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Recombinant DNA Technology

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  1. Recombinant DNA Technology Prof. Elena A. Carrasquillo Chapter 4 Molecular Biotechnology Lecture 4

  2. Cloning • in Plasmid Vector • In bacteriophage vector • Screening the library • DNA probe • Antibody probe Major Steps in building a DNA Library

  3. Genomic Library: Stores a representation of the genome (at least one copy of a gene present) • plasmid, bacteriophage, phagemid, • cosmid vectors • cDNA Library: Stores a representation of the mRNAs expressed at a certain time or stage by a microorganism or organism • plasmid, bacteriophage, phagemid, • cosmid vectors Kinds of Libraries

  4. Restriction Enzyme Mechanisms: • Preparation of DNAs to be joined • (a)Staggered cut: leaves “sticky ends” How is a Library Built:

  5. Restriction Enzyme Mechanisms: Preparation of DNAs to be joined: • (b) Blunt End How is a Library Built:

  6. Staggered “sticky ends” Ligation of DNA cut with a Restriction Enzyme

  7. Role of T4 DNA Ligase Ligation of DNA cut with a Restriction Enzyme

  8. Depends on the size of DNA to be cloned • Is the protein encoded by the DNA going to be expressed in a prokariotic or eukaryotic cell? Choosing the Vector

  9. Allows ordering of DNA fragments Restriction Enzyme Map

  10. We can then build maps of linear and circular molecules Restriction Enzyme Map

  11. a multiple cloning site or MCS an antibiotic resistance gene an origin of replication pBR322 is the basis of most engineered plasmids Plasmid: it’s a circular DNA molecule containing:

  12. Selectable Markers:

  13. F plasmids-transfer information from cell to cell • R plasmids-confer antibiotic resistance • Degradative plasmids-utilization of unusual metabolites • Cryptic plasmids-No apparent function Kinds of plasmids in the wild:

  14. Size range form less than 1 kb to more than 500 kb • Origin of replication-allows plasmid to replicate in the bacteria • Low-copy number: 1-4 per cell • High copy number: 10-100 per cell • Incompatibility groups: different kinds cannot be inside the same cell Other characteristics:

  15. Small size (<15Kb) for optimal efficiency of transformation in bacteria • Unique restriction enzime sites for cloning • One or more selectable genetic markers to allow for differentiation of the plasmids carrying the cloned DNA vs the religated ones. Characteristics of an engineered plasmid:

  16. Plasmid: a cloning vector or vehicle

  17. Characteristics: • Interruption of b-lactamase gene gives rise to white colonies (cloned DNA) vs blue ones (empty) pUC19 another plasmid cloning vector

  18. -Produced by site-directed mutagenesis to alter the DNA sequence but not the protein sequence of b-lactamase -This produced new restriction enzyme sites MCS: multiple cloning site of pUC19:

  19. Creation of a DNA Library: Partial DNA digestion Purpose: To produce overlapping DNA fragments

  20. Vary time of digestion or amount of enzyme units Progress of Reaction: agarose gel electrophoresis

  21. Screening a library:I. Colony hybridization or Southern Blot

  22. Method 1 • Random primers • Enzyme: Klenow Fragment + dNTPs • Non-radioactive: biotin or chemiluminescent labeled dNTPs -Radioactive:32P Preparation of DNA Probe

  23. Method 2: 5’-end labeling • Method 3: 3’end labeling Preparation of DNA Probe

  24. The polymerase (red) adds deoxyribonucleotides to the 3’- hydroxyl groups of the growing chains -The 5’ exonuclease (blue) removes succesive nucleotides from the 5’ phosphate ends -The 3’ exonuclease (yellow) removes succesive nucleotides from the3’ hydroxyl ends Klenow Fragment of E. coli DNA Polymerase I Enzyme Activities

  25. Colony Hybridization Screening a Genomic Library

  26. Procedure: Colony Hybridization:

  27. The probe is an antibody The colonies are grown so that the recombinant protein is expressed Screening a Genomic Library: II.Colony Immunoassay

  28. If chemiluminescense was used, light will be emitted and captured in an X-ray film Screening a Genomic Library: II.Colony Immunoassay

  29. Defective host cells (A-) are transformed with a genomic library derived from cells that are normal with respect to that function (A+) and grown in minimal media. • Those cells harboring a plasmid that corrects the defect will grow in minimal media. Screening a Genomic Library:III.Functional Complementation

  30. Used to obtain functional eukaryotic coding regions. • E. coli does not process introns. • First step: Isolate poly A+ mRNA with oligo (dT) cellulose Another Type of Library:cDNA Library

  31. Second Step:Synthesis of cDNA from mRNA of specific cells cDNA Library:

  32. Third Step:Selecting and Cloning Full length cDNA molecules cDNA Library:

  33. For cloning inserts of 10-20 Kb • Plasmid libraries hold up to 10 kb inserts Genomic Library: Bacteriophageλ (Lambda)

  34. Lytic Cycle:Production of progeny • Lysogenic Cycle: Integration into bacterial chromosome Genomic Library: Bacteriophageλ (Lambda)Life Cycle

  35. Genomic Library: Bacteriophageλ

  36. Genomic Library: Bacteriophageλ

  37. Allows cloning of 45 kb DNA fragments Cosmid library

  38. Based on P1 bacteriophage, the F plasmid and the lacZ region of pUC plasmids • It’s a low copy number plasmid • Carries 50-300kb fragments BACs: Bacterial Artificial Chromosomes

  39. BACs: Bacterial Artificial Chromosomes

  40. YACs:Yeast Artificial Chromosomes

  41. Transformation • Electroporation • Conjugation Methods of Introducing Foreign DNA

  42. Application of an electrical field to cells Electroporation

  43. Conjugation:Tripartite mating