الرحلان الكهربائي الهلامي الأفقي للحمض النووي البلازميدي: فصل الأشكال الملفوفة والمحفوفة والخطية

DNA electrophoresis is a widely used method in molecular biology. One of the applications for which the highest level of separation is required is the determination of the three major topological forms of plasmid DNA, which are supercoiled, nicked (open circular), and linear forms. We will show how the new HE-50 Horizontal Gel System offers plasmid isolation and quality control that allows for greater separation and reproducibility.

[Five Considerations for the Nucleic Acid Gel Electrophoresis Process | Thermo Fisher Scientific - US]

Plasmid Topology: Three Forms

Plasmid DNA is not uniform and is a mixture of three topological isoforms. In the context of isolation, these forms will yield differing rates of migration during DNA electrophoresis.

•Supercoiled (SC) Form: Supercoiled plasmids are considered the fastest migrating species in an agarose gel.

•Nicked (open circular/OC) Form: The circular arrangement of plasmid DNA which has a single strand break is now relaxed and therefore nicked. Due to the larger effective size, this isoform migrates slower than supercoiled DNA.

•Linear Form: Breakage of both covalent strands of the plasmid results in linear DNA. This form of plasmid DNA has a rod shaped structure and its migration is slower than supercoiled, but faster than nicked.

When analyzing the quality of plasmid DNA used for the manufacture of DNA plasmid vaccines, top regulatory authorities require that the preparation consist of at least 80% of the plasmid in the supercoiled form.

Importance of Plasmid Topology in Research and Biopharma

In plasmid DNA, the form of the isoforms affects a number of factors in related processes concerning vaccines:

•Transfection to Mammalian Cells: The supercoiled form of plasmid DNA is the most effective in transfections of mammalian cells compared to relaxed and linear forms.

•mRNA Vaccine Production: The need for linearized plasmid templates for in vitro transcription (IVT) incurs open circular plasmid contaminants, affecting yield and consistency.

•Gene Therapy Vector Manufacturing: High quality supercoiled plasmids are essential for adeno-associated vector (AAV) production.

•Stability Monitoring: The storage conditions and duration of storage affect plasmid stability as the frequency of nicked or linear forms increases.

Recent Research Advances in Plasmid DNA Analysis

Two of the recent studies emphasize the significance of the high-resolution separation of plasmid DNA:

1. ZorE-Mediated Plasmid Nicking (2025)

A study published in Nature Communications (March 2025) aimed at the bacterial defense systems and demonstrated control over the plasmid topologies. The research group under the Zorya Institute described a slow transformation of supercoiled pSG483 plasmid DNA to its nicked and linear forms by ZorE protein. This was noted to be a gradual process, and the transformation was quantitatively analyzed using ImageJ to perform a densitometric study. It was noted that the supercoiled plasmids were gradually nicked by ZorE (768 nM) upon 60 min incubation at 37 °C.

[ZorE-Mediated Plasmid Nicking (2025)]

*"The reaction was stopped by adding EDTA and SDS, and the products were separated using a gel electrophoresis in a 1× TAE buffer, 1.4% agarose gel, and were post stained with eb."* — Nature Communications, 2025

This discovery was found to be critical with respect to the DNA fragment and protein interaction studies conducted using gel systems.

2. Non-B DNA and DNA Repair Proteins (2025)

A PNAS publication from the NIH in January 2025, demonstrated that inserting dinucleotide repeats into pUC18 plasmids had a cis effect on the binding of mismatch repair proteins Mlh1-Pms1. EMSAs tested protein binding at concentrations ranging from 25 nM to 400 nM in both supercoiled and linearized DNA. Non-B form DNA disrupts protein repair functions and adds to the understanding of DNA instability.

[Non-B DNA and DNA Repair Proteins (2025)]

Important Considerations in the Electrophoresis of Plasmid DNA

In the electrophoresis of plasmid DNA, optimization of electrophoretic resolution is critical.

HE-50 Horizontal Gel System: The System of Choice

ال HE-50 Horizontal Gel System from Longlight Technology, employs a uniform design that is beneficial for the electrophoresis of plasmid DNA.

1. High-quality Separation

•99.95% Pure Platinum Electrodes: a critical design element for resolving supercoiled from nicked isoforms.

•Flexible Gel Format: Researchers can choose gel dimensions with any of the four available tray sizes: 60×60 mm, 60×120 mm, 120×60 mm, and 120×120 mm. The largest size offers a long enough migration distance to visualize closely migrating isoforms with higher resolution.

2. User-Oriented Design

•Guided Loading Tray with Black Background: The fluorescent ruler with a dark background enable careful alignment of the pipette tip to the wells when multiple samples are loaded for a direct comparison.

•High-Transparency Polycarbonate Tank: High visibility design with a built in tank shows migration of the dye front and the electrophoresis process without needing to open the tank and interrupt the system.

3. Safety and Efficiency

•Auto Shut-Off Lid: A safety switch cuts power when the lid is opened to eliminate the eletroshock hazard while users load samples or remove the gelatin.

•Low Buffer Consumption: The lower migration chamber incorporates a bridge design which uses less buffer while providing the sufficient ionic environment to perform consistent and reliable DNA electrophoresis.

Steps for Analyzing Plasmid Isoforms

To get the optimal separation of plasmid DNA using the HE-50 system:

• Make the Agarose Gel: Select any of the four tray sizes to cast a 0.8-1.0% agarose gel. Analyze plasmids sized between 3 - 10 kb with 1× TAE buffered agarose gels.

• Sample Preparation: Combine plasmid DNA samples with loading dye. Use 200 - 500 ng of plasmid DNA. The ratios of dye sample: loading dye can be 1:6, 1:3, or 1:1.5. Using a pipette, load the plasmid dye samples into the agarose wells.

• Electrophoresis: A gel with dimension 120 x 120 mm should be run at 100 V. For gels 100 x 100 mm, use 80V. Bromophenol blue is allowed to run for 45-60 minutes. Electrophoresis is stopped when the dye reaches 2/3 of the way to the bottom.

• Visualize and Document: The exposed, transparent cover allows for a gel to be UV'd and imaged while the cover is still in place. When a molecular ruler is present, positive fluorescence is observed.

• Quantify Isoform Distribution: Automated gel documentation systems quantify band intensity. The percentage of supercoiled DNA is determined by the following: [SC intensity] / (SC + OC + Linear intensities) × 100.

Unimpeded Workflow Integration

The HE-50 system is designed for unimpeded integration into existing laboratory workflows:

•Power Supply Integration: The requisite 4 mm banana plugs are compatible with most electrophoresis power supplies (we recommend constant voltage).

•Imaging System Integration: The UV-transparent lid is compatible with standard gel documentation imaging systems, eliminating the need to transfer gels from tanks to imagers.

•Protocol Adoption: The system is compatible with all standard agarose gel concentrations, and electrophoresis buffers (TAE, TBE, SB), enabling adoption of previously published plasmid analysis protocols.

الخاتمة

DNA electrophoresis is the most straightforward and popular method to evaluate the quality of plasmid DNA. Researchers can utilize their knowledge of the migration of supercoiled, nicked, and linear isoforms to optimize separation and interpret results. The HE-50 Horizontal Gel System provides the capabilities — pure platinum electrodes, selectable gel sizes, loading guides, and controls — essential for routine plasmid analysis. As stated in Nature Communications (2025) and NIH (2025), agarose gel electrophoresis is one of the fundamental methods to analyze the interactions between DNA and proteins as well as the topology or structure of DNA.

The HE-50 Horizontal Gel System has the potential to be fully integrated and modular to imaging systems; in this way, it provides customers more options for affordable plasmid DNA imaging systems. To learn more, please send a request for quotation to Longlight Technology.

الأسئلة الشائعة

Q1. Can horizontal gel electrophoresis identify supercoiled and nicked plasmid DNA?

The HE-50 System can identify supercoiled, nicked, and linear DNA plasmids as long as uniformity of the electric field is achieved, and agarose gels of 0.8-1.2% are utilized.

Q2. What are the different migration rates of the various plasmid forms during electrophoresis?

Because plasmid forms are structurally different, they exhibit varying migration rates when subjected to electrophoresis. For instance, linear plasmid forms migrate faster than circular forms. DNA in the linear form has a moderate migration rate, while DNA in the nicked or open circular form has the slowest migration rate.

Q3. What percent of supercoiled DNA is considered high quality for transfection?

A supercoiled form of the plasmid DNA that is greater than 80% is generally considered a high quality plasmid preparation for transfection and gene therapy.

Q4. Can I use TAE buffer instead of TBE for separating plasmid isoforms?

Yes, in most cases TAE buffer can be used for plasmid analysis. However, TBE buffer is optimal for separating plasmid isoforms when the differences are minimal and in extended separations.

Q5. Why do my plasmid samples show multiple bands on the gel?

Multiple bands are usually observed for supercoiled, nicked, and linear forms of the plasmid. Other bands may be a result of poor digestion or degraded DNA and RNA contamination.