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A biotin-labeled primer extension method and its application

A biotin labeling and product technology, applied in the field of biochemistry, can solve the problem of low sensitivity of isotope labeling, achieve the effects of increasing storage difficulty, solving safety problems, and shortening the experimental cycle

Active Publication Date: 2021-07-20
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biotin is basically not affected by decay, so biotin labeling is more stable than traditional isotope labeling, but its relative isotope labeling sensitivity is low, so isotopes are still used as labels in high-precision RNA modification detection

Method used

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  • A biotin-labeled primer extension method and its application
  • A biotin-labeled primer extension method and its application
  • A biotin-labeled primer extension method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 (Saccharomyces cerevisiae RNA m 3 U modification detection)

[0087] Implementation principle:

[0088] The primer extension method utilizes m 3 m 3 U modification for localization detection. m 3 The U modification occurs on the No. 3 N atom of the ribonucleotide guanosine base. This modification replaces the key H atom connected to the N atom for base pairing to form a hydrogen bond with a methyl group (-CH 3 ), hindering the base-pairing process of the guanosine. Taking advantage of this feature, the target RNA sequence is reverse-transcribed using biotin-labeled primers, m3U modification affects the base pairing of its own sites, and part of the cDNA synthesis will be stagnant at m3U 3 The first nucleotide site at the U 3' end forms a stalled cDNA product. Finally, the stalled cDNA product is displayed by gel electrophoresis, membrane transfer, and development. The single-stranded cDNA product prepared by the sanger sequencing method is used as a mark...

Embodiment 2

[0105] Example 2 (Drosophila RNA 2'-O methylation modification detection)

[0106] Implementation principle:

[0107] During primer extension, the 2'-O-methylation modification on RNA has a blocking effect on reverse transcriptase, which is mainly affected by the concentration of dNTP. Under the condition of high dNTP concentration, 2'-O-methylation has a weaker inhibitory effect on reverse transcriptase, while under the condition of low dNTP concentration, 2'-O-methylation has a weaker effect on reverse transcriptase inhibition. Large, partially stalled cDNA products are formed during reverse transcription, and these stalled products can be displayed by biotin labeling and gel electrophoresis to determine the site of 2'-O-methylation modification.

[0108] Implementation steps:

[0109]1. Select 20 adult fruit flies (Drosophila melanogaster), freeze them in liquid nitrogen, grind them into pieces, and use the TRIZOL method to extract the total RNA of the flies.

[0110] 2....

Embodiment 3

[0119] Example 3: Comparison of development accuracy between biotin primer extension method and traditional isotope method

[0120] One, according to the method of embodiment 1, use biotin-labeled primer (5'-GGTAAAACTAACCTGTCTCA-3') to carry out 28S RNA on the Drosophila total RNA sample 3 U modification site detection, the result is as follows image 3 shown.

[0121] 2. Use P32 isotope-labeled primer (5'-GGTAAAACTAACCTGTCTCA-3') to perform 28S RNA on Drosophila total RNA samples 3 U modification site detection. Specific steps are as follows:

[0122] (1) Reagent formula related to isotope-labeled primer extension method (25 μL system)

[0123] A. Reaction solution A

[0124] Primer (1pmol / μL) 2μL

[0125] Total RNA (2000ng / μL) 4μL

[0126] DEPC treated water 4μL

[0127] B. Reaction solution B (using Promega AMV reverse transcriptase, catalog number: M5101)

[0128]

[0129] (2) Implementation steps of the isotope-labeled primer extension method (all operations a...

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Abstract

The invention discloses a biotin-labeled primer extension method and its application. It is to anneal the oligonucleotide primer with the label to the template RNA chain, and the free dNTPs are extended under the action of reverse transcriptase to synthesize the complementary cDNA chain, which is separated and displayed by denaturing gel electrophoresis and label tracking. The transcription product is characterized in that the oligonucleotide primer with a marker is an oligonucleotide primer with a biotin label. The invention optimizes the experimental conditions, uses stable and harmless biotin labels instead of isotope labels, and provides a non-isotope-labeled primer extension method, which solves the safety problem of the isotope method, increases the detection accuracy and shortens the time. Experimental cycle.

Description

Technical field: [0001] The invention belongs to the technical field of biochemistry and relates to a biotin-labeled primer extension method. Background technique: [0002] Primer Extension is an important method to study various properties of RNA. This method is essentially a special reverse transcription method. An oligonucleotide primer is annealed to the template RNA strand. At this time, the free dNTPs are extended under the action of reverse transcriptase to synthesize a cDNA complementary strand, which is passed through a denaturing gel. Electrophoresis and marker tracking techniques separate and display reverse transcription products to achieve the purpose of studying template RNA. [0003] Traditional primer extension methods are mainly used to analyze RNA structure and expression levels. By adjusting the experimental conditions, the primer extension method can meet different research purposes, including but not limited to: according to the principle of nucleic ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686
Inventor 陈洁黄元泰容益康
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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