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Unformatted text preview: 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). Whats 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 youre looking for. The other strand has to be the anti-sense strand or be able to hybridize to the RNA sequence that youre 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 youve 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. Whats 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 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. Whats 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, theres 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 thats the first opportunity for them to bind. At that point, we called this a messenger RiboNucleoProtein (mRNP). This just implies that a mRNA this a messenger RiboNucleoProtein (mRNP)....
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