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Double-stranded DNA binding fluorescent dye, preparation and applications thereof

A fluorescent dye and reaction technology, applied in the field of double-stranded DNA binding fluorescent dyes, can solve the problems of large sequence binding deviation, gene mutation, and small available concentration range, and achieves reduced cytotoxicity, strong amplification signal, and improved sensitivity. and stability effects

Active Publication Date: 2020-01-03
SHANGHAI BIOCHIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SYBRGreen I is widely used because of its high efficiency and low toxicity, but because it cannot be used in common PCR buffers, additional reagents such as DMSO and DBA need to be added; at the same time, although the PCR inhibitory effect can be reduced by increasing the concentration of MgCl2, but Concentration-dependent inhibitory effect persists, so SYBRGreen I is limited in multiplex PCR applications
It was found that when double-PCR amplification was performed, only one product was detected in the melting curve, but electrophoretic analysis clearly showed the presence of two products
Therefore, although SYBRGreen I has a wide range of applications, it still has shortcomings such as a small range of available concentrations and large sequence binding deviations.
[0005] In addition, a variety of existing nucleic acid dyes have high toxicity, mainly because the dye molecules can easily penetrate the cell membrane, thereby combining with the nucleic acid DNA in the cell, resulting in gene mutation and certain carcinogenicity

Method used

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  • Double-stranded DNA binding fluorescent dye, preparation and applications thereof
  • Double-stranded DNA binding fluorescent dye, preparation and applications thereof
  • Double-stranded DNA binding fluorescent dye, preparation and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of double-stranded DNA binding fluorescent dye

[0039] The preparation method of the double-stranded DNA binding fluorescent dye of the present embodiment is as follows:

[0040] 1) The raw material 2-methylbenzothiazole (0.30 g, 2.0 mmol, 1 eq) was slowly added dropwise to an ethanol solution of 1,2-diiodoethane (2.85 g, 10.1 mmol, 5 eq), and the temperature was controlled at 60°C. After the dropwise addition is completed in about 30 minutes, adjust the temperature to reflux temperature and continue for 90 minutes. After the reaction is complete, the solvent is removed and crystallized in acetone to obtain the target product intermediate I:

[0041] .

[0042] 2) Mix intermediate I (1.0 mmol, 1 eq), methanol solution of methylamine (0.48 mmol, 0.48 eq) and diisopropylethylamine (2.5 eq) respectively, add 10 mL of n-butanol as solvent, and react Using sealed tube, the heating temperature was 135°C, prepared and liquid-phase separated to obta...

Embodiment 2

[0049] Example 2 Real-time fluorescent quantitative PCR amplification

[0050] For the target gene GAPDH, using Promega Control DNA as a template and the compound of formula III prepared in Example 1 as a fluorescent dye, real-time fluorescent quantitative PCR amplification was performed using an ABI 7500 fluorescent quantitative PCR instrument. The primer sequences for fluorescent quantitative PCR amplification are as follows:

[0051] Upstream primer: CTGCCAGCCTAGCGTTGAC (SEQ ID NO: 1)

[0052] Downstream primer: CCAATCCTCCCGGTGACAT (SEQ ID NO: 2)

[0053] The 20 μL PCR reaction system is: 2.0 μL of 10×PCR buffer, 1.6 μL of 2.5 mM dNTP, 1.6 μL of 25 mM MgCl, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 0.2 μL of ROX II, 5 U / μL rTap enzyme (TAKARA) 0.125 μL, template DNA (Promega Control DNA) 1.0 μL (containing 80, 40, 20, 10, 5 ng DNA respectively), 1.0 μL fluorescent dye with the structure of formula III (final concentrations are 0.375 nM, 0.75 nM, ...

Embodiment 3

[0057] Embodiment 3 Dyeing effect comparison experiment

[0058] The double-stranded DNA-binding fluorescent dye (final concentration: 0.6 μM) prepared in the above example 1, EB ethidium bromide (final concentration: 5 μM) and 1× Gelred commercial nucleic acid dye were used for agarose gel electrophoresis respectively with DL2000 Marker detection, the detection results are as Figure 13-15 As shown, among them, Figure 13 is the EB dye, Figure 14 for Gelred dyes, Figure 15 For the double-stranded DNA-binding fluorescent dye prepared in Example 1, the loading amounts of the DL2000 Marker corresponding to bands 1-4 are: 200, 100, 50 and 25ng.

[0059] Depend on Figure 13-15 It can be seen that the double-stranded DNA binding fluorescent dye prepared in Example 1 of the present invention, compared with Gelred and EB dyes, can not only obtain dyeing effects equivalent to those of Gelred and EB dyes, but also according to the gel electrophoresis test results obtained under ...

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Abstract

The invention discloses a double-stranded DNA binding fluorescent dye, which comprises a structure represented by a formula i or a stereoisomer thereof. The invention further discloses a preparation method and uses of the dye. According to the invention, two fluorophores are connected through tertiary amine, and the fluorophores easily form pi-pi stacking dislocation, so that the fluorescence intensities of the two molecules before binding is reduced to obtain the strong amplification signal so as to improve the sensitivity and the stability of the double-stranded DNA binding fluorescent dye and reduce the cytotoxicity and the PCR inhibition of the double-stranded DNA binding fluorescent dye.

Description

technical field [0001] The invention relates to the field of nucleic acid dyes, in particular to a novel double-stranded DNA-binding fluorescent dye. Background technique [0002] The most representative of double-stranded DNA (dsDNA)-binding fluorescent dyes is the molecule ethidium bromide, which has long been used to stain nucleic acids in agarose gel electrophoresis. [0003] Double-stranded DNA-binding fluorescent dyes are also used to detect real-time amplification of nucleic acids, such as PCR. The corresponding amplification products can be identified by DNA-binding fluorescent dyes. After the dyes interact with double-stranded nucleic acids, they can emit corresponding fluorescent signals after being excited with an appropriate wavelength. During the PCR reaction, as long as there is double-stranded DNA in the sample to be tested, the dye can be selectively combined, and the fluorescent signal can be detected at the same time. When the double-stranded DNA dissocia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D277/64C09K11/06C09B23/14C12Q1/6876C12Q1/6851
CPCC07D277/64C09B23/146C09K11/06C09K2211/1007C09K2211/1037C12Q1/6851C12Q1/6876C12Q2563/107C12Q2561/113C12Q2531/113
Inventor 徐祎春崔雷韩峻松袁箐周佳菁苏军周欣怡
Owner SHANGHAI BIOCHIP