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What is gel electrophoresis | Types and principles

What is gel electrophoresis | Types and principles

Gel Electrophoresis is a widely used analytical method in molecular biology and biotechnology.

Widely practiced for the separation of particles like viruses, small DNA fragments and nucleic acids and proteins. Also used after the PCR, to separate the DNA copies according to their quality.

Moreover, the electrophoresis method relies on the basic principle that the charged particles in a sample will migrate if an electric field applied to it.

Therefore, once electric field applied, the particle in the solution will migrate according to the charge or size.

Above all, widely used gels like polyacrylamide and agarose are commonly used .

This gel gives sieving or separating effect, and the particle separated by both size and charge.

Small molecules can travel faster than the larger one, also the negative charged particles will move towards anode. Similarly, positive charged particles will move towards cathode, in this way particle gets separated by size and charge.

Electrophoresis of cationic particle is called cataphoresis. Likewise, that of anionic particle called anaphoresis.

what is gel electrophoresis

Types of Electrophoresis

There are different types of electrophoresis but the most commonly and vividly used are mentioned below:

Capillary electrophoresis – molecules separated on the electrophoresis basis by using a capillary tube. To clarify, the molecules separated by running them through the capillary tubes. Accuracy higher but less quantity samples can be loaded at a time.

Native gels electrophoresis – protein run without SDS. Therefore, no proteins will get denatured in this method.

SDS – PAGE – Full form : Sodium Dodecyl Sulfate – Polyacrylamide gel electrophoresis. To clarify, SDS a detergent which can denature the protein and binds to the hydrophobic site of the molecule. Therefore, becoming the most dominant negative charge of the complex.

Here, the proteins are run through gel, polyacrylamide. Therefore, the particle density and size can be varied.

Electrofocusing gel – A gel with a pH gradient used here. As the particle travels through the gradient.

Meanwhile, it will loose protons and finally gets stucked at a point with no pH.

DNA Agarose gels – Here, agarose gels used to separate large fragments of DNA. The method used to separate according to the particle sizes. Here, no SDS needed because, DNA being negatively charged.

Above all, particle size sensitivity can be adjusted by adjusting gel density.

Sequencing gels – method used to separate smaller DNA fragments with a greater resolution. Firstly, the DNA denatured using heat and at the same time passed through polyacrylamide gel. Moreover, DNA which differ by 1 or 2 base can be separated more accurately.

The glowing DNA under the fluorescent light

Gel Electrophoresis applications

Applied for the estimation of DNA molecules.

Helps in mapping cloned DNA.

Analyzing the PCR products.

Gel electrophoresis widely used in forensics, molecular biology and genetic engineering.

Used in verification of amplified DNA using PCR.

Checking the quality of extracted DNA.

Gel electrophoresis a widely used technique, more modernized. Moreover, to determine the DNA fragment quality. In addition, used after the PCR to seperate and extract the DNA products.

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What is RT PCR? |Reverse Transcriptase PCR

What is RT PCR? |Reverse Transcriptase PCR
What is RT PCR

RT PCR or Reverse Transcriptase PCR is a test used to detect retroviral diseases like AIDS. It gained popularity during coronavirus outbreak.

Before knowing about RT PCR, please refer PCR or Polymerase chain reaction first here – https://sciencebirdie.com/2020/06/17/what-is-pcr-polymerase-chain-reaction-explained/

PCR or polymerase chain reaction is the technique in which the DNA is amplified and the billion DNA copies are made. But, it has exceptions like it can only detect only DNA viruses. Therefore, detection of RNA viruses like retroviruses cannot be possible.

What is RT PCR

This is where the RT PCR has the significance.

Working mechanism

Viruses like Coronavirus have RNA as the genetic material, therefore, the detection using the traditional PCR becomes impossible.

So, scientists convert RNA to DNA using an enzyme called Reverse transcriptase, the process known as Reverse Transcription. On the other hand, the conversion of DNA to RNA is called Transcription.

What is RT PCR
Thermocycler or PCR machine used in PCR technique

Steps in RT PCR

Firstly, the RNA converted to DNA using reverse transcription.

The DNA then transferred to the thermocycler machine or the PCR machine.

Afterwards, the steps denaturation, annealing and extension followed and the billion DNA copies extracted.

Please refer the PCR steps here – https://sciencebirdie.com/2020/06/17/what-is-pcr-polymerase-chain-reaction-explained/

Advantages

Coming to advantages, it had got more in addition than the traditional PCR.

Highly sensitive as the RNA involvement in it.

Highly specific, with more accuracy than PCR.

Requires less than 4 hours in diagnosis, moreover, the laboratories take up to 6-8 hours.

As the genetic material placed in a closed tube with enzymes, it has low potential contamination.

RT PCR in Corona test

Nowadays, with Coronavirus being prevalent, the RT PCR use got very much boosted up.

What is RT PCR

Firstly, the victim’s blood sample is taken then using several enzymes the RNA is extracted and converted to DNA and with PCR many copies are made.

Now, the DNA bases are made to pair with another bases tagged with markers. Further, the pairs are examined through fluorescence technique. If there are abnormalities, in the pairs the pairing does not occur. In other words, the person stated positive in the test.

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