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GENETIC TECHNOLOGY 1) GENETIC RECOMBINATION 1. 2. 3. 4. Remove bacterial plasmid with restriction enzymes Add in gene of interest (plasmid is now recombinant DNA molecule) Put back into bacteria Many reproductive cycles later = amplification of gene & protein it makes RESTRICTION ENZYMES Cut up foreign DNA Very specific Recognize short nucleotide sequences (restriction site) cut at specific points within sequence Same sequence found on both strands, running antiparallel RESTRICTION ENZYMES Enzyme cuts phosphodiester bonds of strands These restriction fragments are double stranded with single stranded ends (“sticky ends”) Bacteria’s own DNA is methylated to protect itself RESTRICTION ENZYMES Single strands will hydrogen bond with other complementary “sticky ends” Bonds made permanent with DNA ligase Now we have recombinant DNA FROM RESTRICTION ENZYMES TO PLASMID MAPS PM show how different REs act upon a plasmid Pictorial representation of the different lengths of pieces remaining after the REs worked FROM RESTRICTION ENZYMES TO PLASMID MAPS Procedure Think of plasmid as clock – from 12 to 12 = total # base pairs Approximate location of cut based on base pair fragment length Use logic to solve Double check based on data 2) DNA ANALYSIS GEL ELECTROPHORESIS Sequence of entire genome genomics Begins with gel electrophoresis Sorts DNA based on size & charge Can combine with specific probes to label particular DNA bands 3) POLYMERASE CHAIN REACTION (PCR) Can quickly amplify specific DNA without using cells DNA of interest incubated with DNA polymerase, nucleotides, & ss primer DNA for synthesis DNA heated strands separate Cool primers bond DNA polymerase adds to 3’ end of each primer Repeat 4) GENOME ANALYSIS DNA microarray: ssDNA fragments fixed to slide that are then labeled with fluorescent cDNA Compare genes of species attempt to uncover gene function 5) GENOME ANALYSIS - GENE FUNCTION To determine, turn gene off – see what happens To turn off: RNA interference (RNAi) Synthetic ds RNA matches gene sequence – binds to mRNA Triggers breakdown of mRNA no protein made Remove to turn on again THE FUTURE Proteomics: study of full protein sets Study of variations among the species Form of single nucleotide polymorphisms (SNPs) Single base-pair variations One per 1000 bp DNA TECHNOLOGY APPLICATIONS Disease Diagnosis Use PCR & labeled nucleic acid probes to detect pathogens (ex: HIV) Identification of harmful alleles before birth DNA TECHNOLOGY APPLICATIONS Human Gene Therapy Alteration of genes Replace defective gene with normal one put into cells that keep dividing Appears to be temporary Raises ethical questions DNA TECHNOLOGY APPLICATIONS Pharmaceutical Products Use vector DNA to create human insulin, HGH, TPA, etc Recombinant DNA to make vaccine without using actual pathogen DNA TECHNOLOGY APPLICATIONS Forensics Microsatellite DNA highly variable between individuals Called simple tandem repeats (STR) Environmental Genetically engineered microbes to degrade toxic waste DNA TECHNOLOGY APPLICATIONS Agriculture Transgenic organisms: carry genes from another species Makes “super” species Remove egg fertilize in vitro inject desired DNA into egg nuclei cell will grow & express gene egg put into surrogate DNA TECHNOLOGY APPLICATIONS Plants Vector is recombinant Ti plasmid – inserts into plant genome cell grows into complete plant Can increase nutritional value