G ENETIC T ECHNOLOGY

1) 1) G ENETIC R ECOMBINATION 1. Remove bacterial plasmid with restriction enzymes 2. Add in gene of interest (plasmid is now recombinant DNA molecule) 3. Put back into bacteria 4. 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

R ESTRICTION E NZYMES 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

R ESTRICTION E NZYMES Single strands will hydrogen bond with other complementary “sticky ends” Bonds made permanent with DNA ligase Now we have recombinant DNA

F ROM R ESTRICTION E NZYMES TO P LASMID M APS PM show how different REs act upon a plasmid Pictorial representation of the different lengths of pieces remaining after the REs worked

F ROM R ESTRICTION E NZYMES TO P LASMID M APS 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 A NALYSIS - G EL E LECTROPHORESIS 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) P OLYMERASE C HAIN R EACTION 3) P OLYMERASE C HAIN R EACTION (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) G ENOME A NALYSIS DNA microarrayDNA microarray: ssDNA fragments fixed to slide that are then labeled with fluorescent cDNA Compare genes of species  attempt to uncover gene functiongene function

5) G ENOME A NALYSIS - G ENE F UNCTION To determine, turn gene off – see what happens To turn off: RNA interference (RNAi)RNA interferenceRNAi Synthetic ds RNA matches gene sequence – binds to mRNA Triggers breakdown of mRNA  no protein made Remove to turn on again

T HE F UTURE 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 T ECHNOLOGY A PPLICATIONS Disease Diagnosis Use PCR & labeled nucleic acid probes to detect pathogens (ex: HIV) Identification of harmful alleles before birth

DNA T ECHNOLOGY A PPLICATIONS 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 T ECHNOLOGY A PPLICATIONS Pharmaceutical Products Use vector DNA to create human insulin, HGH, TPA, etc Recombinant DNA to make vaccine without using actual pathogen

DNA T ECHNOLOGY A PPLICATIONS Forensics Microsatellite DNA highly variable between individuals Called simple tandem repeats (STR) Environmental Genetically engineered microbes to degrade toxic waste

DNA T ECHNOLOGY A PPLICATIONS 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 T ECHNOLOGY A PPLICATIONS Plants Vector is recombinant Ti plasmid – inserts into plant genome  cell grows into complete plant Can increase nutritional value