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Kit for extracting blood free DNA

A kit and blood technology, applied in the field of kits for the extraction of free DNA from blood, can solve the problems of low purity and low recovery efficiency, and achieve the effect of improving extraction efficiency, improving recovery efficiency, and reducing material costs

Pending Publication Date: 2020-03-24
SHENZHEN HAPLOX BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different from genomic DNA extraction, existing cfDNA extraction solutions generally have problems such as low recovery efficiency and low purity

Method used

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  • Kit for extracting blood free DNA
  • Kit for extracting blood free DNA
  • Kit for extracting blood free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The kit in this example specifically includes lysate, proteinase K, magnetic bead suspension, binding solution, cleaning solution 1, cleaning solution 2 and eluent.

[0043] Wherein, the lysate is composed of 4M guanidine isothiocyanate, 50mM Tris-HCl, 25mM EDTA, 300mM NaCl and 12% Triton X-100.

[0044] Proteinase K was purchased from Tiangen Biochemical Technology, and the magnetic bead suspension was purchased from Weidu Biotechnology.

[0045] The binding solution consisted of 3.5M guanidine isothiocyanate, 50 mM Tris-HCl, 25 mM EDTA, 12% Triton X-100 and 30% isopropanol.

[0046] Cleaning solution 1 consisted of 2M guanidine isothiocyanate, 50 mM Tris-HCl, 25 mM EDTA, 5% TritonX-100 and 30% isopropanol.

[0047] Wash solution 2 consisted of 10 mM Tris-HCl and 80% absolute ethanol.

[0048] The eluent was nuclease-free sterile deionized water.

[0049] The method of using the kit in this example is as follows:

[0050] (1) Add 40 μL of proteinase K, 1 mL of plas...

Embodiment 2

[0064] In this example, on the basis of Example 1, the components and concentrations of the components of the lysate, the binding solution, the cleaning solution 1 and the cleaning solution 2 were tested, as follows in detail:

[0065] 1. Lysate formulation test

[0066] In this example, different concentrations of the components in the lysate were tested, as shown in Table 2.

[0067] Table 2 Lysate formula test

[0068]

[0069]

[0070] In this example, according to Table 2, the lysates of eight formulations from Test 1 to Test 8 were prepared, and the rest of the reagents, such as proteinase K, binding solution, cleaning solution 1 and cleaning solution 2, etc., were the same as in Example 1, and the same as in Example 1. According to the method, cfDNA was extracted from the plasma sample 1 of Example 1. And measure the concentration and purity of the extracted cfDNA according to the method of Example 1. The results show that the lysates of the eight formulations ...

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Abstract

The invention discloses a kit for extracting blood free DNA. The kit of the invention includes a lysis solution, a binding solution, a cleaning solution 1 and a cleaning solution 2. The binding solution contains 2-5 M guanidine isothiocyanate or guanidine hydrochloride, 10-100mM Tris-HC1, 10-50mM EDTA, 5-15% Triton X-100, and 20-50% absolute ethanol or isopropanol. The cleaning solution 1 contains2-5 M guanidine isothiocyanate or guanidine hydrochloride, 10-100mM Tris-HCl, 10-50mM EDTA, 5-10% Triton X-100, SDS or Tween 20, and 20-50% absolute ethanol or isopropanol. The kit of the invention improves the binding solution and increases the extraction efficiency, improves the cleaning solution 1, increases the binding efficiency of the magnetic beads cfDNA and removes impurities, improves the recovery efficiency and purity of the cfDNA and reduces the material cost.

Description

technical field [0001] This application relates to the technical field of blood free DNA extraction, in particular to a kit for blood free DNA extraction. Background technique [0002] Blood free DNA (cell free DNA, cfDNA) refers to the partially degraded endogenous DNA in the circulating blood that is free from the cells. cfDNA is a complex mixture including benign cells, white blood cells, and pathogenic nucleic acids, of which fetal free DNA (cell free fetal DNA, cffDNA) and tumor free DNA (cell free tumor DNA, ctDNA) only account for a small part. The plasma of healthy people contains a small amount of cfDNA, which is mainly derived from apoptosis, and the fragments are mainly 160-200bp or integer multiples. Tumor cells (CTC) circulating in the blood can also release free DNA, the fragments are mainly 20-200bp, and the large ones can reach more than 1000bp. [0003] cfDNA was first discovered by Mandel and Metais in 1947. With the in-depth study of cfDNA, it is found ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 许明炎张晓妮张生沈广强
Owner SHENZHEN HAPLOX BIOTECH
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