DU5 adaptor, application thereof and cDNA library constructed by dU5 adaptor

An adapter and library technology, applied in the field of cDNA library, can solve the problems of long library construction time, large amount of RNA input, and low PCR efficiency, so as to reduce the formation of adapter-primer dimers, expand the scope of research, reduce The effect of cycle number

Pending Publication Date: 2022-07-19
香港城市大学深圳研究院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the experimental process of rG4-seq sequencing technology requires a large amount of RNA input and takes a long time for library construction. Therefore, we systematically evaluated and optimized some key experimental steps in the entire cDNA library construction process, hoping to improve the efficiency of library construction. Efficiency and quality, this research result has been published in 2019 (Yeung, Zhao et al.2019), and applied for a patent (No.15 / 957,037, USA, under review)
[0004] Based on the above research, we found that the PCR efficiency was low during the library construction process, and the number of reaction cycles required to complete the library construction was high, which affected the quality of the library and resulted in less effective data obtained by subsequent next-generation sequencing analysis.

Method used

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  • DU5 adaptor, application thereof and cDNA library constructed by dU5 adaptor
  • DU5 adaptor, application thereof and cDNA library constructed by dU5 adaptor
  • DU5 adaptor, application thereof and cDNA library constructed by dU5 adaptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] This embodiment provides a dU5' adaptor, which has the following nucleotide sequence: 5'- / 5Phos / -AGATCGGAAGAGCGTCGTGTAGC / dU / CTTCCGATCTNNNNNNNNNN- / 3SpC3 / -3' (SEQ ID NO: 3).

[0057] The dU5' adaptor is a structure containing deoxyuracil specially designed by the inventor. In the newly designed dU5' adaptor, the insertion position of dU is the 24th position from the 5' end, which is more conducive to subsequent PCR The forward primer is paired, and its complementary pairing part is kept intact, which is conducive to the binding of the forward primer and the template, can effectively reduce the formation of adapter-primer dimer, and reduce the number of cycles required for the reaction, thereby improving PCR amplification. Increase efficiency, thereby improving the quality of the library.

[0058] The dU5' adaptor in this example was obtained by commercial synthesis according to its nucleotide sequence, and the commercially synthesized dU5' adaptor nucleic acid sam...

Embodiment 2

[0060] The present embodiment provides a method for constructing an improved cDNA library. The improved part is as follows: figure 1 shown, it includes the following steps:

[0061] The simulated cDNA nucleic acid sample (which has a 3' adaptor, see SEQ ID NO: 4 for the sequence) was mixed with the dU 5' adaptor of Example 1 to carry out a ligation reaction using a rapid ligation kit (NEB, M2200L). The molar ratio of the nucleic acid sample to the dU5' adaptor was 1:10, and the reaction conditions were 37°C for 2 h; the dU5' adaptor-cDNA nucleic acid complex was obtained and subjected to column purification (Zymo Research, R1016);

[0062] Add CutSmart buffer (1X final) USERII enzyme (0.1U / μL final) to the purified dU5' adaptor-cDNA nucleic acid complex to cleave deoxyuracil at a cleavage temperature of 37°C and a reaction time of 15min to form ends Partially complementary adaptor-nucleic acid complexes; excess adaptors were removed by column purification (Zymo Research, R10...

Embodiment 3

[0072] Example 3: Cleavage time optimization of deoxyuracil in dU 5' adaptors by USER II enzyme

[0073] This example investigates the effect of USER II enzyme on the cleavage of deoxyuracil in the cleavage of dU5' adaptors at different cleavage times, and the reaction conditions are set to 37 °C. The experimental results are as figure 2 shown.

[0074] Depend on figure 2 It can be seen that the cracking rate increases with the increase of the reaction time. In order to maximize the cracking rate, a cracking time of 15min is preferably used.

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Abstract

The invention provides a dU5'adapter, application thereof and a cDNA library constructed by the dU5 'adapter. The structure of the dU5'adapter is 5 '-p-S1-S2-S3-S4-SpC3-3', S1, S2, S3 and S4 are gene segments which are sequentially connected through chemical bonds, and p represents phosphorylation modification performed at the 5'end; s1 is a first stem; s2 is a ring part; s3 is a second stem part and contains one deoxidized uracil dU; the 5 '-> 3' sequence of S1 and the 3 '-> 5' sequence of S3 are complementary, S4 is a guide part (N) m, N is any one selected from A, T, G and C basic groups, m is 10, and SpC3 represents C3 interval modification performed at the 3'terminus. The dU5'adapter can reduce formation of adapter-primer dimers, improve the PCR amplification efficiency, shorten the PCR period and improve the library quality, and a new important platform is provided for constructing a cDNA library for wide biological application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a dU5' adaptor and its application and constructed cDNA library. Background technique [0002] cDNA library construction is critical for many high-throughput sequencing applications. In recent years, due to the development of a variety of cDNA library construction methods, the purpose of exploring and researching new properties of RNA has been achieved. For example, the research in the field of RNA structure has been in-depth. [0003] In recent years, our research team has developed RNA G-quadruplex structure sequencing (rG4-seq) technology (Kwok, Marsico et al. 2016), which revealed that the RNA G-quadruplex structure exists in the human body from the transcriptome level. The research on the structure and function of rG4 provides an effective basis. The 5' adaptor used in the library construction can reduce the non-specific binding in the process of ligating cDNA, thereby improving ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/6806C40B50/06C40B80/00
CPCC12N15/1096C12Q1/6806C40B50/06C40B80/00C12Q2531/113C12Q2525/191C12Q2535/122
Inventor 郭骏杰杨沛欣赵洁宇
Owner 香港城市大学深圳研究院
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