Gene Technology

Techniques used to study genes: Polymerase chain reaction (PCR) - produces lots of identical copies of a specific gene. In vivo cloning - also produces lots of identical copies of a specific gene. DNA probes - used to identify specific genes. Is useful to get a copy of the DNA fragment of the gene that you're interested in (target gene). Are 3 ways DNA fragments are produced: 1. REVERSE TRANSCRIPTASE Many cells only contain 2 copies of each gene, making it difficult to obtain DNA fragment containing target gene. But they can contain many mRNA molecules complementary to gene, so mRNA is easier to obtain. mRNA molecules used as templates to make lots of DNA. Enzyme reverse transcriptase makes DNA from RNA template. DNA that's produced is called complementary DNA (cDNA). E.g. pancreatic cells produce insulin protein. They have loads of mRNA molecules complementary to insulin gene, but only 2 copies of the gene itself. So reverse transcriptase could be used to make cDNA from insulin mRNA. To do this, mRNA is isolated from cells and mixed with free DNA nucleotides and reverse transcriptase. Reverse transcriptase uses mRNA as template to synthesise a new strand of cDNA. 2. RESTRICTION ENDONUCLEASE ENZYMES Some sections of DNA have palindromic sequences of nucleotides. These sequences consist of antiparallel base pairs (base pairs that read the same in opposite directions). Restriction endonucleases recognise specific palindromic sequences ( recognition sequences) and cut (digest) the DNA at these places. Different restriction endonucleases cut at different specific recognition sites, because shape of recognition site is complementary to enzyme active site. If recognition sites are present at either side of the DNA fragment that you want, you can use restriction endonuclease to separate it from rest of DNA. DNA sample is incubated with specific restriction endonuclease, which cuts DNA fragment out via hydrolysis reaction. Sometimes cut leaves sticky ends, which can be used to bind (anneal) DNA fragment to another piece of DNA with sticky ends with complementary sequences. 3. POLYMERASE CHAIN REACTION Used to make millions of copies of fragment DNA. Is repeated over and over to make lots of copies. A reaction mixture is set up that contains DNA sample, free nucleotides, primers and DNA polymerase. Primer = short pieces of DNA that are complementary to bases at the start of the fragment that you want. DNA polymerase = enzyme, creates new DNA strands. DNA mixture heated to 95 degrees to break H bond. Mixture cooled to 50-65 degrees so primers can bind (anneal) to strands. Reaction mixture heated to 72 degrees so DNA polymerase can work. DNA polymerase lines up free nucleotides alongside each template strand. Specific base pairing means new complementary strands are formed. 2 new copies of fragment of DNA are formed and 1 cycle of PCR is complete. Cycle starts again, with mixture being heated to 95 degrees and 4 strand are used ( 2 original, 2 new) as templates. Each PCR cycle doubles the amount of DNA - e.g. 1st cycle = 4 fragments ( 2x2), 2nd cycle = 8 fragments (4x2) . 3rd cycle = 16 fragments (8x2) etc.

Gene cloning is all about making loads of identical copies of a gene. Can be 2 different ways: In vitro - gene copies made outside living organism using PCR. In vivo - genes copies made within living organism- As organism grows, and divides, it replicates its DNA, creating multiple copies of a gene. IN VIVO STEP 1 - GENE IS INSERTED INTO A VECTOR DNA fragment inserted into vector DNA. Vector = plasmids, bacteriophages (viruses that infect bacteria). Vector DNA is cut open using restriction endonuclease - sticky ends of vector are complementary to sticky ends of DNA fragment containing gene. Vector DNA and DNA fragment are mixed together with DNA ligase - joins sticky ends of vector DNA and DNA fragment = ligation. New combination of bases in DNA = recombinant DNA. STEP 2 - VECTOR TRANSFERS GENE INTO HOST CELLS Vector with recombinant DNA used to transfer gene into host cells. If plasmid vector is used, host cells have to be persuaded to take in plasmid vector and its DNA. E.g. host bacterial cells are placed in ice-cold calcium chloride solution to make their cell walls more permeable. Plasmids are added and mixture is heat-shocked (heated to 42 degrees for 1/2 mins), which encourages cells to take plasmids. Bacteriophages infect bacterium by injecting its DNA into it. Phage DNA (with target gene in it) then integrates into bacterial DNA. Host cells that take up vectors containing gene of interest are transformed. STEP 3 - IDENTIFYING TRANSFORMED HOST CELLS Maker genes are used to identify transformed cells. Marker genes can be inserted into vectors at same time as gene is being cloned - means any transformed host cells with contain gene to be cloned and marker gene. Host cells are grown in agar plates and each cell divides and replicates its DNA, creating a colony of cloned cells. Transformed cells will produce colonies where all the cells contain cloned gene and marker gene. Marker gene can code for antibiotic resistance - host cells are grown on agar plates containing the specific antibiotic, so only transformed cells that have marker gene will survive and grow. Marker gene can code for fluorescence - when agar plate is placed under UV light, only transformed cells will fluoresce. Identified transformed cells are allowed to grow more, producing lots of copies of cloned genes. ADVANTAGES AND DISADVANTAGES IN VIVO - ADVANTAGES Can produced mRNA and protein as well as DNA because its done in a living cell which contains ribosomes/enzymes needed to produce them. Can also produce modified DNA, modified mRNA, or modified proteins - have modifications added to them e.g. sugar/methyl. Large fragments of DNA can be cloned and inserted into vectors. Can be a cheap method, depending on how much DNA you want to produce. IN VIVO - DISADVANTAGES DNA fragment has to be isolated from other cell components. You may not want modified DNA. Can be a slow process - some bacteria grow really slowly. IN VITRO (PCR) - ADVANTAGES Can be used to produce lots of DNA - not mRNA or protein. DNA produced isn't modified - advantage if you don't want it modified. Only replicates fragment of interest e.g. target gene - don't have to isolate DNA from host DNA or cell components. Fast. IN VITRO - DISADVANTAGES Can only replicate small DNA fragments. Can be expensive. May want modified DNA, mRNA and protein - which aren't made.

Making DNA Fragments

Gene Cloning