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PART 1 CONTENTS Introduction Uses of Gene Technology 1. DNA extraction 2. Restriction Enzymes 3. DNA ligase 4. Reverse Transcription 5. Gel Electrophoresis 6. DNA Probes 7. PCR Polymerase Chain Reaction 8. DNA Sequencing 9. Gene Cloning 10. Plasmids 11. Bacteriophages 12. Transgenesis 13. Tool Kit Vocab Introduction All genetic changes result from * production of random variation * selection pressures Traditional breeding methods selectively choose desirable qualities. By selective breeding the genetic make up of many species has been progressively altered under domestication. These traditional methods of plant and animal breeding had three major limitations. The production of better varieties was largely a matter of luck, since recombination and mutation are both random processes. Gene pools of different species are normally isolated from each other, so geneticists were limited to crossing varieties of the same species, or in some cases, closely related species. It takes much longer to produce new varieties by selective breeding. Molecular genetics has changed all that. Ever since it was discovered that the genetic code is universal, the transplantation of genes between unrelated organisms has been a possibility, and within the last 2 decades a reality. Genes Genes have two main components: The protein coding region- contains the nucleotide triplet codes which code for specific amino acids and the order they are arranged in. This is a universal code so the same protein can in theory be made by any organism. The promoter region- this region controls the gene expression. It regulates in which tissue the gene should be expressed, at what time, and in response to what stimulus the gene is transcribed. These promoters will only work within kingdoms and are not as universal as genes. The promoter and the gene are usually physically separated. USES OF GENE TEHNOLOGY 1. DNA Extraction DNA must be extracted from the blood sample, piece of tissue, bacteria and virus cells. DNA extraction must remove all the unwanted cell debris, membranes and proteins. To isolate your own human DNA... * To a 1/2 cup of water dissolve about 1/2 teaspoon of salt and add a squirt of dishwashing detergent. Save this for step 3. * Swirl about 25 mls of water around your mouth for 30 seconds. This removes some cheek cells. Spit into a disposable cup. * Add about 2cm of the fluid to a test-tube (or a Fuji film canister) and add about 1cm of the saline/detergent solution. Invert gently 3 or 4 times to mix well (but you don't want a lot of froth). This will break open the many cheek cells you spat out, releasing the DNA message that each cell must carry. 2 * Layer on top some ice-cold ethanol (or methanol). Strands of DNA will be seen where the two layers meet. Hook out the strands of DNA that form with a glass hook (or one made from a plastic twist-tie) by slowly dipping up and down through the two layers. To isolate plant DNA * Blend 50g of Cauliflower in 200 mL of water for 30 seconds to get single cells. * Strain through muslin cloth. Pour 15 mL of liquid into a screw cap tube with 3 drops of detergent and shake gently for 5 seconds. * Carefully layer to top of tube with cold alcohol (or meths). Use a hooked glass rod to spool out strands of DNA at the interface. Taken From http://talk.csifiles.com/showflat.php/Cat/0/Number/146627/Main/145305/ How DNA Extraction works… Step 1: isolate the DNA from the rest of the cell This is done by mechanically breaking the cells open, then using detergents and enzymes to break down the cell walls and membranes. The detergents also break down the nuclear membrane releasing the DNA. Step 2: remove the unwanted cell debris This is done by filtering the extract or by centrifuging the mixture. The filtrate (or the pellet at the bottom of the centrifuge tube) will contain the DNA we want and unwanted proteins. Step 3: remove the unwanted proteins This is done by adding a protease enzyme that destroys proteins. Step 4: precipitate out the DNA The DNA can now be precipitated out by pouring a layer of ice-cold ethanol over the surface of the filtrate. The collected DNA can be now be used in some of the other techniques we will look at. 2. Restriction Enzymes Restriction enzymes are used by bacteria to defend themselves against invasion by bacteriophages (viruses that attack bacteria). Restriction enzymes (RE’s) act as chemical scissors and cut DNA at specific sites. The ends created by restriction enzymes are either blunt or sticky. Restriction enzymes are named according to the kind of bacterium from which they are obtained. Each restriction enzyme always cut the DNA at the same place, at the same bases sequence. The two ways of making a cut. 1 A blunt end In this the DNA is cut straight across. 2 A sticky end In this the DNA is cut staggered. They are called palindromic cuts because both ends have sequences which read the same in the 5’ to 3’ direction. Sticky ends are most commonly used. After the DNA has been cut using restriction enzymes The ends produced by either blunt or sticky restriction enzymes can be attached to some other DNA that has been cut by the same restriction enzyme. 3 Fragments can be separated by gel electrophoresis for detailed analysis 3. DNA Ligase DNA ligase acts as a molecular stapler, joining DNA fragments. Its normal role includes the joining of Okasaki Fragments in DNA replication, and in DNA repair. The sticky ends of DNA are joined weakly by hydrogen bonds, the DNA ligase catalyses the formation of phosphodiester bonds. Phosphodiester bonds occur between the phosphates and the sugars on the sides of the DNA ladder. Create sticky-ended DNA fragments from two DNA sources using the same restriction enzyme. Mix the DNA from the two sources and allow complementary ends to form complementary base pairs. This process of forming a loose and temporary join by hydrogen bonding is called annealing. Add DNA ligase to form a permanent link. 4. Reverse Transcription Many viruses do not carry their genetic material as DNA, and carry their genetic material as RNA. 3. Reverse Transcription Reverse transcriptase is an enzyme produced by retroviruses, which include tumour viruses and HIV. Their genetic material is RNA and this is replicated indirectly in the host cell via a DNA template- the reverse of what normally happens. The viral RNA is used as a template to make a complementary sequence of DNA (cDNA), which is then used to make more viral DNA from messenger RNA. 4 5