Basic molecular biology and genetic engineering

1. Basic molecular biology and genetic engineering

2. DNA RNA Protein The central dogma

3. The big three molecules for genetics • DNA (DexoyribioNucelic Acid) • Heredity genetic information of an individual • Encode protein sequences (“genetic code”) • RNA (RiboNucleic Acid) • Transfer short pieces of information to cytoplasm • Provide templates to synthesize protein • Protein • Produced via “translation” of messenger RNA (mRNA) • Each protein has one or more specific functions • Form body’s major components • Carbohydrate and lipid metabolism mediated by proteins

5. Related terminology • Genome • an organism’s complete set of DNA. • Gene • basic physical and functional units of heredity. • specific sequences of DNA encode instructions on how to make proteins. • Chromosome • an organized structure of DNA and protein in the nucleus. • contains many genes and regulatory elements • Gene expression • protein product is being made via transcription and translation

6. DNA RNA Protein The central dogma • Proposed by Francis Crick in 1958 to describe the flow of information in a cell. • Information stored in DNA is transferred residue-by-residue to RNA which in turn transfers the information residue-by-residue to protein. • It has undergone numerous revisions.

7. The Central Dogma DNA RNA Protein Replication duplication of DNA using DNA as the template Transcription synthesis of RNA using DNA as the template Translation synthesis of proteins using RNA as the template

8. DNA & RNA Chains

9. The building blocks for DNA and RNA DNA Adenine (A) Cytosine (C) Guanine (G) Thymine (T) RNA Adenine (A) Cytosine (C) Guanine (G) Uracil (U)

10. DNA: The Basis of Life • Deoxyribonucleic Acid (DNA) • Double stranded with complementary strands A-T, C-G • DNA is a polymer • Sugar-Phosphate-Base • Bases held together by H bonding to the opposite strand

11. Chargaff’s rule A=T(U) and G=C

12. Semi-conservative DNA replication Daughter DNA molecules contain one parental strand and one newly-replicated strand DNA polymerase

13. DNA RNA Protein Residue-by-residue information transfer ATGAGTAACGCG TACTCATTGCGC (DNA) transcription replication (RNA) AUGAGUAACGCG ATGAGTAACGCG TACTCATTGCGC codon + translation ATGAGTAACGCG TACTCATTGCGC (protein) Met-Ser-Asn-Ala Codon: The sequence of 3 nucleotides in DNA/RNA that encodes for a specific amino acid.

14. Human genome are completely sequenced at 2003. What exactly does that mean?

15. Overview of organizations of life • Nucleus = bookshelf • Chromosomes = books • Genes = words • Nucleic acid = letters • We are still learning the meaning of the words • apple = - zxuriguhwefrhqjrnfg =? • Zombie = - manzano =?

16. Similar Known Unknown We know only small part of protein function Any genome

17. DNA RNA Protein Transcription • Making messenger RNA (mRNA) from part of DNA by RNA polymerase • Needs a promoter region to begin transcription.

18. Transcription closed promoter complex Transcription factors + RNA polymerase open promoter complex initiation elongation termination mRNA

19. Where to start Transcription? Different cells have different gene transcription pattern Promoter: A specific DNA sequence for RNA polymerase and transcription factors binding Different transcription factors recognizes different promoters

20. Exon and intron EXON: In protein coding genes,the segment of a gene that consists of a sequence of nucleotides that will be eventually translated into protein. INTRON: Non coding region of eukaryotic gene (transcribed into RNA than spliced) Translation Stop Transcription Stop +1-transcription Start Translation Start DNA Promoter Non - protein coding Intron Protein coding Exon

21. Annotation of eukaryotic genomes Genomic DNA transcription Unprocessed RNA RNA processing Mature mRNA Gm3 AAAAAAA translation Nascent polypeptide folding Active enzyme Function Reactant A Product B

22. Translation tRNA protein Ribosome mRNA

24. Biologic Roles of RNA Messenger RNA (mRNA)carries information from DNA to the ribosomes, and is used as template to synthesize protein. Transfer RNA (tRNA)is a small RNA that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site according to the sequence of a bound mRNA. Ribosome synthesizes polypeptides under the direction of mRNA

25. Mechanism of RNA interference (RNAi): post-transcriptional gene silencing shRNA, dsRNA

26. What is RNA interference (RNAi)? • RNAi – ability of dsRNA to silence gene expression in a sequence-specific manner • Induced by short interfering (si)RNAs • Mechanism: siRNA-directed cleavage of mRNA via RISC complex

27. Analyzing and manipulating a gene or genome

28. DNA or RNA blotting

29. What do we actually see? • Fluorescent chemistries-measurement by either: • Non-specific (SYBR green/EtBr) • Pros: inexpensive, no probe design • Cons: it reports ALL dsDNA formation not just the amplicon • Specific (probe) • Pros: increases specificity of the reaction, multiplex and mutation detection options • Cons: A bit more expensive, takes time to design probes, need a unique probe for each target

30. Hybridization • Single-stranded DNA or RNA will naturally bind to complementary strands. ATGAGTAACGCG TACTCATTGCGC DNA ATGAGTAACGCG UACUCAUUGCGC RNA

31. Probe hybridization Probe: a single-stranded DNA to detect the presence of a complementary sequence among a mixture of other singled-stranded DNA or RNA

32. Labeled DNA/RNA mixture flushed over array of short DNA fragments Laser activation of fluorescent labels DNA Microarrays

33. DNA Microarray • measuring the amount of mRNA bound to each probe on the array. Affymetrix array Each spot (~ 100um) indicates a probe

34. Overview GREEN represents Control DNA RED represents Sample DNA YELLOWrepresents a combination of Control and Sample DNA  BLACK represents areas where neither the Control nor Sample DNA .

35. Polymerase Chain Reaction • Goal: to amplify a low level DNA template for further analysis or manipulation • Need : • Primer: A set of single stranded DNA hybridize to the both end of target region • Taq polymerase: a thermostable DNA polymerase • Template DNA

37. Theoretical Log Target DNA Cycle # Is PCR quantitative? -A linear increase follows exponential -Eventually plateaus Taq polymerase has a half-life of 30 min at 95oC Reality

38. 50-0.005 ng of template- FV Leiden primers Real-time PCR Through the use of fluorescent molecules, real-time PCR allows us to ‘see’ the exponential phase so we can calculate how much we started with. The least the most

39. 5’ 3’ 5’ 3’ 5’ 5’ BD BD BD 5’ BD BD DNA binding dyes 5’ 3’ Annealing 5’ 3’ Taq 5’ BD BD 3’ Extension BD BD BD 5’ 3’ Taq Taq Extension continued apply excitation wavelength 5’ 3’ 5’ Taq 3’ Repeat

40. Emission Filter Detector Excitation Filter Light Source Fluorescence detection

41. What does “cloning” mean? Clone: a collection of molecules, cells or individuals, all identical to an original one • To "clone a gene" is to make many copies of it • Gene can be an exact copy of a natural gene • Gene can be an altered version of a natural gene • Recombinant DNA technology makes it possible

42. Recombinant DNA • taking a piece of one DNA, and combining it with another strand of DNA that would not normally occur together. • In order to do so, we need to - • Copy it (PCR) • Cut it (restriction enzyme digestion) • Paste it (ligation) E. coli vector Human gene

43. Restriction Enzymes • They cut the DNA at specific sequence. • Different restriction enzymes have different recognition sequences.

44. Pasting DNA • Two pieces of DNA can be fused together by adding DNA ligase • Hybridization – complementary base-pairing • Ligation – fixing bonds with single strands

45. Recombinant DNA Techniques

46. Genetic Engineering • To transport a specific segment of DNA from one organism to another Put human gene to E.coli

47. Introducing foreign into organism • The process whereby a DNA sequences are introduced by biologial, biochemical or physical processes. • Transformation - bacteria • Transfection – cell culture or virus • Transgenesis – animal • Biological: virus infection • Biochemical: DEAE-dextran, calcium phosphate, and liposome-mediated transfection methods. • Physical: direct micro-injection of materials, biolistic particle delivery and electroporation.

48. The Power of Recombinant DNA Technology – Human Insulin Production by Bacteria

49. and cut with a restriction enzyme 6) join the plasmid and human fragment Human Insulin Production by Bacteria

50. Mix the recombinant plasmid with bacteria. Human Insulin Production by Bacteria