BSCI222 – Lecture 20- Chapter 19: Molecular Genetic Analysis and Biotechnology- (1) Restriction enzymeso Come from bacteria (in the bacteria, they cut up invading viruses, basically a bacterial immune system). Cut at palindromes (sequence where you want it to cut,sequence that is same on both strands).o Were isolated from many different bacterial species. Each has unique recognition sequence.  A 6 base recognition sequence, like for BamHI, will cut how many times? (1/4)*(1/4) = 1/ (4^6), cuts every 4^6 bases. Does it leave a 3’ overhang ora 5’ overhang? It’s cutting between the last and before-last on the 5’ end  5’ overhang. Called a cohesive or sticky end (extra Hydrogen bonding not satisfied). If no overhang = blunt end.o If you know the first half of the palindrome sequence on one strand, can figure outthe whole thing. Starts GAT, then on the opposite strand must go “TAG” at the end, in order to read the same way. Then, to match the TAG, the original strand must have ATC, and the second strand will have CTA to match the original GAT.GATATC/CTATAG.o Overhangs help the DNA stay together while the ligase is working; much harder to do the ligation if both strands are blunt-ended. And impossible to ligate them together if the overhangs aren’t complementary. - (2) Separation of DNA restriction fragments by gel electrophoresiso Fragments in gel -> run electrical current -> negatively-charged DNA migrates toward the positive pole, with bigger pieces taking longer than smaller ones (limit the time, fragments of different sizes have migrated different distances). For smallpieces and high resolution, use acrylamide (down to base pair resolution). For lowresolution, use agarose (hundred base pairs to twenty thousand base pairs resolution).o DNA is clear; can’t see it in the gel unless it is labeled. Typically use intercalatingdyes (have flat planar structure, insert between the bases in a helix). When they’rein that position, they fluoresce much more brightly. Use ethidium bromide, makesDNA fragments appear orange under UV light (mutagenic, can cause cancer, haveto use gloves).o If you want to see a particular piece of DNA, then need to be probe the DNA that is in the gel. Technique called Southern blot: gel has been run, gets soaked in an alkali solution to denature the double-stranded DNA (! Dr. Kocher said it gets denatured after the transfer!), placed on a platform in a dish containing a buffer, with a membrane on top, and the buffer is draw up into the top layer of plotting paper as it passes through the gel, carries DNA onto the membrane (usually Nylon, positively charged so the DNA will stick to it). The DNA on the membrane is fixed, and then placed in a hybridization bottle with a solution that contains a radioactively labeled probe (floating thingies, trying to find its match inall the DNA, trying to label all the DNA on the filter that has that sequence that you’re looking for) and is gently rotated (DNA is waiting to covalently bond to the probe). The probe molecules bind to the complementary DNA fragments on the membrane, and then use autoradiography (X-ray) to detect the fragments on the membrane that have the probe molecules attached. Get an image of, in how big a piece of DNA, was the sequence we were looking for.  Opened up the possibility for using markers in the human genome. Restriction sites can show Mendellian inheritance. We can only detect the DNA that has the probe; smaller alleles, with smaller restriction sites or more restriction sites, make things more complicated. Don’t know about extra sites because have no way of probing them. RFLPs: restriction fragment length polymorphisms, genotype used to map humans.- (3) A simple cloning vector:o Requires an origin of replication (recognized in the cell that you’re growing it in, so that it is replicated along with the DNA that it carries), restriction site (so that you can put stuff in there), and a selectable marker (take recombinant plasmid andput it into a tube with lots of bacteria, hoping to transform the bacteria; most of them will not pick it up, because it’s not a very efficient process, leaving you with2 kinds of bacteria. Have to kill off the ones without the plasmid, by growing on an antibiotic that the plasmid provides resistance for. Thus, the marker allows youto select for the bacteria that you want, and kill of the ones that you don’t.)o Need a single opening on the plasmid (unique restriction sites), to ligate your piece in. The plasmid and the foreign DNA are cut by the same restriction enzyme(therefore compatible ends), let the sticky ends find each other, and then seal up the sugar-phosphate backbone with DNA ligase. The ratio of plasmid to insert hasto be just right (plasmid size itself doesn’t really matter, can be super long because the circle can get as big as necessary, except for plasmids that you’re trying to transduce via phage head); if you have too much insert, will ligate together before getting into a plasmid, thus the plasmid could have 2, 3, 4, etc. inserts in it. If you have too little insert, the sticky ends of the plasmid might re-ligate with no insert (the sticky ends of the plasmid are always close together because it’s the same piece of DNA). What really matters is the concentration of ends, not DNA. Want equal numbers of plasmid ends and insert ends, therefore getting the greatest number of recombinant molecules.o Problems with a simple vector: relatively few restriction sites available, and there is no way to distinguish recombinant plasmids from non-recombinants.o Modern cloning vectors: engineered to have many more restriction sites, and a copy of the lacZ gene, which allows for blue-white color screening (screening marker, only shows you the difference, but does not kill anything). Cut the plasmid at the restriction site (which is located in the middle of the lacZ gene), add the foreign DNA (making a recombinant plasmid), which is then ampicillin resistant. The original plasmid is lacZ+, while the plasmids with inserts are lacZ- because it was cut in the middle and has foreign DNA in the middle of it. AddIPTG; makes sure that the lac gene is expressed all the time. The resulting molecule from the solution (B-gal, dimerization, more), gives us lots of blue precipitate. If the foreign gene is in the middle of the lac gene, then the solution remains colorless and white. Bacteria with an intact plasmid produces B-gal, which