1 / 13

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:.

liv
Download Presentation

Restriction enzymes (endonucleases)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related