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Method and equipment for constructing sequencing library

A sequencing library and equipment technology, applied in the biological field, can solve problems that need to be further studied

Active Publication Date: 2018-09-04
MGI TECH CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A limitation of current methods is that the initial input RNA yield needs to be between hundreds of ng and μg to meet the losses caused by a series of purification and recovery steps during various enzyme reactions and prior to PCR amplification.
[0004] Therefore, the current research on the construction of cDNA libraries from total RNA still needs to be further studied.

Method used

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  • Method and equipment for constructing sequencing library
  • Method and equipment for constructing sequencing library
  • Method and equipment for constructing sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Source of experimental samples: The material used is human cell RNA standard (Universal Human Reference RNA) purchased from Agilent Technologies.

[0059] Specific experimental steps:

[0060] 1. RNaseH purification of mRNA

[0061] 1.1 Take 100ng of total RNA and anneal to oligo DNA, the reaction system is as follows:

[0062]

[0063] Wherein, Oligo DNA is a mixture formed by mixing the individual sequences shown in SEQ ID NO.1-SEQ ID NO.195 in equal molecular ratios.

[0064] 1.2 React at 95°C for 2 minutes on a PCR machine; cool down gradually, from 0.1°C to 22°C every 1 second; react at 22°C for 5 minutes, and place on ice quickly.

[0065] 1.3 RNaseH enzyme digestion

[0066]

[0067] React at 37°C for 30min.

[0068] 1.4 DNase I enzyme digestion

[0069]

[0070] 1.5 After the reaction, the product was purified with RNA clean XP magnetic beads and dissolved in 10 μl of nuclease-free water.

[0071] 2. mRNA fragmentation

[0072]Add 3.5 μL of 5×fir...

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Abstract

The invention provides a sequencing library construction method and device. The method comprises the following steps: 1, breaking mRNA to obtain fragmented mRNA; 2, dephosphorylating the fragmented mRNA to obtain dephosphorylated mRNA; 3, connecting the dephosphorylated mRNA with a 3' end linker to obtain a linkage product; 4, carrying out inverse transcription on the linkage product to obtain cDNA; 5, separating single-stranded DNA from the cDNA; and 6, cyclizing the single-stranded DNA to obtain annular DNA which forms the sequencing library. The method uses a very tiny amount of RNA to construct the library without PCR.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, to a method and equipment for constructing a sequencing library, and a method and system for sequencing. Background technique [0002] With the application and development of high-throughput sequencing technology, the second-generation sequencing platform is becoming more and more mature and stable. The main problem is that the library preparation steps are cumbersome and the cost remains high. Therefore, the second-generation sequencing cannot meet the market requirements. Walk into thousands of households. The key to solving this problem is to reduce the input amount of starting samples and simplify the library preparation steps. [0003] The traditional construction of cDNA library from total RNA needs to rely on oligo(dT) to capture the information RNA. The information is then synthesized by reverse transcription into the first strand and the second strand, plus adapters,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/6869C40B60/14
Inventor 纪晓钧郭晶祝珍珍耿春雨章文蔚蒋慧
Owner MGI TECH CO LTD