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Module 1: Ferramentas de DNA e Biotecnologia

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Lab Demonstration: Polymerase Chain Reaction

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So, welcome to this lecture, we are going to continue on DNA tools and biotechnology,in the previous lectures we have been discussing about the hereditary unit or DNA, you havealso learnt the theoretical concepts of tools which could help to visualize and amplifyDNA by using agarose gel electrophoresis and polymerase chain reaction or the PCR processes.Today, we are going to show you in the laboratory how these techniques could be performed.So, let us now start the laboratory demonstration for explaining you these techniques and showingyou how these experiments could be actually performed in the laboratory.Hello everyone, I am Srishty, the TA of this course, so now we will be doing agarose gelelectrophoresis to check whether our procedure product has come or not and so for that wewill be making our gel.First of all, we will be making 2% gel, we have weigh, 2 grams of agarose powder andwe will be adding it to TA buffer.So, now we will add 2 gram of agarose to a flask and we will also 100ml of TA buffer,so now they will be dissolving the agarose powder using microwaves.So for 2 minutes, it will be putting it inside and in between, we will be stirring it, sonow a gel is done and we will be pouring it on a gel casting unit.Now as our gel has cool, we will be adding EtBr here in the ratio of 0.5 microgram perml.Why we are adding EtBr, we are adding EtBr, so that the dye will intercalate with ourDNA sample and given fluorescence when observed on the transilluminator, always take carethat you discard the tubes of EtBr in biohazard and as it is a potent mutagen.Now, we will pour our gel, so as you can see these are the types of casting units we canhave, this is the bigger one in which I have already casted a gel, it is a smaller onewhich we casted right now, when we placing a comb, this is the comb of its size in thisbefore the gel solidifies, these are the different types of combs and casting units available,so this is the electrophoretic unit in which we are replacing our gel after it solidifiesfor the run.And this is the voltage jointer.Today, I will be taking you to the PCR process and we will try to learn how the process occursin real time so basically, what is PCR?PCR is as its name suggests it is polymerase chain reaction.In this matter, in this technique what we see is; we have a very small quantity of DNAor any sample then, in this process the sample gets amplified to a million times, so thatsometimes when you are working in a clinical field research or there is a crime scene whereyou have very less quantity of some sample you want to do some research with it or youhave to say give some identification then, you cannot do with such small quantity ofthings, so for that we do PCR.So, this is the machine called thermo cycler which we use for PCR as you can see theseare the well plates, where we put a PCR tubes and run a PCR, here we will be putting a PCRproduct which my colleague has given us and we will checking its result.So, before starting the procedure like you know, the basic components of PCR which weneed, so first of all this is reaction buffer, this reaction buffer we need.It will give proper optimum environment for our enzyme to work, this is our enzyme TaqDNA polymerase.These are the 2 primers for our reaction, this is reverse primer, this is forward primer.We will be adding all these contents to a master mix tube, this is 2 molar dNTP.And you place free water.And this is the amount of reagents we add in each reaction, this is for 1x reaction,this is for 4x.So now, as you know the components of the PCR and we have added into the master mixwill be adding it to a PCR tubes.This tubes I have already added 23 microliter of master mix and 2 microliters of our; sonow as we know, what does a master mix contain; we have already added it in proper amount,so now we will be adding it to a PCR tubes, which I have already added, we have alreadymade a master mix.So, from this master mix, we will be adding 23 micro litre of a master mix to a PCR tubes,in this tubes we already have added 2 micro litre of our DNA which we want to; loop ishere, now we will be putting a tubes into the PCR machine, before putting the tubesin PCR machine make sure you give it a spin, so we will be spinning our tube, so that toensure that our contents are properly mixed.So, now once our tubes are ready, so now as you can see a machine is on, this is a thermocycler, so in this thermo cycler, we will be adding a tubes and in this way and we willbe closing it, make sure you tighten the cap now, as you can see here, we can see manyoptions; run, new, edit, view, files and tools, I have already set the program which is gapDH because we are going to check here for Gap DH gene.So, here sample volume is 25, enter, view the program, this is the program we will beusing as you can see you are able to read multiple temperatures here and number of steps,so the first step is initialization.In initialization, the enzyme will be activated for further denaturation, second step is 94degree for 30 seconds, in this step denaturation of a DNA will occur, in denaturation boththe strength will separate out.In the third step, it is 58 degree for 30 seconds, now this is the underlying step,where the primers are going to anneal, when primers will anneal, the process will go onfurther, next is 72 degree for 30 seconds that is the extension step, so in extensiononce the synthesis, so in this extension step it is for 30 seconds.In extension step, what will occur that synthesis will go on again and again and once a productis formed another product will be taken as template and new product will be formed andthen we will be repeating this cycle for 35 times as you can see here, 35 times go to2, so these 3 steps will be repeated, we will be getting multiple copies.Next is again 72 degrees for 10 minutes, so for final extension if any product is leftfor extension, it will be occurring again.And last is hold; hold at 4 degree, why will be holding to bring down the temperature tonormal to for cooling up the chamber, so now as we know in the program, we will be runninga PCR program.So, as you can see now it has started and here we can see how the steps are going, soit will take this much of time to complete a program, so now we will run the program;run, now we can see a PCR has started and it will take this much of time to complete.So, now as you can see a gel has solidified and we will be putting it on a running unit,as you can see it is properly solidified, so now we will be taking out a combs and puttinga gel in the unit, this is 1x TAE and we have to add it in such a way that our whole gelis immersed in it, so, (FL) so now we will be adding a DNA to the loading dye which havespotted here.This is 1x loading dye, to it we will be adding a PCR product which is, which will be takingas 8 micro litre and after adding it by pipetting, they will be mixing it properly.And will be loading it on a gel, so now to check whether the products; PCR products areof a desired one or not we will check it by loading DNA ladder into it, the size of GapDH here is 595 base pair, so we have loaded 100 base pair ladder and the loading dye weused is this one, so the ladder we have used here is 100 base pair DNA ladder because theproduct size is 595 base pair.Now, we will be running a gel make sure that you make the connections properly and now,so now we will be setting up our voltage is; we will keeping 100 volts, as you can seefor 1 hour and press run, so as you can see here bubbles will start coming out so, thegel has started running.Let us watch the following animation to understand this concept better.Reverse transcription PCR is used to generate multiple copies of DNA with RNA as a startingmaterial.The template RNA molecule is first reverse transcribed into the corresponding cDNA bymeans of the enzyme reverse transcriptase, this enzyme which is commonly found in virusesis capable of synthesizing DNA from an RNA template.Template strand at 72.Traditional PCR is then performed on the cDNA obtained by addition of primers which areallowed to anneal at 54 degrees centigrade.This is followed by addition of nucleotides and taq polymerase which performs elongationof the template strand at 72 degrees centigrade.Second and subsequent rounds of PCR result in further amplification of the cDNA of intereststrand separation is performed at 95 degrees centigrade followed by primer annealing andelongation respectively in this way, the new mRNA transcript originally used is amplifiedin the form of its corresponding cDNA, which can then be studied further.The double stranded DNA that needs to be amplified is heated to 95 degrees centigrade to bringabout strand separation, once the strands are separated primers are added along withthe probe DNA molecules which have the quencher and reporter molecule bound to its ends.Once these have annealed to the template DNA strands at 54 degrees centigrade, taq polymeraseand nucleotides are added and the temperature is again increased to 72 degrees centigradeto carry out strand elongation.The taq polymerase continues to elongate the DNA strand based on the corresponding templateDNA, when it reaches the bound probe molecule, the 5 prime to 3 prime exonuclease activityof taq polymerase degrades the probe into its nucleotide fragments and continues toelongate the DNA strand.The released reporter dye thus gets separated from the quencher molecule during this processand the fluorescence emitted can then be detected using a suitable detector.The increase in fluorescence in real time PCR is directly indicative of the amount ofnucleotide being synthesized and is therefore, a useful tool for measuring gene expression.I hope know your concepts for the tools which are used for the amplification and visualizationof DNA is clear now.In today's session, we have just got a glimpse of how to perform agarose gel electrophoresisand polymerase chain reaction.And actually, whole experiment takes a long time but to give you a demo we have done thethings in a very short time but you can definitely, think about that you know in which way nowyou can use these particularly you know the simple tools to design your experiments andhow you can do the molecular biology to understand some specific biological concepts when itcomes to the research areas, you can now start planning those experiments.And these tools are actually much simpler which you can easily implement in the labsettings, I hope you have enjoyed the lab session, thank you.