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Chapter 9. Messing about with DNA Prepared by Woojoo Choi DNA extraction 1) get a bacterial culture or hack off a small sample 2) Disrupt the cell – using blender or chemicals – Lysozyme: an enzyme that digests the cell wall – Detergent: molecule that dissolves the greasy cell membrane DNA extraction 3) Purify and conentrate DNA – Centrifuge, Phenol(to remove the protein), ribonuclease(an enzyme to get rid of RNA), alcohol(to puch DNA out of solution Cutting up the DNA 1) Restriction enzyme – An enzyme which binds to DNA at recognition site (a specific base sequence), and then cuts the DNA – Recognition site is usually four, six or eight bases long and an inverted repeat – Each restriction enzyme has its own specific recognition site. Where it the DNA cut? 1) Type I restriction enzyme – Cut the DNA a thousand or more bases pair away from its recognition sequence – The base sequence at the cut site is not fixed. – These enzymes are suicidal. – So, not of much use to molecular biologists Where it the DNA cut? 2) Type II restriction enzyme – Cut the DNA within its recognition sequence – Normally used in genetic engineering – There are two different ways of cutting the recognition site. – Blunt ends and sticky ends Where it the DNA cut? 3) Enzymes that generate sticky ends are more useful. – The same sticky ends would be generated on both fragments. – This allows two pieces of DNA to be bound together by hydrogen bonding and matching the sticky ends. – This give time for the permanent covalent bonds. How are fragments of DNA joined together? 1) DNA ligase – An enzyme that joins DNA fragments end to end – DNA with sticky ends tend to stay attached and DNA ligase join them permanently without much trouble – Ligating blunt ends takes a very long time and very inefficient – Bacterial ligase cannot join blunt ends – T4 ligase is used because it is easier to use and can join blunt ends Where do restriction enzymes come from? 1) The bacteria usually fight back by chopping up the DNA of the virus. 2) This restricts the entry of the virus and so the enzymes that chopped up the DNA were called restriction enzyme Protection of the cell’s own DNA 1) Modification enzyme – Whenever a bacterial cell makes a restriction enzyme, this enzyme is also made – An enzyme that chemically alters a base in the recognition site – This protects all the DNA in a bacterial cell from being cut How can individual fragments of DNA be separated out?? 1) Electrophoresis – movement of charged molecules toward an electrode of the opposite charge – Used to separate nucleic acids or proteins or any molecule that has a charge – Agarose gel electrophoresis can be used to purify DNA or it can be used to measure the sized of fragments. How can individual fragments of DNA be separated out?? – Fragments of DNA have the same number of charges per unit length. – They all cruise along at the same speed toward the positive electrode. How can individual fragments of DNA be separated out?? – To separate the fragments we run them through a gel. – Gelatin sets due to a microscopic meshwork formed by its own protein fibers. – Gel for DNA work are made of agarose. How can individual fragments of DNA be separated out?? – The larger molecules find it more difficult to squeeze through the gaps but the smaller ones are slowed down much less. – The result is that the DNA fragments separate in order of size. How can individual fragments of DNA be separated out?? – Both agarose and DNA are naturally colorless, so cannot see where the DNA has ended up. – To find DNA fragments ethidium bromide(Etbr), a dye that stains DNA and RNA when viewed under UV light, is used. How can individual fragments of DNA be separated out?? – After the bands are located, they are cut out of the agarose slab and the DNA is extracted to yield a pure fragment. – To find the size of DNA, we run a set of standard DNA fragments of known sizes alongside, on the same gel. Making a restriction map 1) Restriction map – diagram of DNA showing the cut sites for a series of restriction enzymes – Once a map of all the restriction sites is obtained, DNA can be cut as we wish for analysis and for cloning any useful genes. Making a restriction map Making a restriction map Choosing a vector for our own special purposes 1) Vector – molecule of DNA which can replicate and is used to carry cloned genes or DNA fragments – Bacterial plasmids are the most widely used vector. – ColE1 plasmid: a particular plasmid whose derivatives have been widely used as vectors Choosing a vector for our own special purposes 2) Requirements – The vector should be a reasonably small and manageable DNA molecule. – Moving the vector from cell to cell should be easy. – Growing and purifying large amount of vector DNA should be straightforward. Detecting and selecting vectors 1) To improve the original ColE1 plasmid – First we get rid of the genes for colicin E1, as these are not needed. – Add a gene for resistance to the antibiotic ampicillin, the ampR gene which is gene encoding B-lactamase which degrades penicillin class. – Transform this new vector into bacterial cells. – Those cells containing a plasmid survive, while those which did not get a plasmid are killed. Inserting genes into vectors 1) DNA ligase: enzyme which joins ends of DNA strands together Inserting genes into vectors 2) We have some problems to use this – Our vector would be chopped into pieces, not merely opened coveniently if there were more than one cut site in the vector – We must avoid inserting the cloned gene into any of the genes needed by the plasmid for its own replication and survival within the cell – We need flexibility using many different restriction enzyme in order to have a wide choice Inserting genes into vectors 3) Let’s fix all these problems – We put into ourplasmid a stretch of artificially synthesized DNA about 50 base pairs long which contains cut site for seven or eight of our favorite restriction enzymes, Polylinker or multiple cloning site(MCS) Inserting genes into vectors – One other thing to do is to make sure that there are no extra cut sites on the plasmid for any of the enzymes represented in MCS • The couch potato’s way to do this is to choose for the polylinker only enzymes with zero cut sites in the plasmid • Altenatively, we can get rid of unwanted cut sites by the approach shown in Figure 9.25 Detecting insertions into vectors 1) There are basically three approaches – Brute force and ignorance • Extract plasmid DNA from each of colonies • Cut the plasmid DNA with the restriction enzyme • Electrophoresis and check out the fragments • If the vector contains inserted DNA, we will get two pieces of DNA, one being the original plasmid and the other the inserted DNA fragment Detecting insertions into vectors – Antibiotics • Cells that receive a plasmid without an insert will be resistance to both antibiotics • Those receiving a plasmid with an insert will be resistance to only the first antibiotics Detecting insertions into vectors – Color screening • Plasmids without a DNA insert will produce B-galactosidase and the cells carrying them will turn blue • Plasmids with an insert cannot make B-galactosidase and the cells will stay white Moving genes between organisms: Shuttle vectors 1) Shuttle vectors – a vector able to survive in more than one type of host cell – the detailed requirements for vectors will vary depending on the host organism, but the general ideas will be the same. Yeast artificial chromosomes 1) Genes of higher organisms are often relatively huge. 2) Typical bacterial genes are around a thousand base pairs, the genes for hemophilia may take up a million or more base pairs of DNA. 3) The largest bacterial plasmids that can be manipulated are around 50 kbp. Yeast artificial chromosomes 4) To carry huge pieces of DNA, construct yeast artificial chromosomes(YACs) – linear DNA which mimics a yeast chromosome by having yeast specific origin, centromere and telomere sequences – YACs will survive not only in yeast but also in mice.