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Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same

A technology of oligonucleotides and dumbbells, applied in the field of dumbbell structure oligonucleotides, can solve the problems of unsure of the application technology and should not complement each other, and achieve easy detection and analysis of single nucleotide polymorphisms, increased sensitivity and effect of specificity

Active Publication Date: 2017-08-18
精确诊断有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there are limitations in the technique that a preselected random nucleotide sequence needs to be appended at the 5'-terminal site, and it should not be complementary to any position of the template
This leads to the added inconvenience of not being able to be confident that the technique can be successfully applied when the full genetic sequence of the template is not known

Method used

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  • Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same
  • Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same
  • Dumbbell-structure oligonucleotide, nucleic acid amplification primer comprising same, and nucleic acid amplification method using same

Examples

Experimental program
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Effect test

Embodiment 1

[0121]Example 1: Amplification of Specific Genes for Sexually Transmitted Diseases

[0122] DNA was extracted from samples obtained from 20 patients with suspected sexually transmitted diseases. For the obtained DNA, 2 μl of 10X polymerase chain reaction buffer solution (750 mM Tris-HCl (pH 9.0), 20 mM MgCl 2 , 500mM KCl, 200mM (NH 4 ) 2 SO 4 ), 2 μl of 2.5mM dNTP mixture (2.5mM dATP, 2.5mM dGTP, 2.5mM dTTP, 2.5mM dCTP), 2.0unit of Taq polymerase (Biotools, Spain) and 1 μl of the sequence numbers 1 to 26, 36 and 37 The DS primer (0.5 μM) of the base sequence was added to three distilled water, and after titrating to 20 μl, polymerase chain reaction (10 minutes at 94°C, 30 seconds at 94°C, 60 seconds at 65°C, 60 seconds at 72°C seconds, 35 cycles), the amplification product was obtained. At this time, the base sequences of the respective DSO primers used are shown in Table 1 below. For reference, in the nucleotide sequences recorded in the sequence number 1 to 37 on the se...

Embodiment 2

[0139] Embodiment 2: Utilize the single nucleotide polymorphism analysis of the MTHFR gene of DSO primer, BRAF gene and APC gene

[0140] In order to amplify commercially obtained human MTHFR, BRAF, and APC genes (wild type, hetero type, homo type), human genomic DNA (Invitrogen Inc., USA) was used as a template, and normal After the primers were amplified, the amplified product was confirmed by performing a result confirmation method using restriction enzyme treatment and polymerase chain reaction using the DSO primer of the present invention.

[0141] Table 2

[0142]

[0143] 2μl (50ng / μl) of genomic DNA collected from human blood, 2μl of 10X polymerase chain reaction buffer solution (750mM Tris-HCl (pH 9.0), 20mM MgCl 2 , 500mM KCl, 200mM (NH 4 ) 2 SO 4 ), 2 μl of 2.5mM dNTP mixture (2.5mM dATP, 2.5mM dGTP, 2.5mM dTTP, 2.5mM dCTP), 1.5unit of Taq polymerase (Biotools, Spain) and 1 μl of the mixture of each DSO primer (0.5 μM) Mix, add triple distilled water, titrat...

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Abstract

The present invention relates to a dumbbell-structure oligonucleotide (DSO), a nucleic acid amplification primer comprising the same, and a nucleic acid amplification method using the same and, more specifically, to a method for multiple gene amplification and single-nucleotide polymorphism analysis using a dumbbell-structure oligonucleotide capable of excluding non-specific amplification products prior to binding to a template in a first cycle in performing a polymerase chain reaction. The present invention suppresses undesired amplification products at room temperature using a dumbbell structure oligonucleotide (DSO) generated by adding any nucleotide sequence designed to allow the 5'-terminal oligonucleotide and the 3'-terminal oligonucleotide to complimentarily bind to each other, a 3'-terminal template dependent specific nucleotide sequence, and a universal nucleotide pair for linking the two nucleotide sequences, prior to binding to a template in every first cycle at the time of the polymerase chain reaction (PCR), and as a result, efficiently increases sensitivity and specificity through the reduction in non-specific amplification products, thereby achieving the innovation of the gene amplification method.

Description

technical field [0001] The present invention relates to a dumbbell structure oligonucleotide (dumbbell structure oligonucleotide, DSO), a nucleic acid amplification primer comprising the same and a nucleic acid amplification method using the primer, more specifically, to a method for performing polymerase chain reaction , a method for multiple gene amplification and single nucleotide polymorphism analysis using dumbbell structure oligonucleotides that can eliminate non-specific amplification products before combining with templates in each first cycle. Background technique [0002] Currently, the most common method used by researchers to obtain genetic samples is the polymerase chain reaction method using DNA polymerase. The oligonucleotides utilized in the polymerase chain reaction are designed to bind to opposite side strands of the template DNA. The method arbitrarily adjusts and designs the length and base sequence of the oligonucleotide that can be combined with the te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q2521/301C12Q2525/119C12Q2525/301C12Q2527/107C12Q1/6883C12Q1/6886C12Q1/689C12Q1/705
Inventor 林成植金渊洙
Owner 精确诊断有限公司
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