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Steven Short

BIO215 lecture 3 Today: • restriction endonucleases • electrophoresis serial dilutions can also be used to determine cell concentrations! from: Brock Biology of Microorganisms, 11 edition. Figure 6.11. Bacterial Growth • requires nutrients and specific physical conditions • determining growth stage is important in molecular biology – e.g., for nucleic acid isolations • need to know how bacteria grow, and how growth can be measured Bacterial Growth = Cell Division • aka: binary fission • cells divide into 2 daughter cells • # of cells double each generation Exponential Growth cell number versus time: standard and semi-log plots exponential growth shown by linearity of semi-log plot figures from: Brock Biology of Microorganisms, 11 edition. chapter 6. Bacterial Growth Phases refers to populations, not individuals! from: Brock Biology of Microorganisms, 11 edition. Figure 6.8. Spectrophotometric Measurement Photocell Photocell Nucleases are enzymes that cleave phosphodiester bonds between nucleotides of nucleic acids Exonucleases: cut nucleic acids from the ends of the molecule Endonucleases: cut nucleic acids internally restriction endonucleases are also called restriction enzymes Restriction Endonucleases What are they?  enzymes that cut dsDNA at specific DNA sequences they recognize  “smart DNA scissors” Why are they important?  Nobel Prize in Medicine awarded in 1978 for their discovery  key discovery for the development of recombinant DNA technology Restriction Endonucleases Where do they come from? • bacteria evolved RE for protection from phages • restrict the growth and replication of the phages • but, host DNA must be protected from restriction! DNA Restriction by Type II REs DNA Methylation Blocks RE activity Restriction Endonucleases 3 classes of restriction endonucleases:  Type I: require ATP, cut away from recognition  Type II: most useful for mol. biol., no ATP required, cut at recognition site  Type III: characteristics similar to Type I RE Nomenclature & Conventions e.g., HindIII: Haemophilus influenzae strain RD by convention, recognition sequences are written as 5’ to 3’, with 5’ on the left of the top strand EcoRI:G AATTC Properties of Type II REs Restriction sites are generally palindromic 5’-GAATTC
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