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DNA library, construction method and application thereof

A DNA library and construction method technology, applied in the field of high-throughput gene sequencing, can solve problems such as failure of library construction, low efficiency of library construction, unacceptable operation, etc. Take full advantage of the effect

Inactive Publication Date: 2019-08-09
谢小雷
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0023] (1) The amount of Adapter added is not determined according to the amount of cfDNA, resulting in low efficiency of library construction
[0024] (2) The master mix (ENZ2 and BUF2) is added first during the adapter connection process, resulting in a large number of DNA-DNA invalid connections
[0025] (3) In the actual operation process, due to multiple clinical samples at the same time, this method aggravates the invalid connection between DNA-DNA, resulting in the failure of library construction
Adding the Adapter needs to be fast, but it is not advisable to process multiple samples at the same time, and it is fast and easy to cause sampling errors; and it takes a lot of time to carry out the entire process for each sample separately, which is inefficient

Method used

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  • DNA library, construction method and application thereof
  • DNA library, construction method and application thereof
  • DNA library, construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0055] An embodiment of the DNA library of the present invention and its construction method, the construction method of the DNA library described in this embodiment is:

[0056] (1) Use serum cell-free DNA extraction kit to extract cell-free DNA in peripheral blood of pregnant women;

[0057] (2) End repair: The free DNA obtained in step (1) was subjected to end repair to obtain blunt-ended DNA, and the volume of the blunt-ended DNA was 49 μL;

[0058] (3) Take out the PCR tube used in step (2) from the common PCR instrument (or constant temperature metal bath) and put it into the ice box;

[0059] (4) Calculate the amount of Adapter required for each sample based on the free DNA content, so that the molar ratio of the Adapter to the free DNA extracted in step (1) is 100:1. After adding all the samples; shake and mix well, and spin for 5 seconds bell;

[0060] (5) According to the number of samples, add ENZ2 (T4DNA ligase) and BUF2 according to the ratio in Table 3 to make ...

Embodiment 2

[0070] An embodiment of the DNA library and its construction method of the present invention, the difference between the construction method of the DNA library described in this embodiment and the construction method of the DNA library in Example 1 is only that: in step (4) of this embodiment, the Adapter and the step (1) The molar ratio of extracted free DNA is 50:1.

[0071] In this embodiment, the method of detecting fetal cell-free DNA fragments in maternal peripheral blood by high-throughput sequencing method is the same as that in Embodiment 1.

Embodiment 3

[0073] An embodiment of the DNA library and its construction method of the present invention, the difference between the construction method of the DNA library described in this embodiment and the construction method of the DNA library in Example 1 is only that: in step (4) of this embodiment, the Adapter and the step (1) The molar ratio of extracted free DNA is 300:1.

[0074] In this embodiment, the method of detecting fetal cell-free DNA fragments in maternal peripheral blood by high-throughput sequencing method is the same as that in Embodiment 1.

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Abstract

Belonging to the technical field of gene high-throughput sequencing, the invention relates to a DNA library, a construction method and application thereof, in particular to a DNA library for high-throughput sequencing, a construction method and application thereof. The construction method of the DNA Library includes the steps of: (1) extracting free DNA from the peripheral blood of pregnant women;(2) performing end repair: subjecting the free DNA obtained in step (1) to end repair to obtain bluut end DNA; (3) adding a joint to the bluut end DNA obtained in step (2), mixing the substances evenly, then adding a premixed solution, and then mixing the substances evenly, with the premixed solution including DNA ligase and a buffer solution; and (4) carrying out ligation reaction to obtain theDNA library. In the construction process of the DNA library, the joint is added and is mixed evenly and then the premixed solution is added, thus avoiding DNA-DNA invalid connection caused by the action of DNA ligase before formal ligation reaction, and improving the efficiency of valid connection. After construction of the DNA library by the method provided by the invention, the success rate of library construction is increased to 98.58%.

Description

technical field [0001] The invention relates to a DNA library and its construction method and application, in particular to a DNA library for high-throughput sequencing and its construction method and application, belonging to the technical field of gene high-throughput sequencing. Background technique [0002] Since Lo Yuk-ming and others first discovered the presence of fetal free DNA (cell free DNA, cfDNA) in the peripheral blood of pregnant women in 1997, people have continued to deepen their research on it. The content of cell-free fetal DNA in maternal peripheral blood is between 5% and 30%, and the content of fetal DNA in the population is normally distributed. Fetal cell-free DNA (cfDNA) in maternal peripheral blood may be derived from small molecular DNA fragments produced by placental trophoblast cell apoptosis. Fragments of maternal DNA in peripheral blood are generally larger than fragments of fetal DNA. The length of fetal DNA fragments is about 140-160bp, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/08C12Q1/6806C12Q1/6869
CPCC40B50/06C40B40/08C12Q1/6806C12Q1/6869C12Q2535/122C12Q2521/501
Inventor 谢小雷
Owner 谢小雷