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Method for synthesis of double-stranded DNA corresponding to RNA, and method for amplification of the DNA

A synthesis method and corresponding technology are applied in the field of double-stranded DNA synthesis, which can solve the problems of high-priced instruments, inability to analyze, and inability to obtain cDNA, and achieve the effects of simple operation and low price.

Inactive Publication Date: 2012-04-25
WAKO PURE CHEMICAL INDUSTRIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, regardless of the method, if the RNA does not have a polyA and Cap structure, there are problems that the full-length cDNA cannot be obtained, and the RNA fragment that does not contain the Cap structure cannot be analyzed.
On the other hand, as a method capable of analyzing RNA fragments, there is a well-known microarray method, but this method is not easy to use in the experimental stage because it requires expensive equipment.

Method used

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  • Method for synthesis of double-stranded DNA corresponding to RNA, and method for amplification of the DNA
  • Method for synthesis of double-stranded DNA corresponding to RNA, and method for amplification of the DNA
  • Method for synthesis of double-stranded DNA corresponding to RNA, and method for amplification of the DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Cloning of 3 mRNAs (human albumin, β-actin, GAPDH)

[0054] 1. Reverse transcription reaction

[0055] will respectively contain 1×10 9 Copied human albumin (2122 bases), β-actin (1874 bases), and GAPDH (1380 bases) mRNA (manufactured by NIPPON GENE CO., LTD.) aqueous solution 11 μL , as template RNA in water. To this aqueous solution was added 1 µL of a 20 µmol / L primer solution for reverse transcription (5'-GACCATATGACGAGATCCGAGCTTCTTTTTTTTTTTTTTTTTTTTTT-3'). Next, it was heated at 70° C. for 3 minutes to thermally denature it, and then left to stand in an ice bath for 2 minutes. Then, add: 2 μL reverse transcription reaction buffer (containing 750 mmol / L potassium chloride, 30 mmol / L magnesium chloride, 500 mmol / L Tris hydrochloric acid buffer solution (pH 8.3) of 50 mmol / L dithiothreitol), 4 μL dNTPs ( Respectively 2.5mmol / L of dATP, dGTP, dCTP, dTTP solution mixed solution, produced by Nippon Gene Company), 1 μL ribonuclease inhibitor (20 units / μL, ...

Embodiment 2

[0164] Example 2 Different amplification effects brought about by different reaction temperatures during the synthesis of the second strand.

[0165] 1. Acquisition of Ago2-bound RNA from HeLa cells

[0166] (1) Preparation of human anti-Ago2 antibody immobilized carrier

[0167] 1 ml of PBS (pH 7.4) was added to 20 μL of magnetic carrier Dynabeads Protein G (manufactured by InvitroGen), and after mixing, the carrier was extracted using a magnetic stand. Then, a mixture of 5 μL of 1 mg / mL human anti-Ago2 antibody (manufactured by Wako Pure Chemical Industries, Ltd.) and 95 μL of phosphate-buffered saline (PBS pH 7.4) was added to the carrier, and shaken at room temperature for 1 hour. Next, the carrier was extracted using a magnetic stand, washed three times with 1ml PBS (pH 7.4), and finally suspended in 1ml PBS (pH 7.4), and the resulting solution was the human anti-Ago2 antibody immobilized carrier solution.

[0168] (2) Preparation of HeLa cell extract

[0169] to 1×1...

Embodiment 3

[0190] Example 3 is corresponding to the RNA (derived from the Alu RNA of HeLa cells) that does not have the Cap structure DNA synthesis

[0191] 1. Conversion

[0192] To 2 μL of the PCR reaction solution prepared in 5. of Example 2, 1 μL of 20 ng / μL of pGEMTeasy Vector (manufactured by Promega) and 3 μL of DNA Ligation Kit Mighty Mix (manufactured by TAKARA BIO Co., Ltd.) were added, and reacted at 16°C For 30 minutes, insert the cloned cDNA into the vector. Then, using ECOS TM Competent E.coli DH5α (manufactured by Nippon Genetics Co., Ltd.) was transformed into competent cells using the heat shock method at 42°C for 45 seconds. Next, culture was carried out at 37°C for 16 hours in a Luria-Bertani's broth (LB) agar medium containing 100 µg / mL ampicillin sodium.

[0193] 2. Colony PCR

[0194] Add a part of each of the 96 single bacterial colonies on the culture medium to the reaction solution [full volume 10 μL: 1 μL 10× Universal Buffer (manufactured by Japan Gene ...

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Abstract

An object of the present invention is to provide an inexpensive and simple method for synthesis of a double-stranded DNA corresponding to a particular RNA, and a method for amplification of the aforementioned double-stranded DNA. The present invention relates to a method for synthesis of a double-stranded DNA having a nucleotide sequence corresponding to template RNA having polyA, comprising step 1 in which reverse transcription reaction of template RNA is carried out employing oligo(dT)primer to which DNA fragment having a known sequence has been added at the 5'-terminal, to obtain a single-stranded DNA, and step 2 in which double strand formation reaction of single-stranded DNA obtained in step 1 is carried out employing a random primer to which DNA fragment having a known sequence has been added at the 5'-terminal, in the presence of polymerase which does not have 3'->5' exonuclease activity nor strand displacement activity, to obtain a double-stranded DNA, as well as a method for amplification of a double-stranded DNA having a nucleotide sequence corresponding to template RNA having polyA, further comprising step 3 in which PCR reaction is carried out using the obtained double-stranded DNA.

Description

technical field [0001] The present invention relates to a method for synthesizing double-stranded DNA corresponding to specific RNA and a method for amplifying the DNA. Background technique [0002] At present, in order to obtain full-length cDNA in RNA gene analysis, researched methods include SMART method, Oligo-Capping method, Cap Trapper method, etc. However, regardless of the method, if the RNA does not have a polyA and Cap structure, there are problems such as that it is impossible to obtain a full-length cDNA, and it is impossible to analyze an RNA fragment that does not contain a Cap structure. On the other hand, the microarray method is well known as a method capable of analyzing RNA fragments. However, since this method requires expensive equipment, it is not easy to use in the experimental stage. [0003] Therefore, it is desired to develop a new method for gene analysis that can perform gene analysis even on RNA fragments that do not have a Cap structure, is eas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09
CPCC12N15/1096
Inventor 林田幸信
Owner WAKO PURE CHEMICAL INDUSTRIES
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