3'RACE (rapid amplification of cDNA ends) method for acquiring complete 3' end sequence as 3' end having repetitive sequence

A technology of repeated sequences and terminal sequences, applied in the biological field, can solve the problem of inability to obtain complete terminal fragments

Inactive Publication Date: 2017-05-10
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The main purpose of the present invention is to solve the problem that some species have an A repeat structure at the 3' end of their mRNA sequence, resulting in the problem that the complete fragment of the end cannot be obtained using 3' RACE

Method used

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  • 3'RACE (rapid amplification of cDNA ends) method for acquiring complete 3' end sequence as 3' end having repetitive sequence
  • 3'RACE (rapid amplification of cDNA ends) method for acquiring complete 3' end sequence as 3' end having repetitive sequence
  • 3'RACE (rapid amplification of cDNA ends) method for acquiring complete 3' end sequence as 3' end having repetitive sequence

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Experimental program
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Effect test

Embodiment 1

[0020] The mangrove plant kandelia (Kandelia obovata) iron-type superoxide dismutase (Fe-Superoxide Dismutase) gene was used as the test object, named as FeSOD1. After the total RNA of candela was extracted and passed the quality inspection, it was stored at ultra-low temperature (-80°C) for future use.

Embodiment 2

[0022] Control group: cDNA was reverse transcribed using Thermo Scientific RevertAid First Strand cDNASynthesis Kit (Fermentas EU) according to the general method.

Embodiment 3

[0024] Experimental group: (1) mRNA was tailed with terminal transferase from TaKaRa Company.

[0025] Prepare the following reaction solution in a microcentrifuge tube, the total volume is 50ul (Table 1).

[0026] Table 1 Preparation of TdT reaction solution

[0027]

[0028] Mix the reaction solution evenly, shake slightly and react at 37°C for 30 minutes. React at 80°C for 30 seconds to terminate the reaction.

[0029] (2) Reverse transcription experiments were performed using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Fermentas EU) kit. The preparation of the specific reaction solution is shown in Table 2.

[0030] Prepare the following reactions (Table 2) in microcentrifuge tubes.

[0031] Table 2 Preparation of reaction solution

[0032]

[0033] Note: AP3-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGGGGGGG

[0034] React at 65°C for 5 minutes to open the RNA secondary structure and facilitate primer binding. React on ice for 1 minute.

[0035] ④ P...

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Abstract

The invention discloses a 3'RACE (rapid amplification of cDNA ends) method for acquiring a complete 3' end sequence as a 3' end having a repetitive sequence. The method comprises the following steps: 1) tailing mRNA with terminal transferase, so that a connector sequence is added: tailing the mRNA with dCTP or dGTP, so that a single-base repetitive sequence of 16-22bp is added; 2) synthesizing first chain cDNA: conducting reverse transcription on the tailed mRNA by taking an anchor primer having oligodG or oligodC as a connector sequence; and 3) conducting 3'RACE amplification: with the cDNA having the connector sequence as a template, an internal specific primer GSP2 of the template which is designed in accordance with a RACE primer design principle as well as a corresponding nested primer NGSP2 thereof as forward primers of the amplification, and a complementary sequence (AUAP) of the connector sequence as a reverse amplification primer, conducting 3'RACE on the cDNA sequence having the connector. With the application of the 3'RACE method provided by the invention, the problem that a complete end sequence cannot be acquired in 3'RACE due to a special structure (having the A repetitive sequence) of the sequence can be solved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a 3' RACE method for obtaining a complete 3' end sequence in the case of an A repeat sequence at the 3' end. Background technique [0002] Rapid amplification of cDNA ends (Rapid Amplification of cDNA Ends, RACE) is a method that combines PCR technology and uses known parts of cDNA sequences to obtain complete ends. This method is widely used in many research fields due to its unique advantages. RACE technology has been continuously perfected and improved since it was proposed. Among them, the predecessors found that when the mRNA is reverse-transcribed into the first cDNA strand, the primer often randomly combines with any part of poly(A), which affects the result of reverse transcription. Therefore, in order to solve such problems, researchers introduced two degenerate nucleotides [5'-oligo(dT)16-30MN-3', M=A / G / C, N=A / G / C / T] makes it a lock clocking primer, positioned at the sta...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1096C12Q2521/131C12Q2525/173C12Q2525/191
Inventor 潘铖烺
Owner XIAMEN UNIV
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