Library construction method capable of detecting transcription initiation sites of eukaryotes by using high-throughput sequencing technology

A technology for transcription initiation site and technical detection, which can be used in chemical libraries, recombinant DNA technology, combinatorial chemistry, etc. It can solve the problems of large demand for RNA and cumbersome operation steps, and achieve the effect of simple and easy sequencing methods.

Pending Publication Date: 2019-05-14
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, the method based on Cap-Trapper requires a large amount of RNA, and the operation steps are cumbersome; the disadvantage of Template Switching technology is that template switching will also occur in DNA-RNA complexes, so not all detected sequences are true TSS sites

Method used

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  • Library construction method capable of detecting transcription initiation sites of eukaryotes by using high-throughput sequencing technology
  • Library construction method capable of detecting transcription initiation sites of eukaryotes by using high-throughput sequencing technology
  • Library construction method capable of detecting transcription initiation sites of eukaryotes by using high-throughput sequencing technology

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Experimental program
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Embodiment

[0076] Such as figure 1 As shown, the construction process of the transcription initiation site library provided by this application includes: purification of poly(A) RNA → dephosphorylation of the monophosphate group at the 5' end → removal of the poly(A) RNA cap structure → 5' RNA Adapter ligation→reverse transcription→first PCR amplification→EcoP15I digestion→ligation of double-stranded DNA adapter→first polyacrylamide gel separation and purification of ligation products→transcription initiation site library enrichment (second PCR amplification) → the second polyacrylamide gel separation and purification of the transcription initiation site library → library quality inspection and sequencing analysis. In this example, the leaves of Nipponbare rice seedlings (grown for one month after germination) were used as biological materials, and the construction process of the relevant transcription initiation site library is described in detail as follows.

[0077] (1) Purification...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a library construction method capable of detecting poly (A) RNA transcription initiation sites of eukaryotes by using a high-throughput sequencing technology. The method is used for RNA with poly (A) tail and cap structure in the eukaryotes, and comprises the following steps: purifying to obtain the poly(A) RNA, removing phosphorylation, removing the cap structure, connecting RNA adapter, carrying out reverse transcription, carrying out PCR amplification, carrying out enzyme digestion, connecting double-stranded DNA adapter, carrying out PCR enrichment transcription initiation sites library, carrying out purification and analysis of the library. The method has the advantages of high cap removalefficiency, capability of efficiently constructing a transcription initiation sites library, convenience for accurately positioning the transcription initiation sites, simplicity and convenience in sequencing method, contribution to improving the accuracy of a transcription initiation sites analysis result and the like; the technique can be used for the study of all RNA transcription initiation sites with the cap structure with a slight improvement.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a library construction method capable of detecting eukaryotic poly(A) RNA transcription start sites by using high-throughput sequencing technology. Background technique [0002] The cap structure refers to a special structure formed at the 5' end of eukaryotic mRNA and some non-coding RNAs after post-transcriptional modification, that is, the N7-methylated guanosine and the first nucleotide of the RNA pass through the reverse 5'- The m7GPPPN structure formed by the 5' triphosphate bond connection is also called the methyl guanosine cap. All eukaryotic mRNAs have a cap structure, which plays an important role in mRNA maturation and can serve as a specific marker to recruit protein factors required for mRNA pre-cleavage, poly(A) tail synthesis, and nuclear transport. The cap structure also has important biological significance. mRNA is a template for protein ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6869C40B50/06
Inventor 李用芳王梦蕾赵淼魏康宁王丽
Owner HENAN NORMAL UNIV
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