Cutting the DNA

by Georgina Cornwall, PhD

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    00:01 The first place we should begin is by looking at DNA splicing. How do we get these pieces of foreign DNA to stick together to start with? It involves restriction enzymes or restriction endonucleases. Where do we find restriction endonucleases? We found them in bacteria. Bacteria use restriction endonucleases to essentially chop up invading DNA. Bacteria just like us will be infected by viruses. When a virus lands on a bacteria and tries to inject its DNA, the bacterial immune system goes to work and chops up the DNA destroying the viral DNA so that it can no longer be effective in taking of the bacterial genome and thus the bacteria is not affected by the infected DNA from the virus. That is where they came from. We have captured those restriction endonucleases or restriction enzymes so that we can use them as molecular scissors.

    01:06 Restriction enzymes are molecular scissors and they will cut DNA. There are three types.

    01:11 Type II is the one that we are most interested in because we have a little bit more precision with where they cut. Looking at how this happens on one side here, we have the bacterial chromosome and on the other side, we have the insulin gene that we were looking at in the earlier figure. When we have a restriction enzyme, the type II ones will make staggard cuts and they make staggered cuts because they are looking for a palindromic sequence of particular interest, right, the one that they are targeted to should they have been in a bacterial cell. These palindromic sequences, palindromic meaning reads the same backwards and forwards. Here you see a sequence of AATT and if you read on the other strand in the opposite direction, it also reads AATT. The restriction enzyme EcoR1 comes from E-coli and it was the first restriction enzyme we found so that how it got its name. It will make the staggard cuts on either end of the palindromic sequence, that will reveal sticky ends. The sticky ends will allow these two foreign pieces of DNA to compliment each other and so the base pairs want to stick together and then ligase comes along to stick together the backbones, the phosphate-sugar backbones. Here you can see how the two pieces come together and now we have a recombinant DNA molecule. Again, we are generally looking at a circular chromosome and a foreign piece of DNA being inserted into that circular chromosome and so we'll see two ends of sticky ends and insertion of that sequence.

    About the Lecture

    The lecture Cutting the DNA by Georgina Cornwall, PhD is from the course Biotechnology.

    Included Quiz Questions

    1. Even when the source of the DNA is different
    2. Because they have palindromic sequences
    3. Only when the source of the DNA is the same
    4. But the "sticky ends" will most likely have to be modified
    5. Only if the subunits have been methylated
    1. The EcoRI enzyme helps the eukaryotic cells to become immunized against viral attacks
    2. EcoRI is a useful restriction endonuclease enzyme for gene cloning procedures
    3. The action of the EcoRI enzyme results in the dsDNA fragments having sticky ends with 5’ end overhangs of AATT
    4. EcoRI enzyme cuts G/AATTC recognition sequence on the dsDNA which has a palindromic complementary sequence CTTAA/G
    5. EcoRI enzyme was the first restriction endonuclease enzyme isolated from E. coli

    Author of lecture Cutting the DNA

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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