Identification of Genome Region
Hello everybody this is doctor Vishal Trivedi from department of biosciences and bioengineeringIIT, Guwahati. And in what we were discussing? We were discussing about the electrophoresisand in this particular module we have discuss about the basics of electrophoresis followed by thevertical gel electrophoresis, horizontal gel electrophoresis. And then in the previous lecture’s wehave also discussed about the different variants of the gel electrophoresis.And then ultimately we have also discussed about the some of the research problems where youcan be able to utilize the electrophoresis as a tool to answer those questions. And in thisparticular; series now today we are also going to discuss few more experiments and few moreresearch problems and while discussing these research problems you will be able to understandmore and more potential of the electrophoresis to solve the research problem related to yourwork.(Refer Slide Time: 02:01)
Once you have identified that the transcription factor is interacting the scientist are not going tosatisfy there and they are actually more interested to know more and more question. And that iswhy as I said you know in this course we are not going to only discuss about the one problemwhat we are also going to tell you know the from one problem you are going to see many moreproblems.So what in the previous problem the scientist have identified that the transcription factor isinteracting with DNA. And based on that essay then can be able to identify the transcriptionfactor. Now what they want is to know which region of the genome or which region of the DNAthat transcription factor is binding. So in this particular question what they want is that? Theyhave identified the transcription factor they know that it is binding to the gene of the gene X.But what they want to know is they want to know the; identify the region of the genome wherethis transcription factor is binding. So now this question can be used or this question can besolved by another essay so that we are going to discuss anyway.(Refer Slide Time: 03:14)
So, that can be done in a foot printing but before discussing for the foot printing let me tell you ahow that the idea of foot printing came. So you can have the 2 wooden blocks so 1 wooden blockyou have 1 wooden block and now suppose you use a cutter and cut this wooden block into themultiple pieces. So what you are going to get you are going to get the multiple pieces. And there
will be no issues you will get you will cut this we will able to cut this wooden piece into multiplepieces.Because there is no protection present on to this wooden block now if you rap this wooden blockwith some steel pipe or something. Then what will happen is that you will be able to cut to thiswooden block at the same location with the help of the cutter. And you can be able to cut at thelower side as well but you will not be able to cut at this point because this is already beenprotected from the steel.So what you will in the pattern is that there will be a intact DNA present or intact wooden blockpresent which is going to have the large chunk of the wooden block. Whereas in this case;everything is going to be cutting into the multiple pieces. And that is the exactly way of so if Iask you where the foot print of the wooden block where is the foot print of the steel block, whichyou have added you can easy say that this is the place where I have added the steel block becausethis is the wooden block which is not been cut by the cutter.And that is what is the basic philosophy of doing the foot printing in the same way you canactually cut the DNA and that is how if you protect some region that region is not going to be cutby your cutters and that is actually is going to be indication that it is the region where that proteinis interacting.(Refer Slide Time: 05:21)
So let us see with the DNA so what will happen is that what you do is? You take the genome andyou use a DNAse and ask him to cut. So if you take the genome and ask the enzyme to cut it intomultiple pieces you are going to see the multiple pieces okay. But if you protect some region ofthis DNA or some region you can imagine that at this region we have added a steel block so whatwill happen is?In this case because this is a DNA what we have done is we have added a protein blocks so inthis case this region is now going to be protected from the action of the enzyme. So as a resultthis portion is going to be remains uncut and this portion is not going to appear. So the bandscorresponding to this portion are actually the site where the protein of your where your protein isinteracting and that is what the philosophy of the foot printing of the DNA.Which means you can imagine that you have a very large DNA and in thus DNA at one pointyou have a region where the protein is bound. So what happen is if you cut this it is going to giveyou this pattern because it is not been protected but if it is protected then you will get all otherbands except the band which is corresponding to this region. And that is why this region can beidentified simply by sequencing these bands and you will be able to know that which region ofgenome is been bound by this particular protein. How to perform this?(Refer Slide Time: 07:12)
So to perform you need the different types of reagents, material and equipment’s. So what are thereagents are required? You require a buffer A which actually contains the normal buffers and
you know like 10 millimolar HEPES the NaCl Sucrose, the EDTA, Titon X 100. And then youalso require the protein inhibitor PMSF then you require the buffer B which is exactly the sameexcept that you need a glycerol ETDA and PMSF.Then if you require a buffer C which is the same except that it very high consultation of the NaCland then you require the DNAse binding buffer. So that actually contains the DTT glycerolEDTA MgCl2 and Tris-HClPh 7.6. And then you require the calcium and magnesium solutionwhich is like 5 milli molar calcium 10 milli molar magnesium chloride. Then you require thestop solution which is like 200 millimolar NaCl 30 milli molar EDTA and 1% SDS.And then you require a loading buffer is NaOH formamide 0.1% xylene cyanol and 0.1%bromophenol blue. Apart from that you require a enzyme which is called as the DNAse and forperforming this experiment you require a microfuge as well as the electrophoresis operators.(Refer Slide Time: 08:41)
So in the step 1 you are going to prepare the nuclear extract for preparing the nuclear extract youhave to suspend the 100 mg tissue. In 0.5ml of buffer A then you have to gently homogenous andthen centrifuge at 5000 g for 2 minutes at 4 degree. And remember that all this procedure as to bedone in a, cold conditions like 4 degree. So that you will be able to protect the factors what ispresent in the nuclear extract.
Supernatant can be used as a cytoplasmic extract now once you centrifuge you are going to get apalate and the supernatant can be used as a cytoplasmic extract. Whereas the pellet you willresistment in buffer B centrifuge at 5000g for another 3 minutes. Then you dissolve the pellet soin after this also you are going to get a pellet in a supernatant. Then again you pellet dissolve itinto 50 micro liter of buffer C in ice for 30 minutes with the constant shaking.So after this when you centrifuge at 10000 rpm you are going to get the supernatant as well asyou are going to get the pellet. And that supernatant is known as the nuclear extract you canactually estimate the amount of protein with of the Lowry as well as the Bradford. And thatactually can be used even for you know for a basis to see how much activity you have. Then youhave to use the binding reaction buffers.So the binding reaction buffers for DNase binding buffer 25 micro liter then you require thelabeled DNA 5 micro liter. KCl and then you have to add the nuclear extract and then you havethe make up the volume 250 micro liter with the help of the distilled water.(Refer Slide Time: 10:40)
Because the control is very important you can also run a control where you cannot you shouldnot add the nuclear extracts. So without a nuclear extract is going to tell you that you are going toget all, the fragment in that, particular reactions. Whereas when you add a nuclear extract and ifthe nuclear extract is having some protein which is binding to the DNA that region is going to beprotected from getting the cleaved.
Now you mix the content of both the tubes gently in ice bath for 10 minutes and add 50 microliter calcium magnesium solution; at 18 degree. For 1 minute and then add 3 micro liter ofDNAse incubate this mixtures at 37 degree Celsius for 1 minute. Then you add these stopsolutions so that you will be able to stop the activity of the DNA so you will just going to do alimited DNase activity.Which means you are not to allow a DNase to cut the DNA for you know for very long timeotherwise what will happen is? The DNase is going to cut the DNA completely then you will notbe able to see the fragments. The mixture is then subjected to the phenol chloroform extraction,ethanol precipitation and the re-suspension in the 4 microliter of loading. Which; means oncethis you are going to add this stop solutions then you have to recover those fragments simply bygoing through with the DNA precipitation and you know removal of the protein part.Then you run and this mixture on to a 5% urea DNA sequencing gel for both in the fragmentedDNA samples. So you analyze the DNA patterns obtained for the footprints.(Refer Slide Time: 12:29)
So the results what you are going to see the results you will see that this is the sample for thestandards right. So this is your control samples so what you see is you see the bands from top tobottom all the bands what you see is actually corresponding to the different amount different
sizes of the nucleotides. Whereas in this 4 lanes where you have actually added the nuclearextract what you see is that a large junk of the region is not having any fragments.And these are actually the footprint bands because that is what exactly happens these are thebands which are disappeared from your treated sample means the sample where you have addeda extracts.(Refer Slide Time: 13:19)
So the next problem is that the scientist, have isolated a old rock sample with DNA sample fromthe Dinosour’s fossil. They have isolated the DNA and did PCR amplification with the randomprimers. Now they want to determine the size of the amplified DNA. So what they have done isthey have isolated a old rock and that old rock was containing a small dinosaur fossil. So whatthey have done is they have isolated this particular fossil and then by the help of some advancetechnique they could be able to isolate the very small fragment of the DNA.But this amount of the DNA was not enough so that you can be able to do any kind of analysislike sequencing and you know the looking at the size of this DNA and what will be thecomposition of the nucleotides in the particular DNA. So for this purpose they have to amplifyso what they have done? They have done a PCR and then they got the amplified DNA so thisamount of DNA was good enough.
So now the question is how to determine the size of the DNA so for this they can be able to usethe agarose gel electrophoresis. So let us see how to do that.(Refer Slide Time: 14:41)
So in the size of a DNA can be determined by comparing the size of the known DNA molecules.The DNA of known sizes are resolved on to 0.8% agarose with the along with the unknownsample.(Refer Slide Time: 14:58)
What you have to do is first resolve the DNA on the agarose gel along with the DNA moleculemarkers. Then you calculate the relative Rf values with the help of the migration of the DNAversus the migration of the DNA dye. So migration of DNA versus the migration of the tracking
dye and then you plot the log molecular weight versus relative Rf value. So that actually is goingto give you a standard curve.Then what you do is you perform a linear regression to calculate the equations and that equationis going to be in the form of y = mx+c so that equation can be used to calculate the molecularweight of your unknown sample. Which means if suppose I have this is a you know if I have theRf value of this I can just simply go either conventional way of going with the intercept and I cancalculate the log molecular weight and then I can easily go and calculate the molecular weight bytaking the antilog.Or I can just simply go with the you know so what will be the result in this essay is that is youhave first run the sample with the molecular weight occur and this is your sample. So what youcan do is simply calculate the Rf value for this molecule weight markers and calculate themolecular weight of you and then samples. So if you have the software’s you have the imageanalysis software’sThis image analysis software can easily be able to trained; with the help of these moleculeweight markers. So you know the size of this molecular and then with the help of the software’syou can be able to do the calculation automatically without even plotting this. Because thisplotting will always been done by the software itself and that actually is going to give you thesize of the DNA.(Refer Slide Time: 16:57)
Let us move on to the next problems so the next problem is a PhD students have isolatedmessenger RNA from the cancer sample. And he wants to use this messenger RNA for northernblotting. Now he wants to check the quality of the messenger RNA. So you know when you wantto do the northern blotting or that actually anyway we are going to discuss in our subsequentlectures and you the first thing is you have to understand the; or you have to know the quality ofthe messenger RNA.Which means how; the messenger RNA is whether it is intact or whether it is degraded all that.Because if you use the degraded messenger RNA then you northern blotting is results not goingto be accurate and they will be not going to be conclusive.(Refer Slide Time: 17:48)
First is that RNA gels are always be performed under the de-naturating conditions. And they arebeen perform in the de-naturated condition because you have the secondary structures in RNA.You have the different types of secondary structures like stems you have the Hairpin Loop, youhave the Pseudo loop, Pseudoknot you have the bulges, you have the internal loops and you havethe multiple loops.And how this secondary structures are not good because they allow the RNA to run fast on to theagarose gel. And if they will fast if they will run fast it will get less time for the molecule tointeract with the agarose gel. And consequently there will be less resolution within the differentRNA species. Destruction of secondary structures in the RNA structure minimizes these; effectand allow the better separation on the agarose gel.So because it as a secondary structure it becomes very compact structure and because of that itactually runs very fast within the pores what is present in the agarose gels. And as you know andI think we have discussed in the past also that when you are doing a horizontal gelelectrophoresis with the agarose as a matrix. The pores sizes; within the agarose is very bigcompared to the pores sizes within the polyacrylamide gel electrophoresis.(Refer Slide Time: 19:13)
How to do that the RNA samples and the agarose gel contains formaldehyde to denature thesecondary structure present in the RNA and then that prevents the reformation of the doublestandard region in the RNA structures. And that is actually going to let you to run these RNAinto a linear structure’s and that is how they will be able to get resolves nicely from the otherRNA species and it will give you the better pattern.(Refer Slide Time: 19:45)
The materials and the equipment’s what you required to run the RNA gels what require is youneed a agarose you need the you require 10X mops buffer the composition of the mops buffer isgiven. Then you required the 37% formaldehyde solutions then you require a RNA molecular
mark ladder. So this is different from the DNA ladder because here you are going to have thedifferent sizes of the RNA.Then you required the 0.5 molar ammonium acetate then you require the staining dye which the0.5 micro grams per ml ehidium bromide and that is prepared in the 0.5 molars ammoniumhesitate. Then you require the RNA’s free water formamide formaldehydes loading buffers andthat is the composition of the formaldehyde loading buffer. And the loading buffer can befilterized by the 0.2 micro filter and then it can aliquoted into a small pieces and store at -22degree.What you require the autoclave gloves you require the water bath you require the horizontalelectrophoresis system. You require the power supply then you require a container where youcan be able to store the RNA’s free staining and de-staining gels. Then you require the shaker’syou require the UV chamber’s you require a gel Doc and then you require a flask for preparingthe agarose gel.(Refer Slide Time: 21:14)
It is a multistep process so in the step 1 itself you have to do the isolation the messenger RNA.So that you are going to do with the help of the affinity purification and that anyway we aregoing to discuss when we will discuss about the northern blotting. Then you have to prepare thede-naturating agarose gel and that is also a multiple steps. So in the step 1 you have to do thepreparation of RNA’s free water.
So the RNA free water is prepared by simply dissolving the diethylpyrocarbonate or DEPC indeionized still water to a final concentration of 0.1%. And DEPC is a strong inhibitor of strongRNA’s so that actually is a inhibitor of RNA which means it is actually going. If you prepare oruse the DEPC treated water it is actually going to protect your RNA from degradation because itis going to inactivate the RNA’s what is present in the buffers or in the reaction mixtures.You stir the solution for 12 hours and then autoclave to disintegrate the DEPC and then you canstore this solution at room temperature for very long time.(Refer Slide Time: 22:35)
Then the third step you have to do the casting of the agarose gel so in a flask at the 1 gram ofagarose to the 75 ml of RNase free water. Heat the solutions to melt the agarose and observe thedisappearance of the agarose flask this I think we have already discussed when we discussingabout the agarose gel formation, when we were discussing about how to resolve this for the DNAas well. Then you allow the solution to cool down up to 55 degree Celsius inside a fuming hoodat 10 ml of 10 X more buffer and 18 ml of 37% formaldehyde.Setup the casting tray with a comb and pour the gel in the fuming hood at this stage you have tokeep the very care that formaldehyde it very toxic and can be easily absorbed through skin wearglass and you have to use the flask. Because formaldehyde is very evaporate very easily so that it
actually get into your body through the breathing. So that is why you have to use the mask aswell as you have to wear the cloves so that you have not get a, absorb through skin as well.Now in the step 3 you have to prepare the RNA samples so take the RNA samples and make upto the makeup to the 6.5 micro liter with a appropriate quantity. So whatever the RNA sampleyou have you just prepare a 6.5 micro liter of sample. And to each sample you add the 2.5 microliter 10X mops running buffer 4.5 micro liter or 37% formaldehyde and 11.5 micro liter offormaldehyde formamide.So that actually is going to make a reaction mixture of 20 micro liter mix it by vortexing andbriefly spin to collect the sample at that bottom. Then you inside the hood at 5 micro liter ofRNA loading buffer mix it by vertexing and briefly spin it to let the sample at the bottom. As youcan see we are doing all this procedure under the fuming hood so that you should not get exposedto the fumes what is coming from the formaldehyde as well as the formamide.(Refer Slide Time: 24:54)
In the step 4 you do the loading of the RNA samples so fill the agarose denatured gel preparedwith the 1% 1X mops running buffer and load the RNA sample in to the lane. So the loading isexactly the same as what we have discussed of the loading of the DNA into the agarose gel.Except that you have to be little careful that all these tips and all other instruments and thingswhat you are using should be sterilized.
Because RNA is very sensitive for the degradation so because the RNA is present everywhere sothat it actually get degraded with very fast. So you have to sterilize everything and then you haveto use the filter tips instead of the normal tips.(Refer Slide Time: 25:46)
Now in the step 5 you have to do the running of the de-naturating agarose so place the lead on tothe buffer chamber and perform the electrophoresis and 5 volt per centimeter until the dye frontreaches to the 2 third of the length. So this is exactly the same like you have to do the negativeelectrode positive electrode and then you run the RNA. And you have to run up to the 2 thirdlength of the gel. After this you have to do a staining and destaining to visualize the RNA.(Refer Slide Time: 26:16)
Then you have to do the staining of the agarose gel so in a RNAse free container agarose gel isdipped into the 0.5 molar ammonium acetate for 40 minutes at a room temperature. This is forfollowed by so you remove the solution and dip the block in a, 0.5 molar ammonium acetatecontaining 0.5 micro gram per ml ethidium bromomide. So this is the staining dye which isprepared in the 0.5 molar ammonium sulphate.Then you incubate the gel in room temperature for 30 to 40 minutes and if required and the stainis too intense which means if you are not been able to see a better background or better contrast
between the RNA as well as the background then what you can do is you can simply do the de-stain by the 0.5 molar ammonium acetate for another 50 minutes to 2 hours. Then what you do is
the transfer the gel to a UV chamber and captures the image with a gel documentation unit. Atypical RNA profile is look like this.(Refer Slide Time: 27:28)
So here what you see is this is the RNA ladder so what you see is the bands of the different sizesand the typical sample will look like this where you have the RNA of different sizes. And this isgoing to be resolved so these are the messengers RNAse of different sizes. Whereas normallywhen you run the DNA or agarose del which is not the denaturating gel what you see is that thefull RNA is present at the bottom of the gel and not been resolved.So this is what you see is actually a resolution of this which is where all the RNA bands are beenseparated into the multiple messenger RNAse. And that is how you can be able to perform the
northern blotting by transferring into a membrane and with probing with the radioactive probes.So with this I would like to conclude our lecture here, thank you.