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Lecture 12 - Lecture12 04:37 Slide2 .Howdoyou ?...

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Lecture 12 04:37 Slide 2 Northern blotting separates molecules based on molecular weights. How do you  look at such result and get information out of it? So you have a complex mixture  of nucleic acids (DNA or RNA). You load it into an agarose gel. You  electrophorese it and you separate the nucleic acids based on size (small=fast).  You transfer this gel to a membrane and you have a mirror image of the agalrose  gel on this membrane. You probe this membrane with a radiolabeled probe  (DNA). What’s a radiolabeled probe? DNA can hybridize with RNA  complimentary sequences. This is a duplex DNA that contains a RNA sequence  that you want to look for. Its got a sense and anti-sense strands. So one of these  has to be the DNA equivalent of the sequence you’re looking for. The other  strand has to be the anti-sense strand or be able to hybridize to the RNA  sequence that you’re looking for. So very conveniently, you denature both  strands, add primers and appropriate enzymes to synthesize the complimentary  DNA to these denatured probes strands and the reds indicate that you’ve used  radiolabeled nucleotides and you can actually visualize this nucleic acids by  autoradiography and importantly you notice that only one of the two strands that  you made probes from is going to be able to hybridize to your RNA. Then you  add these probes to your northern blot. What’s the information do you get out of  this? What do different sizes mean? What do different intensities mean? These  are the two kinds information you get out of northern blotting.     Slide 6
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After transcription, the mRNA gets post-transcriptionally modified in two very  important ways: cap is added to the 5’ end and the 3’ end has a very long poly A  tail. What’s diagramed here is that there are proteins that specifically recognize  the cap as well as proteins that specifically recognize poly A tail. In the nucleus,  there’s a complex called Cap Binding Complex (CBC) and this joins to the mRNA  very shortly after transcription (serves to protect the mRNA in the cytoplasm) and  poly A binding protein binds to the poly A tail ( once in the cytoplasm, it is  replaced by a cytoplasmic poly A binding protein). The mRNA is coded with lots  of other different proteins. All these other proteins are binding to unique  sequences in the mRNA. There are many different classes, the two (SR and  hnRNP) that are shown here are very large. A lot of them are present in the  nucleus and so that’s the first opportunity for them to bind. At that point, we called  this a messenger  RiboNucleoProtein (mRNP). This just implies that a mRNA 
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