Biotechnology and Recombinant DNA

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1 Biotechnology and Recombinant DNA Recombinant DNA procedures - an overview Biotechnology: The use of microorganisms, cells, or cell components to make a product. Foods, antibiotics, vitamins, enzymes Recombinant DNA technology = Genetic Engineering: Insertion or modification of genes in an organism to produce desired proteins/outcomes. e.g. gene(s) from one organism can be placed into another organism s DNA - including between species - to produce: recombinant proteins e.g. insulin, hepatitis B vaccine enough copies of the recombinant DNA for further studies Recombinant DNA procedures - an overview Recombinant DNA procedures - an overview Clones Clones Vector must be self-replicating 1

2 Tools of Biotechnology 1. Selection: Natural selection - survival of the fittest Artificial selection - selection by humans of organisms with desirable characteristic for breeding/cultivation e.g. Culturing of a naturally occurring microbe that produces a desired product 2. Mutation (a change in the DNA): Mutagens cause mutations that might result in a microbe with a desirable trait Select and culture microbe with a desired mutation Site-directed mutagenesis: Change a specific DNA gene sequence to change a protein Tools of Biotechnology (cont.) 3. Restriction enzymes (RE) Cut specific sequences of DNA Occur naturally in some bacteria in vivo role - destroy bacteriophage DNA in bacterial cells Cannot digest (host) DNA with methylated cytosines Purified REs used in genetic engineering A specific RE always recognizes and cuts DNA at a very specific DNA nucleotide sequence. e.g. enzyme EcoRI - GAATTC Know what DNA SEQUENCE means Tools of Biotechnology (cont.) 3. Restriction enzymes (RE) - cont. 100s of different REs The cuts made by some REs are staggered, producing sticky ends The role of restriction enzymes in making recombinant DNA The sticky end of one DNA fragment can hydrogen bond with a complimentary base sequence (sticky end) of another fragment. Note: recognition sites are on both strands of DNA, but run in opposite directions DNA from another source, cut with the same RE Covalent linkage by DNA ligase 2

3 The role of restriction enzymes in making recombinant DNA Recombinant DNA procedures - an overview Vector must be self-replicating Tools of Biotechnology (cont.) 4. Vectors Role - transport recombinant DNA into the desired cell Example - plasmids, viruses Important characteristics: self-replicating small resist destruction by the recipient cell Carry a marker gene: to make retrieval of recombinant clones carrying the vector easy e.g. gene coding for antibiotic resistance, or a specific enzyme Tools of Biotechnology (cont.) 4. Vectors (cont.) Shuttle vectors can exist in several different species Use of vectors in gene therapy: to insert functional genes into human cells with a defective gene 3

4 Vectors - example of a plasmid vector Tools of Biotechnology (cont.) Gene for ampicillin (antibiotic) resistance Gene for β- galactosidase enzyme Restriction enzyme sites Origin of replication 5. Polymerase Chain Reaction (PCR) DNA "photocopier" Enzymatic technique to make multiple copies of (amplify) a piece of DNA. Results in over a billion copies of the original target DNA! Used to: Clone DNA for recombination Amplify DNA to detectable levels Sequence DNA Diagnose genetic disease Detect pathogens PCR PCR 1st cycle 2nd cycle 1st cycle 4

5 PCR Recombinant DNA procedures - an overview Vector must be self-replicating 2nd cycle 3rd cycle 4th cycle etc.. usually cycles in total Techniques of genetic engineering: Inserting recombinant DNA into cells After recombinant (foreign) DNA is constructed in vitro (i.e. outside of a cell), it needs to be inserted back into a cell Can be accomplished by: Transformation Electroporation Protoplast fusion Microinjection Gene gun Techniques of genetic engineering: Inserting recombinant DNA into cells Transformation: Few species are naturally competent (i.e. able to spontaneously be transformed by DNA) Chemical treatment (e.g. CaCl 2 ) can make them competent: e.g. treated E. coli mixed with DNA and heat shocked Electroporation: Electric current used - pores form in plasma membrane, through which DNA enters If substantial cell wall present (e.g. G+) - need to first convert cells to protoplasts by enzymatic treatment 5

6 Techniques of genetic engineering: Inserting recombinant DNA into cells Protoplast fusion: In solution, protoplasts fuse to form hybrids at a slow rate frequency increased by polyethylene glycol A fused hybrid cell contains two chromosomes that can undergo natural recombination Protoplast fusion with bacterial cells Techniques of genetic engineering: Inserting recombinant DNA into cells Microinjection: Glass micropipette used to inject DNA directly into a cell Gene gun: Techniques of genetic engineering: Inserting recombinant DNA into cells DNA used to coat microscopic gold or tungsten particles These then shot into a cell (eukaryotic) using a burst of helium 6

7 Obtaining DNA Gene/Genomic libraries How do genetic engineers obtain the genes they are interested in? Two main sources of genes used by genetic engineers: 1. Gene libraries: composed of pieces of an entire genome stored in bacterial plasmids or in phages. 2. Synthetic DNA: DNA is synthesized by a machine - but only short sequences (not much > 100 nucleotides long). The cloning of prokaryotic DNA is straightforward The cloning of prokaryotic DNA is straightforward Cloning of eukaryotic DNA is generally more involved this part is more complicated 7

8 Complementary DNA (cdna) the cloning of eukaryotic DNA Let's clone a gene! cdna is made from mrna by using the enzyme reverse transcriptase Need a vector with an marker gene (e.g. antibiotic resistance) Selecting the desired clone from a gene library Generally a two (or more) step procedure: FIRST STEP: Blue-white screening of the gene library - selects for cells containing foreign (cloned) DNA 8

9 Blue-white screening Blue-white screening Selecting the desired clone from a gene library Colony hybridization We know which bacterial cells contain foreign DNA but WHICH ones have the gene/dna we are interested in?? Uses DNA probes: Short pieces of single-stranded DNA SECOND STEP: If an identifiable gene product produced: simple culturing required Otherwise.. gene identified by colony hybridization screening: Used to identify cells that carry a specific cloned gene Complementary to the gene of interest Can be labelled with fluorescent dye or radioactivity Will bind to the gene of interest by complementary base pairing Are essentially gene-seeking missiles next slide SDS lysis of cells Treat with NaOH 9

10 Colony hybridization (cont.) E. coli - the genetic engineering workhorse Advantages: easily grown, fast grower genomics well studied Disadvantages: Need to eliminate endotoxin from products Cells must be lysed to get product Alternatives: some G+ species (e.g. Bacillus subtilis) eukaryotes - yeast, mammalian and plant cells Genetic engineering - therapeutic applications The Human Genome Project Subunit vaccines: Production of only part of a pathogen - a protein e.g. nonpathogenic viruses carrying genes for pathogen's antigens as vaccines DNA vaccines Gene therapy to replace defective or missing genes Gene silencing All 3 billion (!) nucleotides sequenced by one of methods used called random shotgun sequencing everydaybird.blogspot.com What do the results look like????? 10

11 The Human Genome Project Sequencing of complete genomes by random shotgun sequencing Figure 9.14 everydaybird.blogspot.com Sequencing of complete genomes by random shotgun sequencing Sequencing of complete genomes Sequence data analysed, and DNA sequences assembled using computer software... new field: Bioinformatics: the science of analysing genetic (and protein) sequence data by computer-assisted analysis 11

12 Pharmaceutical products of genetic engineering Scientific applications of recombinant DNA technology Apart from producing useful products, other examples are: 1. The study of DNA 2. DNA sequencing - e.g. genes or complete genomes of organisms; even ancient DNA (neanderthal; mammoth) Scientific applications of recombinant DNA technology Agarose gel electrophoresis Apart from producing useful products, other examples are: 1. The study of DNA 2. DNA sequencing - e.g. genes or complete genomes of organisms; even ancient DNA (neanderthal; mammoth) 3. Genetic screening: testing for the presence of genetic diseases 4. DNA fingerprinting (forensic microbiology): - identification of pathogens - epidemiology of infectious outbreaks - forensic medicine - parentage testing 12

13 Genetic screening Genetic screening Genetic screening enables the identification of a particular DNA sequence among many others. Genetic screening enables the identification of a particular DNA sequence among many others. An example of a technique used in genetic screening is Southern Blotting and subsequent hybridisation with a probe targeted to the gene of interest: NOTE: probe made from the DNA sequence of the DEFECTIVE form of the gene Southern blotting and Hybridisation Southern blotting and Hybridisation 13

14 Southern blotting and Hybridisation Southern blotting and Hybridisation Southern blotting and Hybridisation Southern blotting and Hybridisation NOTE: In the case of genetic screening - the probe will bind (hybridize) with the defective gene ONLY IF IT IS PRESENT in the sample tested (= positive result). 14

15 Genetic engineering: safety issues and ethics Avoid accidental release of genetically engineered (modified) organisms - GMOs Genetically modified crops must be safe for consumption and for the environment Labelling of genetically modified food - proposition 522 Who will have access to an individual's genetic information? What to do with a positive genetic test if there is no treatment or cure? Biological weapons 15

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