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Restriction enzymes (endonucleases)

Restriction enzymes (endonucleases). Cleave a specific DNA sequence Protect bacteria from phage infection by digesting phage DNA after infection. Cellular DNA is protected by methylases - block restriction enzyme activity. Each organism has a specific set of restriction enzymes:.

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Restriction enzymes (endonucleases)

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  1. Restriction enzymes (endonucleases) • Cleave a specific DNA sequence • Protect bacteria from phage infection by digesting phage DNA after infection. • Cellular DNA is protected by methylases - block restriction enzyme activity

  2. Each organism has a specific set of restriction enzymes: • EcoRI from Escherichia coli • BamHI from Bacillus amyloliqueraciens • PvuI and PvuII are different enzymes from same strain. Ch. 3-1

  3. Restriction enzymes are used for cloning and analyzing DNA fragments

  4. Sequence Recognition and cleavage: a) 5' overhang EcoRI GAATTC G AATTC CTTAAG CTTAA G b) 3' overhang KpnI GGTACC GGTAC C CCATGG C CATGG c) Blunt end SmaI CCCGGG CCC GGG GGGCCC GGG CCC Ch. 3-2

  5. Sticky ends • The overhangs on cleaved DNA can serve as “sticky ends” or unpaired bases that can be used to link pieces of DNA. • Use the same enzyme, or one that leaves the same overhang to cut two DNA sources. • Complementary bases will pair. • Ligase will seal.

  6. Vectors In order to study a DNA fragment (e.g., a gene), it must be amplified and eventually purified. • Do this by cloning the DNA into a vector, generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli. Ch. 1-1

  7. Cloning Scheme Digest Ligate Amplify and Prep 1-1

  8. Vector Types There are three commonly used types of vectors: 1) plasmid vectors (e.g., pUC plasmids) • These are the most common in biotechnology 2) bacteriophage vectors (e.g., phage ) 3) phagemid (hybrid)vectors Each has a different use, and there are many derivatives of these basic building blocks. Ch. 1-1

  9. Plasmids • Circular DNA molecules found in bacteria • Replicated by the host’s machinery independently of the genome. This is accomplished by a sequence on the plasmid called ori, for origin of replication. • Some plasmids are present in E. coli at 200-500 copies/cell

  10. Plasmid Engineering • Plasmids also contain selectable markers: • These are genes encoding proteins which provide a way to rapidly and easily find bacteria containing the plasmid. • Commonly- provide resistance to an antibiotic like ampicillin. • Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. Ch. 1-2

  11. Safety Features • Modern cloning plasmids have been engineered to be incapable of transfer between bacterial cells • Provide a level of biological containment. • Naturally occurring plasmids with their drug resistance genes have produced antibiotic-resistant bacteria. Ch. 1-2

  12. Transforming plasmids Into bacteria Ch. 1-2

  13. Screening for Inserts • Transform bacteria with plasmids containing gene for ampicillin resistance; small number will transform. • Spread bacteria on plates containing nutrient agar and ampicillin. • Only transformed cells will survive and form colonies. 1-3

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