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Plasma DNA library and construction method thereof

A DNA library and construction method technology, applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., can solve the problems of long time and low efficiency of plasma DNA library construction, shorten the time for library construction, and improve the efficiency of library construction Effect

Inactive Publication Date: 2019-11-19
BEIJING USCI MEDICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] The main purpose of the present invention is to provide a plasma DNA library and its construction method to solve the problems of long construction time and low efficiency of plasma DNA library in the prior art

Method used

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  • Plasma DNA library and construction method thereof
  • Plasma DNA library and construction method thereof
  • Plasma DNA library and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The preparation method of embodiment 1 plasma dna library

[0048] 1. Plasma separation and preparation

[0049] ①The first plasma separation

[0050] After placing the peripheral blood of pregnant women in Streck blood collection tubes, centrifuge at 1600g, 4°C for 10min;

[0051] Check the properties of the upper plasma, which is transparent and yellowish, without hemolysis and other abnormal phenomena, which is qualified.

[0052] ②Pipe the upper layer of plasma into a new centrifuge tube, then centrifuge at 16000g at 4°C for 10 minutes, and take the upper layer of plasma for use.

[0053] 2. Plasma processing

[0054] Add Tween 20 reagent, mix thoroughly with the plasma and centrifuge briefly, and centrifuge the plasma to the bottom of the tube.

[0055] The plasma processing system is shown in Table 1:

[0056] Table 1:

[0057]

[0058] Pretreatment system: Tween 20 is used to treat plasma, and the final volume concentration of Tween 20 ranges from 0.5% t...

Embodiment 2

[0087] The selection of embodiment 2 emulsifiers

[0088] Using the same steps as in Example 1, the same initial amount of plasma (both 45 μL), using different amounts of emulsifiers to emulsify the plasma, using the same rTaq enzyme (Takara R500Z) for end repair and adding "A" operation , when the subsequent steps are the same, the influence of emulsifier dosage gradient on the constructed library storage capacity is detected, see Table 7 for details.

[0089] Table 7:

[0090]

[0091] In addition, emulsifying reagents such as Triton-100 or NP40 were also tested. The test results are similar to Tween 20, which can emulsify and increase fluidity, and improve the convenience of operation and library yield to a certain extent.

Embodiment 3

[0092] Example 3 Screening of Taq enzymes

[0093] Use the same initial amount of plasma (45 μL), use the same emulsifier (Tween 20) 3.00 μL to emulsify the plasma, use different enzymes to repair the end and add "A", and the subsequent steps are the same , to detect the effect of different end repair and "A" added enzymes on the capacity of the constructed library, see Table 8 for details.

[0094] Table 8:

[0095]

[0096] According to the results in Table 7 and Table 8, Tween 20 was selected as the emulsifier, and rTaq was used as the end repair plus "A" enzyme for subsequent detection of different sequencing platforms. Among them, the library test quality inspection results of the illumina sequencing platform are shown in Table 9.

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Abstract

The invention provides a plasma DNA library and a construction method thereof. The construction method comprises the following steps: adding an emulsifier into plasma to obtain emulsified plasma; performing end repair / dA on the emulsified plasma to obtain repaired plasma; performing adapter ligation on the repaired plasma to obtain a ligation product; and performing PCR amplification on the ligation product to obtain the plasma DNA library. According to the construction method, plasma is directly used as an object without the need for extracting free DNA in the plasma, and the steps of performing end repair / dA, adapter ligation and PCR amplification after the plasma is subjected to emulsifying pretreatment to improve the fluidity and operability of the plasma, and construction of a plasmaDNA library without DNA extraction can be truly implemented. The method omits the step of plasma DNA extraction, the whole experimental procedure of library construction can be completed in 3 hours, and the library construction efficiency is greatly improved. Therefore, the construction method can meet the requirement for quick batch library construction in the modern time.

Description

technical field [0001] The invention relates to the technical field of high-throughput sequencing, in particular to a plasma DNA library and a construction method thereof. Background technique [0002] DNA sequencing technology, that is, the technical method of determining the DNA sequence. In molecular biology research, DNA sequence analysis is the basis for further research and modification of target genes. The original generation sequencing technology mainly includes the dideoxy chain terminal termination method invented by Sanger et al. (1977) and the chemical degradation method invented by Maxam and Gilbert (1977). These two methods are very different in principle, but they are all based on the fact that the nucleotide starts at a fixed point and ends at a specific base at random, resulting in four groups of A, T, C, and G with different lengths. A series of nucleotides are then detected by electrophoresis on a urea-denatured PAGE gel to obtain the DNA sequence. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C40B50/06
CPCC12Q1/6806C40B50/06C12Q1/686
Inventor 王建伟孙广欣王冬伍启熹张静波石露王伟伟刘倩唐宇
Owner BEIJING USCI MEDICAL LAB CO LTD
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