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DNA technology Genetic engineering biotechnology = gene cloning Use of : Plasmids Restriction enzymes DNA vectors Plasmids Insert gene of choice in plasmid Plasmid = circular DNA in bacteria Plasmid in host bacteria Option Bacteria makes copies of gene product (insulin) Option Use gene in new or other organisms Oil spill bacteria Freeze resistant produce Restriction enzymes Open / cut DNA at restriction sites Insert DNA with desired gene sequence DNA ligase ‘seals’ gene in place = recombinant DNA restriction enzymes Note: Restriction enzymes discovered in bacteria as their defense against viruses electrophoresis Method of separating DNA fragments Restriction enzymes ‘cut’ DNA into fragments ‘cut’ DNA placed in agarose gel Electrodes apply voltage to gel DNA negatively charged so moves from negative to positive Smaller fragments move further than larger fragments VNTRs Variable number tandem repeats (VNTR) Human DNA contains MANY repeat segments ATTAGGATTAGGATTAGG Repeat segments variable in number in RFLP segments Number of repeats unique to RFLP segments Used in forensic science to ID DNA at crime scenes Can only ‘rule out’ RFLP Restriction fragment length polymorphism (RFPL) Restriction enzymes cut DNA at restriction sites Restriction sites occur at different locations on chromosomes DNA fragments are of different lengths Electrophoresis = band patterns unique to individuals PCR Polymerase chain reaction (PCR) Add targeted DNA sequence Add DNA polymerase + nucleotides + primers ‘amplify’ targeted DNA by making copies Repeated in incubation periods to increase number of copies of target DNA DNA probes Used to identify presence of target DNA Tagged probes with matching DNA to target DNA Identify if target DNA exists Increasing use as diagnostic procedures for presence of target DNA (ie STDs) Quicker TAT PCR amplifies results requiring small initial sample More technology microarrays Used in gene identification/expression Opened the field of pharmacogenetics DNA microarrays Southern blotting Southern Blotting Used to identify if certain DNA segments are present In a persons DNA DNA vectors Vector = transporter of ‘something’ from one organism to another Use plasmids in vectors to ‘deliver’ genes Isolate plasmid (tag with antibiotic resistant gene) Insert desired gene using restriction enzymes Uptake of recombinant DNA (new plasmid) via transformation ID transformed bacteria by growth on antibiotic media More biotechnology Inserting DNA into eukaryotic cells electroporation Electric shock opens cell membrane Uptake of DNA via transformation Known DNA sequences (genes) kept in ‘libraries’ Kept as cDNA (complimentary DNA strands) cDNA kept in phages OR plasmids proteonomics proteomics/sequencing proteins Identifying amino acid sequences in proteins Proteins do the metabolic work SNPs (small nuclear polymorphisms) Single nucleotide changes Used to ID whether patients respond differently to treatments Or diseases Used to compare groups with and without diseases In research Human genome project 15 year project to identify ALL genes in human genome Completed approx 7 years early (2001) Today approz 90% of genome identified ‘short cut’ used to ID genome of Haemophilus influenza And Drosophila genome sequencing Results of short cut confirmed by gene mapping Short cut used to ID human genome ID confirmed by mapping J. Craig Venter and his company Celera Genomics (CA) Feb 2001 ID of DNA sequences Sanger method (page 378) 1. Unknown DNA = template sanger sequencing 2. Add radioactively (or fluorescence tagged) primers Primers = repetitive DNA sequences ? Noncoding sequences 10-25% or DNA Usually at ‘ends’ of coding sequences 3. Add dATP, dGTP. dCTP, dTTP 4. Add DNA polymerase 5. incubate 6. electrophorese 7. Bands = DNA sequence Cloning genes and agriculture Transgenic organisms Genes from one organism placed in another organism Growth hormone genes into animals used for human food plants Freeze resistance Herbicide resistance Pest resistance Vitamin production Amino acid production Ethics of cloning Some countries ban engineered US agriculture Unknown long term effects of ingestion Effect on ecological balance? Gene therapy and human disease? Selective breeding ‘for pleasure’ ?
