Method for dyeing short-chain oligonucleotide through fixation of methanol and anthocyanidin

An oligonucleotide and anthocyanin technology, which is applied in the field of short-chain oligonucleotide methanol-fixed anthocyanin staining method, can solve the problem that short-chain oligonucleotides cannot be effectively detected, oligonucleotides cannot be detected, and The problem of short oligonucleotide fragments, etc., achieves the effect of less sample amount, improved sensitivity, and fewer operation steps.

Inactive Publication Date: 2014-10-15
GUANGXI MEDICAL UNIVERSITY
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Problems solved by technology

However, except for the silver staining method, none of the above-mentioned staining methods can effectively detect short-chain oligonucleotides, because the oligonucleotide fragments are relatively short, especially when the length is only 11 bases or less, the oligonucleotides cannot be detected effectively. Nucleotides tend to diffuse out of the gel during staining, causing staining failure
However, the silver staining method has many operating steps, takes a long time, and cannot detect some oligonucleotides.

Method used

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  • Method for dyeing short-chain oligonucleotide through fixation of methanol and anthocyanidin
  • Method for dyeing short-chain oligonucleotide through fixation of methanol and anthocyanidin
  • Method for dyeing short-chain oligonucleotide through fixation of methanol and anthocyanidin

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Embodiment 1

[0029] Embodiment 1 dyeing effect experiment

[0030] Experimental reagent: oligonucleotide to be tested is oligo C 6 G 2 , oligo C 5 G 3, oligo C 4 G 4 , oligo C 3 G 5 , oligo CGCG 5 , oligo (CG 3 ) 2 , oligo (ACG) 3 、oligo T 9 、oligo A 7 C 4 , oligo(A 5 T), oligo (ACGT) 2 , oligo C 8 , oligo C 7 , oligo C 6 , oligo C 5 、oligo T 7 、oligo T 6 and oligo T 5 , synthesized by Shanghai Sangon, its sequence is shown in Table 1. Acrylamide, methylene bisacrylamide, urea, Tris, Na 2 EDTA·2H 2 O, boric acid, methanol, acetic acid, tetramethylethylenediamine (TEMED), ammonium persulfate, formamide and bromophenol blue were all of analytical grade and were purchased from Shanghai Sangong. SDNAClear Nucleic Acids Stain Dye and SYBR Green II RNA gel stain were purchased from Shanghai Sangon and Beijing Suolaibao Technology Co., Ltd., respectively.

[0031] Experiment 1: on oligo C 6 G 2 , oligo C 5 G 3 , oligo C 4 G 4 , oligo C 3 G 5 , oligo CGCG 5 , oli...

Embodiment 2

[0059] Example 2 Dyeing Sensitivity Experiment

[0060] The staining result of embodiment 1 shows that the present invention can detect the oligonucleotide of 5-11 base length, now with oligonucleotide oligo A 11 As a standard sample, the sensitivity of the method of the present invention is further detected: the experiment is divided into an experimental group and a control group. Experimental group oligo A 11 The loading amount of the sample was 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 μg; the control group oligo A 11 The sample loadings were 2 μg, 4 μg, 6 μg, 8 μg and 10 μg in sequence. The concentration of the denaturing polyacrylamide gel was 15%, the voltage was 200V, and the electrophoresis was performed for 1 hour at 25°C. After the electrophoresis, the experimental group first fixed the oligonucleotides with methanol fixative, and then stained with SDNAClear Nucleic Acids Stain Dye, while the control group did not fix with methanol, and directly ...

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Abstract

The invention discloses a method for dyeing short-chain oligonucleotide through fixation of methanol and anthocyanidin, and belongs to the field of molecular biology nucleic acid non-radioactive isotope dyeing methods. The method provided by the invention comprises the following steps: first, conducting polyacrylamide gel electrophoresis on the oligonucleotide; subsequently, fixing the oligonucleotide on the polyacrylamide gel through methanol stationary liquid, rinsing through deionized water, and dyeing through anthocyanidin and other nucleic acid dyes; finally, observing results through an ultraviolet scenograph or a gel imager. According the method provided by the invention, the radioactive isotope is not required, so that the method is simple, convenient and feasible, the dyeing effect is excellent, and the short-chain oligonucleotide with the length of 5-11 bases can be detected; meanwhile, the using amount of a sample is less, the analysis can be performed only by using 0.04-3 micrograms of the oligonucleotide, and the detection sensitivity is high, and compared with the dyeing method directly using the anthocyanidin, the dyeing method provided by the invention has the advantage that the detection sensitivity is improved more than 200 times.

Description

technical field [0001] The invention relates to the field of molecular biological nucleic acid non-radioactive isotope staining methods, specifically, after electrophoresis, methanol is used for fixation, and then one of two anthocyanin nucleic acid dyes, SDNAClear Nucleic Acids Stain Dye and SYBR Green II RNA gel stain, is used for staining , a method for detection of oligonucleotides 5-11 bases in length. Background technique [0002] Oligonucleotides mainly refer to short-chain nucleotides with less than 20 bases, including deoxyoligoribonucleic acid (DNA) and oligoribonucleic acid (RNA), which exist widely in nature and can also be artificially synthesized products. Synthetic oligonucleotides are mainly used in processes such as PCR, nucleic acid molecule hybridization, nucleic acid ligation reaction, and RNA interference. The quality of oligonucleotides can directly affect the success or failure of the above process, so the length and quality of oligonucleotides often ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N27/447
Inventor 李卫张小龙
Owner GUANGXI MEDICAL UNIVERSITY
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