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Method and device for constructing sequencing library

A technology for sequencing libraries and sequences, which can be used in chemical libraries, library creation, combinatorial chemistry, etc., and can solve problems that need to be improved.

Active Publication Date: 2019-08-09
MGI TECH CO LTD
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] Therefore, the current method for the preparation of small molecule RNA samples still needs to be improved.

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  • Method and device for constructing sequencing library
  • Method and device for constructing sequencing library
  • Method and device for constructing sequencing library

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Experimental program
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Embodiment 1

[0065] Source of experimental samples: standard universal human reference RNA (Agilent), humanbrain reference RNA (Ambion)

[0066] According to the following experimental steps, four groups of parallel experiments were carried out:

[0067] 1. 3' joint connection

[0068] 1) Take 1 μg of total RNA (1110ng / μl, not less than 200ng / μl), add RNase-free water to 2.5μl (not more than 5μl), add 1μl of 10μM adenylylated 3' linker (the sequence of the 3' linker is : 5'-GTCTCCAGTCGAAGCCCGATCNNNNNNNNNNGAGCTTGTCT-3' (SEQ ID NO.13), wherein, N represents a tag sequence, and the tag sequence is any one of SEQ ID NO.1 to SEQ ID NO.8). Denature in a PCR instrument at 70°C for 2 minutes.

[0069] SEQ ID NO. tag sequence 1 CTGTTTGGAC 2 AGTTGCCAAT 3 TTATCAGGTC 4 TAAGAGACCC 5 ACTCTCTACC 6 GGTCTAGTCA 7 TAGCTAAGTG 8 TACAAAAGGA

[0070] 2) Add the following reaction mixture to the tube of RNA and adapter in the previous step:

[00...

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Abstract

The invention provides a method and a device for constructing a sequencing library. The method comprises the following steps: (a) ligating a first adaptor at the 3' terminal of an RNA molecule, so that a first ligation product is obtained; (b) ligating a second adaptor at the 5' terminal of the first ligation product, so that a second ligation product is obtained; (c) carrying out inverse transcription on the second ligation product, so that an inverse transcription product is obtained; (d) carrying out amplification on the inverse transcription product by utilizing a pair of PCR primers, so that a PCR amplification product is obtained, wherein one of the pair of PCR primers contains streptavidin; (e) separating a single-stranded DNA molecule from the PCR amplification product; and (f) carrying out cyclization on the single-stranded DNA molecule, so that a cyclization product is obtained, and the cyclization product forms the sequencing library. The method can be effectively used for constructing the RNA molecule sequencing library, the operation is simple, the effect is good, and the relatively high stability and repeatability are obtained.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a method and device for constructing a sequencing library. Background technique [0002] Small RNA (small RNA) is a class of RNA molecules with a length of 18-30 nt in organisms, mainly including miRNA, siRNA and piRNA. Small RNA can regulate gene expression, play an important role in basic biological processes such as cell growth, development, and metabolism, and even play a key role in the formation of cancer and other related diseases. [0003] At present, with the application and development of next-generation high-throughput sequencing technology, small molecule RNA sequencing technology is becoming more and more mature. Usually, the small molecule RNA sample preparation method is mainly based on the ligation method, but in this method, in order to improve the ligation efficiency, an excessive amount of adapters will be invested in the ligation process, and these excess...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06
Inventor 张春燕祝珍珍耿春雨章文蔚蒋慧
Owner MGI TECH CO LTD