By: Mohsen Naeemipour

1. In the name of God Molecular Biotechnology By: MohsenNaeemipour

2. Timetable of Course • The Development of Molecular Biotechnology • DNA, RNA, and Protein Synthesis • Recombinant DNA Technology • Chemical Synthesis, Amplification, and Sequencing of DNA • Manipulation of Gene Expression in Prokaryotes • Heterologous Protein Production in Eukaryotic Cells • Directed Mutagenesis and Protein Engineering • Molecular Diagnostics • Protein Therapeutics • Nucleic Acids as Therapeutic Agents • Bioinformatics, Genomics, and Proteomics • Transgenic Animals

3. Lecture 4: Chemical Synthesis, Amplification, and Sequencing of DNA

4. Chemical Synthesis of DNA 4

5. Chemical Synthesis of DNA • Assembling whole genes or parts of genes • Amplifying specific DNA sequences • Introducing mutations into cloned genes • Screening gene libraries • Sequencing DNA • Facilitating gene cloning

6. Flowchart for the chemical synthesis of DNA oligonucleotides

7. Starting complex for the chemical synthesis of a DNA strand Spacer molecule controlled-pore glass (CPG) bead, dimethoxytrityl (DMT)

8. A Phosphoramidite Molecular

9. An anhydrous reagent (acetonitrile) Argon Trichloroacetic acid (TCA) (Detritylation) Acetonitrile Argon

10. Activation and coupling phosphoramidite tetrazole

11. Acetic anhydride and dimethylaminopyridine are added to acetylate the unreacted 5′ hydroxyl groups

12. The phosphite triester is oxidized with an iodine mixture to form the more stable pentavalent phosphate triester

13. Flowchart for the chemical synthesis of DNA oligonucleotides

14. Overall yields of chemically synthesized oligonucleotides with different coupling efficiencies

15. Uses of Synthesized Oligonucleotides

16. Typical linker and adaptor sequences

17. Cloning with a linker

18. Creating a restriction endonuclease site in a vector with an adaptor

19. Enzymatic DNA synthesis of a gene 19

20. Assembly of a synthetic gene from short oligonucleotides

21. Assembly and in vitro enzymatic DNA synthesis of a gene

22. Gene Synthesis by PCR

24. DNA-Sequencing Techniques 24

25. Blocked DNA synthesis

26. A. dideoxynucleotide B. deoxyribonucleotide

27. Primer extension during DNA synthesis in the presence of dideoxynucleotides

28. Simulated autoradiograph of a dideoxynucleotide DNA-sequencing gel

29. Automated fluorescent-dye terminator Sanger DNA sequencing

31. DNA sequencing by primer walking

32. Timetable of Course • The Development of Molecular Biotechnology • DNA, RNA, and Protein Synthesis • Recombinant DNA Technology • Chemical Synthesis, Amplification, and Sequencing of DNA • Manipulation of Gene Expression in Prokaryotes • Heterologous Protein Production in Eukaryotic Cells • Directed Mutagenesis and Protein Engineering • Molecular Diagnostics • Protein Therapeutics • Nucleic Acids as Therapeutic Agents • Bioinformatics, Genomics, and Proteomics • Transgenic Animals

33. Lecture 5: Manipulation of Gene Expression in Prokaryotes

34. Manipulation of Gene Expression • Promoter and transcription terminator sequences • Strength of the ribosome-binding site • Number of copies of the cloned gene • Gene is plasmid borne or integrated into the genome of the host cell • Final cellular location of the synthesized foreign protein • Efficiency of translation in the host organism • Intrinsic stability within the host cell of the protein • encoded by the cloned gene.

35. Regulatable Promoters • lac and trp (tryptophan) operons • promoters are commonly used hybrid constructs • the ratio of the number of repressor protein molecules to the number of copies of the promoter sequences • two different plasmids repressor gene is placed on a low-copy-number (1-8) high-copy-number plasmid (30-100)

36. Regulation of gene expression controlled by the pL promoter

37. A portion of the DNA sequence of the E. coli lac promoter (plac) and its mutated, more active, form (pmut).

38. pPLc2833 plasmid + pKN402 pCP3 vector Increasing Protein Production

39. Dual-plasmid system for controlling the λ pL promoter by regulating the cI repressor with tryptophan Large-Scale Systems

40. Uses of Fusion Proteins

41. Some protein fusion systems used to facilitate the purification of foreign proteins in E. coli and other host organisms

42. Schematic representation of the genetic construct used to produce a secreted fusion protein • reducing the degradation • enabling the product to be purified

43. Immunoaffinity chromatographic purification of a fusion protein

44. Purification of a protein

45. Surface Display

46. outer membrane protein A (OmpA). peptide-glycan-associated lipoprotein (PAL) from E. coli, Pseudomonas aeruginosa outer membrane protein F (OprF).

47. Translation Expression Vectors A ribosome-binding site is a sequence of 6 to 8 nucleotides (e.g., UAAGGAGG) in mRNA

48. Rarely codons used by the host cell • If the target gene is eukaryotic, it may be cloned and expressed in a eukaryotic host cell • (2) A new version of the target gene containing codons • that are more commonly used by the host cell may be chemically synthesized (codon optimization) • (3) A host cell that has been engineered to overexpress several rare tRNAs may be employed

49. Increases in gene expression that result from altering the codon usage of the wild-type gene (or cDNA) to more closely correspond to the host E. coli cell

50. Overexpress several rare tRNAs the Ara h2 protein, approximately 100-fold over the amount that was synthesized in conventional E. coli cells AGG, AGA, AUA, CUA, and CGA