What Is Capillary Electrophoresis?

Promega Corporation

1 Aug 201802:15

Summary

TLDRCapillary electrophoresis is a high-speed, high-throughput technique for separating DNA fragments using thin, polymer-filled capillaries. It allows for single-base resolution with minimal sample size. The process involves loading fluorescently labeled DNA into a capillary array, applying an electric field to move the fragments, and then detecting them as they pass a laser, with the fluorescence captured by a CCD camera. This method, ideal for STR analysis and Sanger sequencing, is showcased in the video by the Spectrum Compact CE system, emphasizing its efficiency and utility in life science and forensic labs.

Takeaways

🌟 Capillary electrophoresis is a technique for separating and identifying fluorescently labeled DNA fragments.
🧪 It uses thin polymer-filled capillaries instead of slab gels, allowing for stronger electric fields and faster separations.
🔋 The process offers high throughput and single base resolution, requiring only small sample quantities.
🔬 Applications include STR analysis, Sanger sequencing, and various other uses in life science and forensic labs.
🚀 The method begins with preparing and loading fluorescently labeled DNA samples into a capillary array.
🌐 An electric current is formed when the capillary array enters the sample wells, moving DNA fragments electrokinetically.
🔌 The ends of the capillary array are inserted into cathode and anode buffers, with a specific voltage and temperature applied for controlled migration.
📊 Fragments are separated by size, with the smallest moving the fastest, allowing for size-based analysis.
🔦 Detection occurs as fragments pass a detection window, where a laser excites the fluorescent dyes, and the emitted light is recorded.
📷 A CCD camera captures the fluorescence signature and migration time of each fragment, providing raw data for analysis.
💻 The data is sent to collection software as peaks, ready for analysis with various software programs.
🛠️ The animation is presented by Spectrum Compact, promoting a compact and powerful CE instrument for laboratory use.

Q & A

What is capillary electrophoresis?
-Capillary electrophoresis is a method used for separating and identifying fragments of fluorescently labeled DNA by passing them through very thin polymer-filled capillaries.
How does the use of capillaries in capillary electrophoresis differ from conventional slab gels?
-Capillaries allow for the application of a much stronger electric field, resulting in faster separation and higher throughput compared to conventional slab gels.
What are the key features of the capillary electrophoresis process?
-The process features single-based resolution and requires small sample quantities.
What are some applications of capillary electrophoresis in laboratories?
-Applications include Short Tandem Repeat (STR) analysis, Sanger sequencing, and many other everyday applications used by life science and forensic laboratories.
How are fluorescently labeled DNA samples prepared for capillary electrophoresis?
-The samples are prepared and loaded into the capillary electrophoresis instrument, where a capillary array is filled and placed into the sample wells.
What happens when the capillary array enters the sample wells?
-An electric current is formed, and the DNA fragments are moved electrokinetically into the capillaries.
What is the role of the cathode and anode buffer in the process?
-The ends of the capillary array are inserted into the cathode and anode buffer, which helps in controlling the sample migration through the capillary array.
How does the instrument control the sample migration?
-The instrument applies a specific voltage and temperature to control the sample migration through the capillary array.
What is the final step in the capillary electrophoresis process?
-The final step is sample detection, where fluorescently labeled fragments are detected as they move past the detection window.
How are the fluorescent signatures of each fragment recorded?
-As the fragments move past the detection window, the laser excites the fluorescent dyes, and the emitted light is spectrally separated and recorded by a CCD camera.
What happens to the raw data after it is recorded by the CCD camera?
-The raw data is sent to the collection software in the form of a peak and is then ready for analysis using a chosen software program.
Who brought this animation about capillary electrophoresis?
-The animation is brought to you by Spectrum Compact, a company that provides a small but mighty capillary electrophoresis instrument for laboratories.

Outlines

00:00

🧬 Capillary Electrophoresis: DNA Fragment Separation

Capillary electrophoresis is a technique used for the separation and identification of fluorescently labeled DNA fragments. This method employs thin polymer-filled capillaries instead of traditional slab gels, allowing for a stronger electric field, which in turn accelerates the separation process and increases the rate of analysis. The process is characterized by single-base resolution and requires minimal sample volumes. It is widely used in STR analysis, Sanger sequencing, and various other applications in life science and forensic laboratories. The script describes the preparation of DNA samples, their loading into the instrument, the application of an electric current to move the DNA fragments into the capillaries, and the subsequent separation based on size. The smallest fragments travel the fastest. The final step involves the detection of the fragments as they pass a detection window, where a laser excites the fluorescent dyes, and the emitted light is recorded by a CCD camera, capturing the fluorescence signature and migration time of each fragment. The data is then sent to collection software for analysis, as demonstrated by the Spectrum Compact CE system.

Mindmap

Keywords

💡Capillary Electrophoresis

Capillary electrophoresis is a technique used to separate and identify different molecules, such as DNA fragments, based on their size. It is central to the video's theme as it is the main method discussed. In the script, it is described as being performed using a thin polymer-filled capillary, which allows for a stronger electric field and faster separation compared to traditional slab gels.

💡Fluorescently Labeled DNA

Fluorescently labeled DNA refers to DNA molecules that have been marked with fluorescent dyes, enabling their detection and analysis under specific light conditions. This concept is crucial in the video as it allows for the tracking of DNA fragments during capillary electrophoresis. The script mentions that DNA samples are prepared with fluorescent labels before being loaded into the instrument for separation.

💡Capillaries

Capillaries in this context are the thin tubes used in capillary electrophoresis to carry the DNA samples. They are filled with a polymer and are key to the process described in the video. The script explains that using capillaries instead of a conventional slab gel allows for a stronger electric field, leading to faster separation and higher throughput.

💡Electric Field

An electric field is a region around a charged particle where an electric force is exerted on other charged particles. In the video, the electric field is used to drive the movement of DNA fragments through the capillaries during electrophoresis. The script emphasizes that a much stronger electric field can be applied in capillary electrophoresis compared to other methods.

💡Separation

Separation in the script refers to the process of distinguishing and moving apart DNA fragments of different sizes within the capillary. It is a fundamental aspect of capillary electrophoresis and is directly related to the video's theme. The smallest fragments are described as moving the fastest during this process.

💡Sample Quantities

Sample quantities refer to the amount of DNA needed for the analysis. The script mentions that capillary electrophoresis requires small sample quantities, making it an efficient method for DNA analysis, especially in fields like forensics where sample sizes might be limited.

💡STR Analysis

STR analysis, or Short Tandem Repeat analysis, is a type of DNA profiling technique used in forensic science. It is mentioned in the script as one of the applications of capillary electrophoresis, highlighting its utility in identifying individuals based on DNA patterns.

💡Sanger Sequencing

Sanger sequencing is a method of DNA sequencing that was first developed by Frederick Sanger. It is cited in the script as another application where capillary electrophoresis is used, particularly for determining the order of nucleotides within a DNA molecule.

💡Laser

A laser is a device that emits light through a process of optical amplification. In the context of the video, a laser is used to excite the fluorescent dyes on the DNA fragments as they pass the detection window during capillary electrophoresis. This process is essential for detecting and recording the fragments.

💡CCD Camera

A CCD camera, or Charge-Coupled Device camera, is a type of image sensor used in photography and video recording. In the script, it is mentioned as the device that records the fluorescence signature of each DNA fragment, capturing the data necessary for analysis.

💡Collection Software

Collection software is the program used to collect and analyze data from experiments, such as the data generated by capillary electrophoresis. The script describes how raw data from the CCD camera is sent to the collection software in the form of peaks, ready for further analysis.

Highlights

Capillary electrophoresis is a method for separating and identifying fluorescently labeled DNA fragments.

It uses thin polymer-filled capillaries instead of conventional slab gels.

Allows for a stronger electric field, resulting in faster separation and higher throughput.

The process offers single-based resolution and requires small sample quantities.

Applications include STR analysis, Sanger sequencing, and other everyday lab uses.

Fluorescently labeled DNA samples are prepared and loaded into the instrument.

A capillary array is filled and placed into sample wells to form an electric current.

DNA fragments are moved electrokinetically into the capillaries.

The capillary array ends are inserted into cathode and anode buffers.

Instrument applies specific voltage and temperature to control sample migration.

Fragments are separated by size, with the smallest moving the fastest.

Sample detection involves a laser exciting fluorescent dyes and a CCD camera recording the fluorescence.

The CCD camera records the fluorescence signature and migration time of each fragment.

Raw data is sent to collection software as peaks ready for analysis.

The animation is brought to you by Spectrum Compact, a compact CE system for labs.

Spectrum Compact is described as a small but mighty CE instrument.