weber uiuc edu 07 December 2007 MCB150 Lecture 40 Lecture 40 07 December 2007 Announcements Announcements for the rest of the semester will be made on the announcements page only Genetic Engineering Genetic engineering the creation of recombinant DNA molecules o This is possible because DNA can be digested Cut DNA with restriction endonucleases o Digest the DNA in the lab using one enzyme E g elephant DNA digested with BamHI E g pig DNA also digested with BamHI BamHI is looking for the same sequence in both samples Still in a test tube in vitro in a laboratory o Can then put the pieces together o Important to know that this is in a tube in the lab Cells do not make recombinant DNA molecules on purpose o Remember DNA is DNA eh So the DNA can be from any organism o If they form sticky overhangs that are complementary to one another then the strands form complementary base pairs There is nothing keeping the original two fragments from rejoining unless care is taken to prevent it from happening o It is possible to weed out those unwanted results later Use ligase to splice them back together o The sticky ends are compatible throw in ligase and the pig and elephant DNA are now combined Must cut with the same enzyme to get complementary overhangs o If the restriction sites do not match then the two fragments will not join up o That is if the sticky ends are not complementary they will not form base pairs They have to be complementary o Ligase cannot work around lack of complementary pairing What about blunt cutters o In theory easier because there are no sticky ends No complementary base pairing is required o In organism A B can cut with a different blunt cutters o In theory just bring the ends together and ligate them o Blunt cutters are harder to work with in practice which is why the sticky end cutters are the ones typically used Recognition sequences occur randomly throughout the genome o If you know the size of the genome and how often the restriction sites occur on average can predict how many sites there are in a given DNA molecule o Statistically a six base palindrome say BamHI would be a 1 4096 4 6 chance of finding that Page 1 of 9 weber uiuc edu 07 December 2007 MCB150 Lecture 40 o On average therefore a six pair base cutter has a 1 4000 chance of finding a cutting site o Now digest with BamHI an entire E coli chromosome all the way to completion The E coli genome has about 4Mbp o Doing more math each fragment would be about 4000bp in length The fragments will differ in size o In an E coli genome there would be 1000 fragments produced 4000 x 1000 fragments This is on average Why does this matter o Because the big molecules like an intact chromosome are hard to work with Eukaryotes would be even harder than E coli o If we want to study the DNA isolate purify amplify and work with then it is easier to work with smaller fragments Digest into smaller fragments It is easier to isolate genes if the pieces are smaller o Also because we are frequently interested in single genes only it is easier to work with the fragments that have the genes only How do we get enough of that DNA to do something useful with it o One copy of a gene is not usable o How do we make millions of copies Cloning the DNA Use cloning to produce large amounts of DNA Cut the source DNA with a particular restriction enzyme to get the fragment of interest o Tube A contains E coli chromosome digested to completion o Tube B contains a plasmid into which we can insert small fragments of DNA then returned back into the vector Insert the smaller fragments into the vector molecule o E g a plasmid or a phage o Which you choose depends upon the size of the inserts Insert a piece of DNA into the vector to create a recombinant vector o We understand the vector plasmid The insert we don t know much about o Vectors are called vectors because they differ from the wild type they have been modified to stop them from being virulent Plasmids are used most often but phage are typically used for larger inserted fragments o Small inserts work better with plasmids 100 s to 1000 s of base pairs are best for plasmids o If the inserts are 10 000 s of base pairs then it would be better to use a phage o For this lecture 100 s to 1000 s in plasmids o Which one doesn t really matter One insert into one vector How do we make larger amounts of DNA Page 2 of 9 weber uiuc edu 07 December 2007 MCB150 Lecture 40 Cloning the DNA Introduce these vectors into a living host cell o Everything up to this point has been done in a test tube o Only now are we using living organisms Hope for now that our vectors have the recombinant DNA o We don t know for certain yet whether the recombination was successful E coli is already set up to replicate the plasmid in huge copy numbers for genetically engineered plasmids 300 500 copies o Use E coli as a machine to crank out our product The process of isolating a region of DNA of interest and replicating recombinant DNA in a high copy number vector is called cloning o Cloning literally refers to the process of making identical copies of a DNA molecule amplification o Can take an unknown organism pull out its genome replicate and analyze it Requirements of plasmid vector molecules o It has to have an ori Without an ori the plasmid will not be replicated This is the signal for replication Without one the plasmid will not replicate defeating our purpose o It has to have restriction sites for the insertion of new DNA Without a restriction site we cannot open it to insert new DNA There has to be a restriction site for the enzyme we wish to use Also some enzymes are less effective at cutting than others Helpful if there are multiple restriction sites in a good plasmid As the researcher with multiple restriction sites on the plasmid can work with a large variety of restriction enzymes o It has to have some way to select for vector in cell a selectable marker o Have to have some way to distinguish vector along from vector insert a reporter gene This is different from distinguishing between taking up a vector and taking up a vector along with the insert That is the plasmid can close down without the insert Don t want to study this want the ones that had an insert Typical Plasmid Vectors Many commercially available vectors DIAGRAM the diagram contains all of the requirements for a good vector o Ori restriction enzyme recognition site a polylinker selectable for vector antibiotic resistance is the selectable …