[email protected] MCB150 07 December 2007 Lecture 40 Page 1 of 9 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 [email protected] MCB150 07 December 2007 Lecture 40 Page 2 of 9 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 [email protected] MCB150 07 December 2007 Lecture 40 Page 3 of 9 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