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Preparation method and application of oligonucleotide with closed dideoxyribonucleoside

A nucleotide and nucleic acid technology, applied in biochemical equipment and methods, microbiological measurement/testing, etc., can solve problems such as poor sensitivity and accuracy, time-consuming and cumbersome operations, and complicated operations

Inactive Publication Date: 2019-05-10
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of these traditional detection methods are complicated to operate, time-consuming and cumbersome, and have poor specificity, sensitivity and accuracy.

Method used

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  • Preparation method and application of oligonucleotide with closed dideoxyribonucleoside
  • Preparation method and application of oligonucleotide with closed dideoxyribonucleoside
  • Preparation method and application of oligonucleotide with closed dideoxyribonucleoside

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 utilizes terminal transferase to prepare ddNTP-blocked primers

[0116] The inventors selected and cloned a genomic DNA fragment of human acetaldehyde dehydrogenase (ALDH2) rs671, which contains a common point mutation in Asians and is often used as a target for hangover gene detection. The dominant homozygote in this region is the GG allele, the heterozygote is the GA allele, and the recessive homozygote is the AA allele. We named the dominant mutation sequence as ALDH-G, and the recessive gene mutation as ALDH-A. Two primers were designed according to the template, the upstream primer F2 and the downstream primer R1. The upstream primer F2 is completely identical to the sequence before the mutation site, and the downstream primer is completely complementary to the template. Use terminal transferase (TdT) to transfer ddGTP to the end of the F2 primer, and then use this blocking primer for normal PCR amplification. Under normal circumstances, TaqDNA polymer...

Embodiment 2

[0128] Embodiment 2 phosphatase can be inactivated by heat

[0129] In this implementation example, the same ALDH-A template plasmid as in Example 1 was selected, and an upstream primer F1 and a downstream primer R1 were designed according to the selected sequence.

[0130] The primer sequences are as follows:

[0131] Upstream primer F1: 5'-ttcaaattacagggtcaact-3' (SEQ ID NO.1);

[0132] Downstream primer R1: 5'-cgagccaccagcagaccctc-3' (SEQ ID NO.2)

[0133] Reaction system: ddATP (10mM) 2ul, ordinary phosphatase and thermosensitive phosphatase 20U each, 10*buffer 2ul, add water to 10ul.

[0134] Reaction conditions: first react at 37°C for one hour, then heat at 95°C for 10 minutes for the experimental group containing common phosphatase, and heat at 80°C for 10 minutes for the experimental group with thermosensitive phosphatase.

[0135] Carry out the reaction on the PCR instrument according to the above-mentioned reaction system and system, wherein ddATP (#12068) in the...

Embodiment 3

[0141] Example 3 Thermosensitive phosphatase exerts effect on ddATP

[0142] Using the same pair of primers and templates as in Example 2, the ddATP treated with phosphatase in Example 2 was used to perform a onestep RT-PCR fluorescence quantitative experiment to prove that the thermosensitive phosphatase acts on ddATP.

[0143] The reaction system is as follows:

[0144]

[0145] The reaction thermal cycling conditions are as follows:

[0146] Reaction conditions:

[0147]

[0148] According to the above reaction system and reaction conditions in ROCHE The reaction was performed on 96 real-time fluorescent quantitative PCR instrument, and then the amplified product was identified by agarose gel electrophoresis. The gel used was 2% agarose gel, and the molecular weight marker was DL 2000 DNA Marker (3427A) purchased from TaKaRa Company. PCR amplification curve and product electrophoresis results are as follows: image 3 As shown, the amplification curve of the exper...

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Abstract

The invention provides a preparation method and application of an oligonucleotide with a closed dideoxyribonucleoside. The method specifically comprises the steps that a terminal transferase (TdT) isfirstly used for catalyzing deoxyribonucleotides (dNTPs) or dideoxynucleotides (ddNTPs) in combination with the characteristics of a 3'hydroxyl terminal of a DNA molecule, so that a primer is mixed with any one of four dideoxynucleotides (ddATP, ddTTP, ddCTP or ddGTP), the TdT can add the dideoxynucleotides to the 3'hydroxyl terminal of the primer, and the obtained primer modified by the ddNTP cannot be catalytically extended by a DNA polymerase. The prepared oligonucleotide (primer) with the closed dideoxyribonucleoside can be used in combination with amplification methods such as allele PCR,proof-reading PCR for detecting mutation.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and more specifically relates to a preparation method and application of a dideoxynucleoside-blocked oligonucleotide. Background technique [0002] Point mutation, also known as single base substitution, refers to a mutation caused by a single base change, which can be divided into two types: transition and transversion. A transition refers to a substitution between a purine and a purine or a pyrimidine to a pyrimidine; a transversion refers to a substitution between a purine and a pyrimidine. Single-base mutations in DNA molecules widely exist in the encoding of genetic information in organisms, and are one of the important causes of genetic diseases. As far as the human genome is concerned, among the sense mutations of many genes that cause human diseases, pathogen subtypes, and drug resistance genes, single-base mutations account for a considerable proportion. The important to...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
Inventor 张弛宇张梦玲胡轶红
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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