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Molecular Biology

Molecular Biology. Working with DNA. Topics. Genomic vs. Vector DNA Purifying plasmid DNA Restriction enzymes R estriction maps. DNA. Genomic Prokaryote vs. eukaryote Circular or linear One or more chromosomes Extra-genomic Vectors Plasmids. Vectors Vs Plasmids. Vector:

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Molecular Biology

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  1. Molecular Biology Working with DNA

  2. Topics • Genomic vs. Vector DNA • Purifying plasmid DNA • Restriction enzymes • Restriction maps

  3. DNA • Genomic • Prokaryote vs. eukaryote • Circular or linear • One or more chromosomes • Extra-genomic • Vectors • Plasmids

  4. Vectors Vs Plasmids • Vector: • DNA vehicle that allows the cloning, maintenance and amplification of a DNA sequence • Plasmids • Virus • Chromosomes • All plasmids are vectors • Not all vectors are plasmids

  5. Plasmids • Small circular DNA molecules maintained and amplified in eukaryotic or prokaryotic cells • Amplification in bacteria • Used as vector for cloning or expression of DNA of interest

  6. Characteristics of plasmid vectors • Restriction sites for cloning • Origin of replication (Ori) • Selection marker • Genes conferring resistance to antibiotics

  7. DNA Isolation • Goals • Isolation of DNA of interest • Chromosomal or plasmid? • Eliminate other components • Chromosomal or plasmid DNA? • Proteins • RNA • Chemicals • Salts, detergents, etc.

  8. DNA isolation (cont’d) • Cell lysis • Cell wall and membrane • Enzymatic • Chemical • Mechanical • Isolation of DNA of interest • Differential sedimentation • Chromatography • Removing other components • Enzymatic • Differential sedimentation • Chromatography

  9. Plasmid DNA isolation by alkaline lysis (E.coli )

  10. Solutions Used • Sol. I – Resuspension buffer • TrisHCl – Buffer that protects nucleic acids • EDTA - Chelates Mg++, prevents nucleases from working • Sol. II – Lysis solution • NaOH - ^pH lyses cells, denatures DNA • SDS – Dissolves membranes, denatures and binds proteins

  11. Solutions Used (Cont’d) • Sol. III- Potassium acetate • Renaturation of DNA • Precipitates SDS • Precipitates genomic DNA and proteins • Isopropanol / Ethanol • Precipitates nucleic acids (plasmid and ?) • Salts remain soluble • TE-RNase - Tris & EDTA again; RNase??

  12. Quantification of DNA • Determining Conc. of DNA • A260 of 1.0 = 50µg/mL or 50ng/µL • Determining Amount of DNA • 1mL of a solution with an A260 of 1.0 contains 50µg DNA • 1µL of a solution with an A260 of 1.0 contains 50ng DNA • Do not forget to account for the DILUTION FACTOR

  13. Restriction enzymes • Endonuclease • Cleaves internal phosphodiester linkages. • Recognize specific double stranded DNA sequences • Different endonucleases recognize different sequences • Recognize palindrome sequences

  14. Palindromes • The same sequence is read in the 5’ » 3’ direction on both strands 5’-G G A T C C-3’ 3’-C C T A G G-5’

  15. 5’-G G A T C C-3’ 3’-C C T A G G-5’ • The same phosphodiester linkages are cleaved on both strands!

  16. 5’-G G A T C C-3’ 3’-C C T A G G-5’ Different ends are generated Blunt ends

  17. 5’-G G A T C C-3’ 3’-C C T A G G-5’ Different ends are generated 5’ overhangs

  18. 5’-G G A T C C-3’ 3’-C C T A G G-5’ Different ends are generated 3’ overhangs

  19. Blunt ends HO O OH P P P P O Compatibility of ends Compatible

  20. Overhangs HO HO OH O P P P P Incompatible Compatibility of ends

  21. Overhangs GATC-P GATC-P HO P-CTAG OH O O P-CTAG Compatibility of ends Annealing Compatible

  22. Overhangs GATC-P GATC-P HO P-TCCA OH OH HO P-TCCA Compatibility of ends Annealing Incompatible

  23. Restriction Maps

  24. Restriction maps • Determining the positions of restriction enzyme sites • Linear DNA maps • Circular DNA maps (plasmids) • Maps of inserts withinvectors

  25. Approach • Determinewhether the DNA has digested • Is the digestion complete or partial? • How manycuts? • Determine the relative positions

  26. Is the DNA digested? • Compare to the undigested control • Whichsampleswere not digested? • 1 and 4 • Whichsamplesweredigested? • 2 and 3 Ladder Control 1 2 3 4

  27. Is the digestion complete? • Complete digestion • All the DNA molecules are cleavedat all the possible sites • Partial digestion • A fraction of the molecules are not digested • Partial undigested • A fraction of the moleculesweredigested, but not at all the possible sites • Partial digestion

  28. Complete digestion Digestion

  29. Partial digestion: Partial undigested Non digested Digestion

  30. Partial digestion partial Digestion partial

  31. Is the digestion complete or partial? • Compare to control • Verify the intensity of the bands • Verify the sizes Ladder Control 1 2 3 4

  32. How manycuts? • Number of sites • Circular DNA = number of bands • Linear DNA = Number of bands – 1 • Determine the relative positions • The fragment sizes represent the distances between the sites

  33. Linear DNA maps 7.0 HindIII 4.0 3.0 HindIII + SalI 3.0 2.0 2.0

  34. Circular DNA maps (plasmids) 7.0 1.0 1.0 2.5 1.5 10.0 10.0 2.0 9.0 3.0

  35. Insertion maps Insertion site MCS MCS Recombinant plasmid Vector

  36. Approach • Determine the total size • Determine size of the insert • Total size – size of vector • Determine the insertion site within the MCS • Determinewhich enzymes cutwihin the insert • Relative mapping in relation to the sites atknown positions

  37. Insertion maps • Total size • 7.7Kb • Insert size • 7.7 – 2.7 = 5.0Kb • Insertion site • Generates 2 fragments of which one is the size of the vector • XbaI

  38. Insertion maps Sites to map

  39. Map of PstI : 2 and 5.7Kb 5.7 Kb 2.0 Kb 5.0

  40. Map of EcoRI: 1, 3 and 3.7Kb P 3.7 1.0 3.0 1.0 1.0 3.0

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