1 / 25

Bacterial cloning

Learn about the process of bacterial cloning to amplify DNA and create identical clones, through the use of plasmid vectors, PCR products, and restriction endonucleases. Understand the role of restriction enzymes and their applications in cloning, DNA storage, and genotyping.

williamsv
Download Presentation

Bacterial cloning

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. Bacterial cloning Especially of PCR product DNA

  2. PCR recap

  3. PCR gel product

  4. Gel 1 and 2 Gel 1 and 2 Gel 3 and 4 Gel 3 and 4

  5. Cloning • Cloning is the way in which we can take a single molecule, and make lots of bacterial cells that contain an identical molecule. • These cells are clones, hence the name • This used to be the only way to amplify DNA. It is still by far the most accurate.

  6. Plasmid vectors – circular, autonomous bacterial DNA

  7. Cloning PCR products • When we amplify DNA using PCR, it is often necessary to “clone” this DNA • We do this in order to replicate it without errors • Also, by cloning a protein coding sequence into E. coli, we can then produce the protein in the bacterium.

  8. The vector is made with a “T” overhang

  9. Taq polymerase leaves an “A” overhang • Taq is the thermostable DNA polymerase from Thermus aquaticus we used for PCR. • When Taq synthesizes a new strand, it always puts an extra “A” at the end • This can be useful, but note: other polymerases do not do this, only Taq polymerase

  10. Restriction Endonucleases Also called restriction enzymes 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA 3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially

  11. Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: ROrder discovered: 1

  12. “Able was I, ere, I saw Elba” 5’-GGATCC-3’ 3’-CCTAGG-5’ Bam H1 site: Recognition sites have symmetry (palindromic)

  13. Restriction enzymes recognize specific 4-8 bp sequences Some enzymes cut in a staggered fashion - “sticky ends” EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’ Some enzymes cut in a direct fashion – “blunt ends” PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’

  14. Why don’t bacteria destroy their own DNA with their restriction enzymes?

  15. Methylation

  16. Uses for Restriction Enzymes RFLP analysis (Restriction Fragment Length Polymorphism), CAPS (Cleaved Amplified Polymorphic Sequence) markers Cloning and construction of transgene “cassettes” DNA storage – libraries Genotyping by sequencing (“GBS”).

  17. Complementary base pairing 5’-A-C-G-G-T-A-C-T-A-GA-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’ + DNA Ligase, + rATP 5’-C-G-G-T-A-C-T-A-G-OH 3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4 5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’ recombinant DNA molecule Restriction Enzymes for Cloning Bacterial DNA cleaved with EcoRICorn DNA cleaved with EcoRI + PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’ HO-G-T-C-G-A-T-G-C-5’

  18. Electroporation

  19. When the electric field is applied, • the ions move according to their • charge. • ii) Pathways are formed across the • cell membrane allowing DNA • to enter. • iii) When the electric field is • deactivated, the membrane heals.

More Related