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Overview

Overview. 5’…GAATTC…3’ 3’…CTTAAG…5’. 5’…G -OH P- AATTC…3’ 3’…CTTAA -P HO- G…5’. Restriction Endonucleases. A Restriction Endonucleases will cut both strands of a DNA duplex at a specific place These “places” need not be directly opposite:.

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Overview

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  1. Overview

  2. 5’…GAATTC…3’ 3’…CTTAAG…5’ 5’…G -OHP-AATTC…3’ 3’…CTTAA -PHO-G…5’ Restriction Endonucleases • A Restriction Endonucleases will cut both strands of a DNA duplex at a specific place • These “places” need not be directly opposite: 5’…GAATTC…3’ 3’…CTTAAG…5’ 5’…G -OHP-AATTC…3’ 3’…CTTAA -PHO-G…5’ • Note that the above enzyme is EcoRI, the first restriction endonuclease characterized

  3. Sticky Ends

  4. Ligation

  5. More RE Enzymes

  6. Most R.E. RecognitionSequences are Palindromes G^AATT-C C-TTAA^G G^GATC-C C-CTAG^G Nodeba Bob Abedon A^GATC-C T-CTAG^G GC^GGCC-GC CG-CCGG^CG

  7. Ligation into Plasmid Upon Ligation, we now have Recombinant DNA!

  8. Transformation

  9. Transformation Note that plasmid is vector that carries DNA into recipient cells Other vectors include viruses (transduction) as well as otherwise inert projectiles

  10. Cloning a Gene

  11. Agrobacterium tumefaciens

  12. Gene Therapy Alternatively, DNA can be cloned directly into the germ line…

  13. PCR DNA Amplification • Cloning allows the amplification of genotype (DNA) as well as phenotype (proteins) • If all you really need is the DNA, then PCR (polymerase chain reaction) is an easy way to amplify DNA without cloning

  14. PCR DNA Amplification

  15. Complementary (c)DNA

  16. Note that the reverse transcriptase is primed by the poly-A found at the end of most eukaryotic mRNAs Note that there are issues (& variation) as to how one makes the second DNA strand Here that strand is generated using a second enzyme that is primed via generation of a hairpin of DNA by reverse transcriptase Complementary (c)DNA

  17. Making a Phage Library

  18. Clone Identification • Probing for correct DNA sequence • Probing for correct protein product • Antibiotic resistance • β-galactocidase expression • Etc. • Once you have the correct clone, then what? • Subcloning • Expression vector • Protein characterization • Protein purification • Etc. Have Transformant, then What?

  19. Gel Electrophoresis (1/2)

  20. Gel Electrophoresis (2/2)

  21. Southern Blotting

  22. RFLP Analysis

  23. DNA Sequencing

  24. DNA Microarray

  25. Protein Characterization • Function • Isoelectric point • SDS PAGE (1D & 2D) • Western blotting • Epitope analysis (monoclonal antibodies) • Etc.

  26. Monoclonal Antibodies (Mabs) Note that though clonal, Monoclonal Antibodies are not (or at least used to be not) produced by processes that have anything to do with Gene Cloning

  27. Visualized proteins Western Blotting w/Mabs or w/Pabs

  28. Visualized proteins 2-D Protein Electrophoresis

  29. Online Tools • Buying primers (custom oligos): http://www.qiagen.com/ • 2x2 sequence comparisons: http://www2.igh.cnrs.fr/bin/align-guess.cgi • n x n sequence comparisons: http://www.genebee.msu.su/services/malign_reduced.html • Sequence manipulation: http://arbl.cvmbs.colostate.edu/molkit/manip/index.html • Sequence translation: http://arbl.cvmbs.colostate.edu/molkit/translate/index.html • Restriction Enzymes: http://rebase.neb.com/rebase/rebase.html • Restriction sites: http://www.ccsi.com/firstmarket/cutter/cut2.html • Sequence searches: http://www.ncbi.nlm.nih.gov/blast/ • Other tools: http://molbiol-tools.ca/

  30. Link to Next Presentation

  31. Acknowledgements http://www.bbchs.k12.il.us/Teacher_Pages/Hammond/Powerpoint/Chapter_20.ppt

  32. In Situ Hybridization

  33. Using Probes to Map DNA

  34. rep-PCR

  35. Full-Genome Sequencing

  36. Gene Mapping using RFLP

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