysate: 35mmol / L Tris-Hcl, 100mmol / LNaCl, 10mmol / L disodium edetate, 5mol / L guanidine isothiocyanate, 2mol / L potassium acetate, 1.5wt% triton and 3wt % Tween 20, pH value is 5.0;

[0040] ②Magnetic bead pretreatment solution: 500ppm non-polar amino acid and 1000ppm fructooligosaccharide, wherein the non-polar amino acid is composed of leucine and valine in a weight ratio of 1:0.5.

[0041] ③ Rinse solution Ⅰ: 100mmol / L Tris-Hcl, 50mmol / LNaCl, 10mmol / L disodium edetate, 3mol / L guanidine hydrochloride, 2wt% triton, 5wt% Tween 20 and 30wt% isopropanol , the pH value is 5.2;

[0042] ④ Rinse solution Ⅱ: 75% ethanol;

[0043] ⑤ Conjugate solution: 0.2mmol / L polysorbate 80 degradation product, 16mmol / L polyethylene glycol 8000 and 1.5mmol / L sodium chloride;

[0044] ⑥Nucleic acid eluent: 15mmol / L Tris-Hcl and 1mmol / L EDTA...

Embodiment 3

[0046] Embodiment 3 A kind of human cell-free DNA extraction kit

[0047] The difference between Example 3 and Example 2 is that ② magnetic bead pretreatment solution is not included, and other parameters and operations are as shown in Example 2.

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Abstract

The invention belongs to the field of biotechnology, and specifically relates to a method for extracting human free DNA and a kit thereof, including magnetic bead pretreatment liquid, cell lysate, binding liquid, rinsing liquid I, rinsing liquid II, nucleic acid eluting liquid and carboxylation For magnetic beads, the binding solution includes components at the following concentrations: 0.1-0.3 mmol/L polysorbate 80 degradation product, 12-26 mmol/L polyethylene glycol 8000 and 1-2 mmol/L sodium chloride. The extraction efficiency, extraction purity and CT value of the kit of the invention are significantly better than those of the existing kits, and have the advantages of low cost and easy operation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human free DNA extraction method and a kit thereof. Background technique [0002] Nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biological macromolecules with genetic information. The DNA of eukaryotes mainly exists in the nucleus in the form of chromosomes, but fragmented free DNA (Cell-free DNA, cfDNA) is also free in human peripheral blood, and these free DNA can be single-stranded or double-stranded DNA Most of the free DNA is double-stranded DNA in the form of nucleoprotein, and the fragments are small, generally between tens or hundreds of bp in length. [0003] Studies have found that cell-free DNA contains very important genetic information. In 1997, YMD Lo et al. detected the existence of Y chromosome in the peripheral blood of pregnant women who were pregnant with male fetuses, which proved the existence of fetal cel...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 周煌凯钟诗龙邓美英陆嫚云徐毓璇姚啟聪
Owner 广州海思医疗科技有限公司
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