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Reagent used for ex vivo expansion and method thereof

A technology for in vitro amplification and reagents, applied in the reagents and fields for in vitro amplification of RNA, which can solve the problems of unknown RNA and RNA mixtures without amplification or effective detection

Inactive Publication Date: 2009-06-03
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For the amplification and synthesis technology of RNA of known sequence, it is relatively mature, and the detection of RNA of known sequence such as microRNA (miRNA) of known sequence also has method introduction (C.K.Raymond, et al., RNA 2005, 11, 1737 -1744; C.Chen, et al., Nucleic Acids Res.2005, 33, e179; S.Ro, et al., Biochem.Biophys.Res.Commun.2006, 351, 756-763), but for unknown RNA and RNA Mixtures with no existing method for true amplification or efficient detection

Method used

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  • Reagent used for ex vivo expansion and method thereof
  • Reagent used for ex vivo expansion and method thereof
  • Reagent used for ex vivo expansion and method thereof

Examples

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Embodiment 1

[0085] The experimental process of the restriction endonuclease cutting site at the 3' end of the recognition sequence is as follows (see figure 1 and figure 2 ). This example uses the restriction endonuclease NlaIII Electrophoresis results see Figure 4 .

[0086] A. Ligation of appropriate SL1 to target RNA, the resulting product is called P1.

[0087] In order not to be self-ligated, it is first necessary to dephosphorylate the 5' end of the target RNA, and then make the 5' phosphorylated SL1 under the action of a ligase such as T4 RNA ligase (NlaIII is used in the embodiments of the present invention, so SL1 The 5' end contains the site of endonuclease NIaIII) and dephosphorylated target RNA. SL1 can be DNA or RNA, or a DNA-RNA hybrid strand with RNA at the 5' end. Generally speaking, the ligation efficiency of RNA single strand to DNA single strand is lower than that of two RNAs. Since the 3' end of SL1 is blocked or in base pairing, T4 RNA ligase, which can only ...

Embodiment 2

[0108] The experimental process of the restriction endonuclease cutting site at the 5' end of the recognition sequence is as follows (see figure 1 and image 3 ). This embodiment uses restriction endonuclease DPNII, and its enzyme cutting site is: Electrophoresis results see Figure 5 .

[0109] Steps A-D are the same as in Example 1 except that the 3' end of SL1 contains a site for endonuclease DPNII.

[0110] E. Perform PCR on P4 to obtain P5.

[0111] Using SLP1 with free hydroxyl at the 5' end and LLP1 with biotin at the 5' end as primers or SLP1 with biotin at the 5' end and LLP1 with free hydroxyl at the 5' end, PCR amplification was performed on P4 to obtain the product P5. There are promoter-associated sequences in LLP1.

[0112] F. Separation of the two chains of P5 using biotin at the 5' end of one of the chains.

[0113] Biotin can specifically combine with Streptavidin or Avidin, so the two chains of P5 are separated after the magnetic beads containing Stre...

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Abstract

The invention discloses a reagent used for ex vivo expansion and a method thereof, comprising an adaptor, a primer, a promoter, a restriction enzyme, and the like. The aim of RNA ex vivo expansion is achieved. Target RNA in the invention is connected with the adaptor and is expanded after RT-PCR under the existing of the primer. Then, a stem-loop structure is used for forming a unique site of the restriction enzyme. A product obtained through the restriction enzyme is promoted by the promoter and is transcribed to obtain a sequence which is totally the same with the target RNA sample except a G is added on 5' end and an A or C is added on few 3' ends. The RNA product can be directly used as the original sample RNA. The reagent used for the ex vivo expansion and the method thereof can be applied to various aspects related to the application of RNA, and the like, including general scientific research, medical assay and forensic identification.

Description

technical field [0001] The present invention relates to the design of reagents for amplifying RNA in vitro and the in vitro amplification method for RNA, especially unknown sequence RNA and RNA mixture. Background technique [0002] As endogenously produced non-coding small RNAs (non-coding small RNAs) are found to play an important role in the body's signal transduction and gene regulation, there is an increasing demand for their detection and application. However, due to the limited source of the sample and the possible sample size, plus the large number of nucleic acid species in each sample and the limited content of each nucleic acid and the relative content difference is too large, when the specific sequence of the nucleic acid that plays a role is not known, Samples from limited sources not only limit the number of experiments that can be performed but also may not be able to replicate important experiments of the past. Therefore, when the specific sequence of the nu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 周国春杨方义成浩万军庭
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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