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Method for amplifying nucleic acid sequence

A technology of nucleotide sequence and nucleic acid, applied in the field of synthetic DNA

Inactive Publication Date: 2006-08-02
TAKARA HOLDINGS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If the reaction is performed routinely, for example for the purpose of carrying out genetic experiments, the cost problem associated with the use of modified deoxyribonucleotide triphosphates becomes a serious problem

Method used

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  • Method for amplifying nucleic acid sequence
  • Method for amplifying nucleic acid sequence

Examples

Experimental program
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Effect test

Embodiment 1

[0258] (1) Synthetic template DNA and primers

[0259] A single-stranded DNA of 99 bases used as a template and primers used in this example were synthesized using a DNA synthesizer (Applied Biosystems). The nucleotide sequence of the 99-base single-stranded DNA is shown in SEQ ID NO: 1 in the Sequence Listing. The basic nucleotide sequences of the upstream primer and the downstream primer are respectively shown in SEQ ID NO: 2 and 3 of the sequence listing. The structure of the primers used in this embodiment is described in detail below:

[0260] Primer pair 1: a combination of primers having the nucleotide sequence shown in SEQ ID NO: 2 or 3 of the sequence table and all consisting of deoxyribonucleotides;

[0261] Primer pair 2: wherein in each primer of primer pair 1, the first and second deoxyribonucleotides at the 3'-terminus are replaced with ribonucleotides and the second ribonucleoside at the 3'-terminus A combination of primers in which the phosphate bond on the ...

Embodiment 2

[0279] (1) Preparation of RNA

[0280] RNA used as a template in this example was prepared from cultured human cells HT29 (ATCC HTB-38) (Dainippon Pharmaceutical) using TRIzol reagent (Life Technologies). The concentration of the obtained total RNA was adjusted to 1 µg / µl. The OD260 / OD280 value is 1.8, which indicates the spectrophotometric purity of the RNA.

[0281] (2) Amplification reaction

[0282] Bca BEST DNA polymerase with reverse transcription activity and DNA polymerase activity and RNase H endonuclease were used to determine whether cDNA was amplified from RNA.

[0283] A reaction mixture having the composition described in Example 2 was prepared by adding 1 μg of the above total RNA. The target region encoding human transferrin receptor (gene accession number X01060) was amplified using primer pair 2 of Example 1 as primers.

[0284] The reaction mixture was incubated at 55°C for 60 minutes, then heated at 90°C for 2 minutes to inactivate the enzyme. When 8 μ...

Embodiment 3

[0286] (1) Synthetic primers

[0287] The amplification method of the present invention was studied using double-stranded DNA as a template. The primers used were synthesized with a DNA synthesizer (Applied Biosystems). The basic nucleotide sequences of the primers are shown in SEQ ID NO: 5-13 of the Sequence Listing. The structures of the primers used in this example are described in detail below. pUC19 DNA (Takara Shuzo) was used as a template for primer pairs A-F. The nucleotide sequence of pUC19 can be obtained from a database (gene accession number L09137). The amplified double-stranded DNA fragment was used as a template for primer pair G. According to the attached standard method, primers and TaKaRa RNA PCR kit (AMV) Ver.2.1 (Takara Shuzo) with the sequence shown in SEQ ID NO: 14 or 15 of the sequence listing were used to prepare from the human total RNA obtained in Example 2 fragment.

[0288] Primer pair A (length of amplified fragment: about 450bp): having the ...

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Abstract

The present invention provides a convenient and effective method for amplifying a nucleic acid sequence characterized by effecting a DNA synthesis reaction in the presence of chimeric oligonucleotide primers; a method for supplying a large amount of DNA amplification fragments; an effective method for amplifying a nucleic acid sequence by combining the above method with another nucleic acid sequence amplification method; a method for detecting a nucleic acid sequence for detecting or quantitating a microorganism such as a virus, a bacterium, a fungus or a yeast; and a method for detecting a DNA amplification fragment obtained by the above method in situ.

Description

[0001] This application is a divisional application of an invention patent application with an application date of March 14, 2000, an application number of 00807534.4, and an invention title of "Method for Amplifying Nucleic Acid Sequence". technical field [0002] The present invention relates to a method for synthesizing DNA, which can be used in the field of genetic engineering. It relates to a method of amplifying a nucleotide sequence used as a template and a method of detecting nucleic acids amplified by said method. Background technique [0003] DNA synthesis is used for various purposes in research in the field of genetic engineering. With the exception of the synthesis of short strands of DNA such as oligonucleotides, most DNA synthesis is accomplished by enzymatic methods in which DNA polymerases are used. An example of an enzymatic method is the polymerase chain reaction (PCR) method, which is described in detail in US Pat. Nos. 4,683,195, 4,683,202, and 4,800,15...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
Inventor 向井博之山本纯子武田理三宅一惠上森隆司佐藤好美森山麻里子椹木治久萩屋道雄浅田起代蔵加藤郁之进
Owner TAKARA HOLDINGS
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