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Agarose Gel Electrophoresis, Practice Problems and FAQs

Agarose Gel Electrophoresis, Practice Problems and FAQs

Do you know how a jigsaw puzzle works? It is the tiling puzzle that needs the construction of often irregularly shaped interlocking and mosaiced pieces, each of which often contains a section of a picture. When put together, the pieces form a whole picture. In order to solve a jigsaw puzzle, it is absolutely necessary that we find the correct matching or interlocking puzzle blocks.

Did you know that scientists also have to solve a similar puzzle while creating a recombinant DNA? 

You must be aware that for creating a recombinant DNA, the gene of interest is isolated and inserted into a vector. To achieve this, both the chromosome and the vector are cut using the same restriction enzyme so that they have complementary ends that can be joined to form a rDNA. But for this to happen, the gene of interest has to be identified out of the pool of DNA fragments obtained due to digestion of the chromosome carrying the gene of interest. This is much like finding the correct piece in a jigsaw puzzle but more complicated as the DNA fragments cannot be distinguished using our naked eyes. So what technique can be used for identifying and separating our desired gene of interest?

The principles of such a technique should be based on the properties of DNA. The technique of agarose gel electrophoresis comes into play here which helps us to find the correct DNA fragment carrying the GOI. In this article we will discuss the principle of agarose gel electrophoresis and how it is carried out to separate DNA fragments.

Table of contents

  • Gel electrophoresis
  • Agarose gel electrophoresis
  • Practice Problems
  • FAQs

Gel electrophoresis

The word ‘gel’ in the name gel electrophoresis suggests that the process is carried out in a gel medium, ‘electro’ suggests the involvement of electricity and ‘phoresis’ means being carried. So, gel electrophoresis involves charged molecules being carried in an electric field in a gel medium. The charged molecules referred to here can be nucleic acids, nucleotides, enzymes, proteins and amino acids. They are separated based on the difference in their sizes when they travel in the gel medium under the influence of an electric field. The smaller molecules travel faster through the pores of the gel and reach farther while the larger molecules travel slowly and are left behind. This can be understood better from the given illustration.



                  GIF: The smaller kitten passes through the holes faster than the bigger one

Gel electrophoresis can primarily be of two types - agarose gel electrophoresis and polyacrylamide gel electrophoresis. Agarose gel electrophoresis is used for the separation of medium and large sized DNA fragments ranging from 100 base pairs to 20 kilobase pairs. Polyacrylamide gel electrophoresis is primarily used for the separation of proteins. It can be used to obtain a higher resolution of DNA separation by separating fragments which differ only by a few base pairs.

However, agarose gel electrophoresis is favoured for the separation of DNA fragments because non-polymerised polyacrylamide DNA fragments can be toxic to the DNA and preparation of polyacrylamide gels is more complicated compared to agarose gel electrophoresis.

Agarose gel electrophoresis

Agarose is a naturally occurring polysaccharide that is obtained from the polymer agar that is extracted from red algae. It is composed of alternating units of β-D-galactose and 1,4-linked 3, 6-anhydrogalactose. As the name suggests, in agarose gel electrophoresis agarose is used as the gel medium. The gel is porous with same sized pores as seen in the image.



                                                 Fig: Pores in an agarose gel

It is the most commonly used technique for the separation and identification of the GOI from the many DNA fragments generated due to digestion by restriction enzymes. But before we get into the details of the process, let us ask ourselves a question, what makes DNA a charged molecule?

We know that DNA is a polymer made up of two helically wound strands composed of repeated units of nucleotides. Nucleotides are composed of a pentose sugar (deoxyribose), a phosphate and a nitrogenous base. A phosphodiester bond connects the phosphate group linked to the 5’ carbon of the pentose sugar of one nucleotide with the hydroxyl group linked to the 3’ carbon of the pentose sugar of the adjacent nucleotide.


 
                          Fig: Phosphodiester bond between nucleotides

Thus, the backbone of the DNA strands are made up of pentose sugar and phosphate moieties and the phosphate groups bear a negative charge which makes DNA a negatively charged molecule.



             Fig: Negatively charged DNA

An electric field is generated by connecting a positive and a negative electrode to the two ends of the gel and allowing electric current to pass through it. When placed under the influence of an electric field, DNA loaded onto the wells of the gel tends to move towards the positively charged electrode or anode. As they move across the gel, the DNA fragments with smaller size travel faster and reach farther while the ones with larger size will lag behind. This is the underlying principle behind separation of DNA fragments by agarose gel electrophoresis. 



                     Fig: Movement of DNA in agarose gel

Steps of agarose gel electrophoresis

We can divide the process of gel electrophoresis into five steps. After the completion of these steps we obtain the DNA fragment carrying the gene of interest (GOI).

Step 1: Preparation of the agarose gel

The first step is to make or set the agarose gel. For this we take agarose powder in a conical flask. In this solid agarose, we add a buffer to make a solution.



                 Fig: Mixing of agarose with buffer solution

Then, the agarose in the buffer solution is heated in a microwave to dissolve the agarose completely. It is then allowed to cool down.



                                   Fig: Melting of agarose in microwave

To this melted agarose, a chemical called ethidium bromide is added. It helps in visualisation of DNA. 



   Fig: Addition of of ethidium bromide to agarose

Once all of this is done, the agarose mixture is poured into a plastic casting tray. In a casting tray, a comb will be attached. This forms wells for our DNA sample to be loaded. The agarose mixture is then poured into the casting tray and it is allowed to cool and solidify. After cooling, the agarose polymers bind non-covalently to form a mesh/ network of bundles with pores which help in sieving the DNA fragments based on size. Once the gel is solidified, the comb is carefully removed from the gel, which leaves the wells or cavities in which the DNA can be loaded.



                                             Fig: Transferring agarose gel into casting tray



                             Fig: Agarose gel with combs

Step 2 : Setting the apparatus

When the whole apparatus is set up, the casting tray with the gel can be placed in a gel electrophoresis box. A running buffer is added to this box to help in conduction of electricity across the gel. The buffer helps to maintain the pH of the medium and allows DNA to migrate properly. It also protects the PO3- groups of DNA from the H+, produced due to dissociation of water, in the medium by binding to them. Buffers also contain chelating agents like EDTA (Ethylenediaminetetraacetic acid) which help to chelate the Mg2+ ions in the medium that are needed for the action of DNAse enzymes. Thus, it also protects the DNA from degradation.



                                             Fig: Addition of running buffer

Step 3: Loading a DNA sample 

The next step is to load the sample in the gel. DNA sample is taken in a centrifuge tube and mixed with a DNA loading dye as DNA is a colourless molecule. The dye is mainly composed of bromophenol blue. This dye helps to keep track of the DNA in the gel.



                                                              Fig: DNA loading dye

Now we can load the DNA and the loading dye mixture into the first well of the gel using a micropipette. 

1

The first well is loaded with a DNA ladder. DNA ladder is nothing but a scale, which is used as a reference to find the GOI based on size. It is a solution having DNA molecules of varying lengths that can be used as a reference to estimate the size of the separated DNA fragments. 



          Fig: Loading of DNA ladder in the well

In the second well we can load our PCR amplified DNA sample having the GOI.



          Fig: Filling the well with PCR sample

Step 4 : Run the DNA sample on the gel

The next step is to run the DNA sample on the gel under the influence of an electric field. The electrophoresis box is connected to electrodes and current is passed through it using a power source.



                                         Fig: Running the DNA in the gel



     GIF: Electricity passing through the gel

Step 5 : Visualise the DNA sample

The next step is to visualise the DNA. We had already added the chemical called ethidium bromide (EtBr) into the DNA sample. Ethidium bromide binds to DNA by inserting itself between the stacked bases in double-stranded DNA. 



                                 Fig: Insertion of ethidium bromide between the DNA

We can visualise the DNA bands under UV light as ethidium bromide fluoresces under UV light. When the UV transilluminator is turned on and the gel is placed under UV light, DNA can be seen as orange bands. 



                                     Fig: UV transilluminator

Now, though we can see DNA bands, we cannot identify the GOI just by looking at the bands. Do you remember we loaded a DNA ladder into the first well of the gel? So the GOI can be recognized by comparing the position of the sample DNA bands to the ladder. A thick band confirms the amplification of the DNA sample by PCR as it tells that the amount of DNA has increased. From this we can find our GOI by comparing the values with the DNA ladder.

For example, if our GoI is the human insulin gene which is around 1425 bp long, the unknown DNA fragment which lies just below the 1500 band in the DNA ladder will be our GOI.



                                                         Fig: DNA Ladder

Excision of GOI

Now we have the GOI with us, but we have to cut the GOI from the gel, then remove the selected gel slice.



                                 Fig: Excision of GOI

Finally, the gel slice is squeezed quickly and firmly into a sterile tube.



                                   Fig: Squeezing the GOI into tube

Elution of the GOI

The next step is elution of the gel. In this step a gel dissolving buffer is added to the tube containing our gel piece with the GOI. Then the tube is heated to dissolve or melt the agarose. 



                                                                 Fig: Elution of GOI

Then the DNA is precipitated by adding chilled alcohol and is separated out. Now the DNA we have is the GOI with some extra sequences flanking it. It can now be inserted into the cloning or expression vector and introduced into a host organism either for cloning purposes or for the purpose of expressing the gene product. 



                                               Fig: Addition of alcohol

Practice Problems

Q 1. In her practical exams, Asha was provided with a sample of scraped cells from the human buccal cavity. She, very carefully, extracted the genomic DNA, amplified the desired DNA and ran gel electrophoresis. However, she was utterly disappointed as she could not find the DNA bands herself. However, she scored full marks in her practical. What can be the possible reason as to which Asha was not able to see the bands that the teacher could find out?

a. She did not use ethidium bromide
b. She was observing the gel by exposing it to incandescent bulb
c. She placed the gel on the electrophoresis unit such that the wells were towards the positive terminal of the power supply 
d. She did not isolate the DNA properly

Answer: Ethidium bromide is the stain that acts as an intercalating agent in nucleic acids.The bands on the gel were observed by the teacher under UV light. Hence the experiment must have been conducted appropriately by Asha, justifying the full marks.Asha might not have observed the gel under ultraviolet radiation. She must have observed it by exposing it to an incandescent bulb. Incandescent light is characterised by a very low ultraviolet fraction. So, ethidium bromide molecules exhibited negligible fluorescence. Hence, the bands were not observed by her. Since the gel was perfect in all aspects, hence, option a, c and d might not have occurred.

Hence the correct option is b.

Q 2. Assuming that DNA fragment A contains more base pairs than fragment B, which of the following statements will not hold true when they are separated using agarose gel electrophoresis?

a. Both A and B will travel at the same speed across the gel. 
b. B will migrate faster compared to A towards the anode.
c. A will migrate faster compared to B towards the anode.
d. Both A and B will appear as distinct orange bands under UV light, corresponding to their molecular weights.

Answer: The speed at which DNA migrates across the agarose gel is determined by the size of the DNA molecule. The larger the size of a DNA fragment, the greater is the resistance that it experiences to move through the pores of the agarose gel. Thus a smaller DNA molecule with fewer base pairs and a lower molecular weight will travel faster and further compared to a larger molecule with a higher number of base pairs and higher molecular weight. As A has a greater number of base pairs than B, A will migrate with less speed compared to that of B which will reach the anode faster than A. DNA being negatively charged, travels from cathode towards the anode when current is passed through the gel.

The DNA fragments will form discrete bands based on their molecular weights as they have different molecular weights. They will appear as distinct orange bands under UV light due to intercalation of ethidium bromide between the stacked bases in double-stranded DNA. Ethidium bromide fluoresces under UV light and appears as an orange band.

Hence the correct option is c.

Q 3. Suppose while performing agarose gel electrophoresis, you observe that DNA samples remain static. However, he used the amount of the sample that was left to start another experiment after making some adjustment. This time he saw that the fragments started moving. The adjustment is most likely to be

1. Running another gel with bigger pores
2. Running another gel with smaller pores 
3. Slightly increasing the potential difference between the electrodes
4. Slightly decreasing the potential difference between the electrodes

a. I only
b. I or III
c. I or IV
d. IV only

Answer: The technique of gel electrophoresis is influenced by certain factors such as potential difference between the electrodes and the pore size of the gel. The probable reasons due to which the DNA fragments were not moving could be very small pores in the gel or very weak electric field which is not enough to drive the migration of the DNA molecule.

The small size of the gel pore size might be due to high concentration of agarose in the gel which might lead to excessive cross linking and hence smaller pores. Running another gel with optimum agarose concentration such that the pores become bigger can help the DNA fragments to pass through the gel.

Higher the potential difference, the higher is the strength of the electric field between the two electrodes. Thus, a greater force acts on the negatively charged DNA molecules to drive their movement from the negative electrode (cathode) to the positive electrode (anode). Hence, if increasing potential difference or voltage speeds up the movement of DNA molecules, provided all other experimental parameters are kept constant. 

Hence the correct option is c.

Q 4. What is the most likely outcome of a gel electrophoresis conducted with the DNA loading well placed towards the anode?

a. DNA fragments would be separated with respect to their size
b. DNA fragments would move towards the cathode
c. DNA fragments did not show usual migration towards anode
d. Both a and b can be possible

Answer: Agarose gel electrophoresis is a laboratory technique based on the principle of movement of charged particles (here DNA) in agarose matrix towards oppositely charged electrodes (anode) under the influence of electric field. Normally, the gel is placed in the electrophoresis chamber such that the wells are located towards the cathode. The negatively charged DNA molecules migrate from the cathode towards the anode depending on their size under the influence of the electric field. If the gel is placed in the chamber such that the wells are located towards the anode, the scenario would be completely different. The DNA molecules which are negatively charged, would not be able to migrate towards the cathode. This is because the cathode is also negatively charged. Hence, usual migration and separation of DNA fragments would not occur.

Hence the correct option is c.

FAQs

Q 1. Can agarose gel be used to separate proteins?
Answer: Recently agarose gel mixed with sodium dodecyl sulphate (SDS) are used to separate large proteins ranging from 200-4000 kDa.

Q 2. What is SDS PAGE?
Answer: SDS-PAGE or sodium dodecyl sulphate–polyacrylamide gel electrophoresis is invented by Ulrich K. Laemmli. It is a discontinuous electrophoretic technology and it is used to separate proteins with molecular masses ranging from 5 to 250 kDa. The use of sodium dodecyl sulphate (SDS, also known as sodium lauryl sulphate) in combination with a polyacrylamide gel eliminates the influence of structure and charge, allowing proteins to be sorted entirely on the basis of molecular weight differences.

Q 3. What are the different types of gel electrophoresis depending upon the number of dimensions of the proteins extracted?
Answer: There are three major types of electrophoresis. They are 1D, 2D and 3D gel electrophoresis. The key difference between 1D and 2D gel electrophoresis is the properties used for the separation of proteins on gel electrophoresis. 1D gel electrophoresis only separates proteins based on the molecular weight while 2D gel electrophoresis separates proteins based on its iso-electric point and molecular weight. Three-dimensional (3D)-gel electrophoresis is a new method for protein analysis in a separation medium that extends substantially in all three spatial dimensions. Proteins can be analysed according to one, two, or three independent separation parameters, i.e., native size, pI (isoelectric point), and molecular mass (MM).

Q 4. Who discovered gel electrophoresis?
Answer: Arne Tiselius, a Swedish biochemist, pioneered gel electrophoresis with his publication "A New Apparatus for Electrophoretic Analysis of Colloidal Mixtures" in 1937, and was awarded the Nobel Prize for his work in 1948.

YOUTUBE LINK: https://youtu.be/qSeuqV_lDwY

Related Topics

Principles of biotechnology 

Cloning vectors 

Process of recombinant DNA technology (Upstream processing) 

Process of recombinant DNA technology: (Downstream processing) 

Transformation 

Restriction enzymes 

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