[Music] this kind of an image is not new to us especially for people who love forensic science but what exactly is this it's an image of the separated DNA fragments on a piece of gel and the process of separating them is called gel electrophoresis doesn't the sound very difficult separating DNA fragments on a piece of gel but actually the process of gel electrophoresis is not very difficult in fact it's quite easy let's have a look at how the process works and also the principle behind it let's begin with the definition first gel electrophoresis is a technique to separate the different DNA molecules based on their sizes can you guess why is it called gel electrophoresis okay let me help you with this this separation technique is based on the movement of charged molecules when exposed to an electrical field and this movement occurs in a gel medium hence it's known as gel electrophoresis now let's understand the process step by step but before that since the term charge is used by us here can you guess the charge present on a DNA molecule let me tell you that a DNA is negatively charged and this is due to the presence of phosphate groups these phosphate being negatively charged and present in the backbone of the DNA double helix impart a negative charge to the complete molecule and what is the gel used interestingly the gel which is generally used for the DNA is agarose gel and it's obtained from the seaweeds now getting back to a process we said that the DNA molecules get separated on the basis of their sizes so if we have a sample of DNA which is containing fragments formed with the activity of restriction enzymes then separating the fragments based on the difference in their sizes would be possible with gel electrophoresis let's have a look at this in detail do you understand the principle and working let's take the analogy of a sieve we note that if a mixture of different sized particles is poured onto a sieve then only the particles smaller in size than the pores will escape while those bigger in size will be retained back here the size of the particles is the only parameter used for the separation same is the case with DNA fragments when our DNA sample containing various sized fragments is loaded onto a gel then the charge applied across the gel helps in the mobility of these fragments however not all fragments move at the same pace those which are smaller move ahead and those which are larger in size find it difficult to move to understand this better let's zoom into the gel that is prepared for the experiment here at the microscopic level we find that the gel appears to be a mesh-like structure and the pore size is nearly constant throughout so now imagine what will happen if different sized DNA fragments are made to pass through these pores it's obvious that the small sized fragments will escape the pores faster while the larger fragments will find it difficult to come out they will take a lot of time to cross the smaller pores and dust will lag behind this is how we can understand the principle of separation of the DNA fragments based on their sizes now let's see how the procedure works the first requirement is the complete set up this will include the casting tray gel a comb for making wells in the gel electric supply and most importantly DNA sample with different sized DNA fragments here's an illustration of the casting tray containing the gel in which we will load the DNA mixture we usually opt for a comb to form well like structures into the gel and why do we do that because it's in these worlds in the agarose gel that we load the DNA mixture now tell me where should the wells be formed at the ends or at the centre think about it we know that the DNA fragments being negatively charged will travel from a point of negative charge to the point of positive charge does we need to form the wells to load the DNA samples near the negative terminal or the cathode to be precise so that the DNA will move towards the positively charged anode this movement of the DNA molecule will be promoted by the electric field but we cannot track the movement of the DNA in the gel as it's colorless for this we use a colored loading dye to track the movement of the DNA the mixture of the DNA samples with the colored loading dye is now all set to get into the gel but before loading the DNA mixture we will fill the apparatus with a buffer as shown the buffer is used to provide better conductivity of electricity next we will load the DNA mixture into the wells and we're ready to turn on the power supply once the power supply is turned on the DNA mixture will move through the gel which can be tracked with the help of the loading dye the loading dye is selected in such a way that it travels a bit faster than the DNA segments present in the mixture and why is that so that's because we want the loading dye to reach the terminal end faster than the DNA once the dye reaches the anode we get an indication that the DNA must have reached somewhere near and does the power supply has to be turned off moving ahead with the next step now we know that the DNA molecules have got separated but can we really see them no that's not possible so how will that be possible now how do we observe the separated segments to observe the DNA molecules we treat the agarose gel with helium bromide solution the major reason for using ethidium bromide is that it easily binds to the DNA molecules and when the agarose gel containing the DNA is observed under ultraviolet light bright orange colored bands are clearly seen these are nothing but the bands of the DNA the separation has been successful based on the size of the DNA fragments the larger molecule are the ones found here which means these are the ones that moved slowly on the other hand the smaller molecules which moved faster are the ones spotted here now how do we know the size of any DNA fragment the bands obtained are compared with a standard chart known as the DNA ladders on comparing the positions of the bands with the ones from the chart we can easily make out the length of the fragments obtained once we know the length of the fragments we can easily identify the desired DNA now the desired DNA fragment can be first manually cut out from the agarose gel and then extracted this process is called illusion the extraction of the DNA is done in such a way that it can be used for further downstream processing this was the simple explanation of how any gel electrophoresis technique works it's considered as one of the very important techniques used in recombinant DNA technology to learn more about such interesting processes stay tuned to our Channel and do not forget to subscribe happy learning you