Kit for DNA library building and application of kit

A technology of kits and DNA molecules, which is applied to kits for DNA library construction and its application fields, and can solve problems such as complex operations, long time-consuming, and low output of library construction

Active Publication Date: 2018-10-16
TIANJIN MEDICAL LAB BGI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of KAPA-LTP kits for DNA library construction has problems that are not conducive to production, such as high cost of single library construction, low yield of library construction, long time-consuming, and complicated operations.

Method used

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  • Kit for DNA library building and application of kit
  • Kit for DNA library building and application of kit
  • Kit for DNA library building and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, the preparation of kit

[0062] The composition of 5×Fast Library Repair Mix 1 is shown in Table 1.

[0063] Table 1

[0064] components

[0065] For 10×T4PNK buffer and ddH 2 For O, the data in the second column of Table 1 is the amount added; for other components, the data in the second column of Table 1 is the concentration of the component in 5× Fast Library Repair Mix 1.

[0066] Wherein, dNTP is formed by mixing dATP, dCTP, dTTP, and dGTP in a ratio of 1:1:1:1. For example, a dNTP concentration of 1.25 nmol / μL means that the dATP, dCTP, dTTP and dGTP concentrations are all 1.25 nmol / μL.

[0067] The composition of Adaptor Ligation Mix 2 is shown in Table 2.

[0068] Table 2

[0069] components

[0070] For 10×T4PNK buffer, the data in the second column of Table 2 is the amount of the component added in the 30 μL system; for other components, the data in the second column of Table 2 is the amount of the component in the 30 μL ...

Embodiment 2

[0071] Embodiment 2, establishment of method

[0072] 1. Extract free nucleic acid from plasma (plasma comes from normal population or tumor patients)

[0073] 1. Take EDTA anticoagulated peripheral blood, centrifuge at 4°C and 1600g for 10 minutes, and collect the upper plasma.

[0074] 2. Take the plasma obtained in step 1, centrifuge at 12,000g at 4°C for 10 minutes, and collect the upper layer of plasma.

[0075] 3. Take the plasma obtained in step 2 and extract it with the QIAamp Circulating Nucleic Acid kit to obtain a free nucleic acid solution.

[0076] Take the free nucleic acid solution, using dsDNA BR Analysis Kit (Thermo Fisher Scientific, Cat. No. Q32850) was used to detect the concentration of dsDNA (the instrument was Agilent 2100 Bioanalyzer).

[0077] For the detection results of the free nucleic acid solution in a single test, see image 3 .

[0078] 2. Establishment of plasma cfDNA library

[0079] 1. Take the free nucleic acid solution obtained in s...

Embodiment 3

[0145] Application of the kit prepared in embodiment 3, embodiment 1 and the method established in embodiment 2

[0146] The EDTA anticoagulated peripheral blood of volunteers (normal population or tumor patients) with informed consent was collected, and the kit prepared in Example 1 was used for detection according to the method established in Example 2 (X=0.5).

[0147] 1. Different T4PNK concentrations were used in the end repair and A addition reactions

[0148] In the initial reaction system of end repair and A addition reaction, the concentration of T4PNK is 0.2U / μL, 0.4U / μL or 0.6U / μL, the concentration of Klenow Fragment is 0.6U / μL, and the concentration of LA Taq DNA Polymerase is 0.05U / μL, the concentration of T4DNAPolymerase is 0.12U / μL, the concentration of dNTP is 0.25nmol / μL, and the concentration of dATP is 0.2nmol / μL.

[0149] In the initial reaction system for adapter connection, each component adopts the best reference value.

[0150] The results are shown...

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Abstract

The invention discloses a kit for DNA library building and an application of the kit, and in particular relates to a kit for peripheral blood cfDNA library building and an application of the kit. According to the invention, the kit for DNA library building is firstly provided, wherein the kit comprises a component A and a component B; the component A consists of the following ingredients at a ratio as follows: 1-3U of T4 PNK to 1-4.5U of Klenow Fragment to 0.25-0.5U of LA Taq DNA Polymerase to 0.3-0.9U of T4 DNA Polymerase to 0.5-2.5 nmol of dNTP to 0.5-2.5nmol of dATP; and the component B consists of the following ingredients at a ratio as follows: 2.5-8nmol of ATP to 26.5 132U of T4 DNA Ligase to 0.213 0.534[mu]L of PEG 8000. According to the kit and the application method provided by the invention, DNA loss in a library building process can be reduced to the greatest extent, the usage amount of plasma can be reduced, library building cost can be reduced, library building time can beshortened, operations can be simplified and more stable and reliable results can be obtained. The kit provided by the invention is significant for improving library building efficiency and reducing labor cost.

Description

technical field [0001] The invention relates to a kit for building a DNA library and its application, in particular to a kit for building a cfDNA library in peripheral blood and its application. Background technique [0002] Peripheral blood free DNA is the free DNA (cell-free DNA, cfDNA) existing in the peripheral blood circulation. There are two main sources of cfDNA: the first one, the "DNA ladder" fragmented nucleic acid produced during the process of cell apoptosis, the size is basically 150-200bp; the second one, the cell is lysed (this cleavage is both It can be derived from necrosis caused by physical stimulation, or it can be the nucleic acid produced by immune cell killing), and its size is close to that of genomic DNA. Of course, no matter which way the cfDNA is produced, it will be further rapidly cleared in the peripheral blood (this time may range from tens of minutes to several hours, but the mechanism is still unclear). [0003] Compared with healthy people...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2521/501C12Q2527/125C12Q2525/191C12Q2531/113
Inventor 刘军赵强王玉秋朱红梅茅矛叶明芝魏汉敏席阳安德烈·阿莱克谢耶夫
Owner TIANJIN MEDICAL LAB BGI
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