Chapter 5 Exploring Genes and Genomes

1. Chapter 5 Exploring Genes and Genomes

2. Basic Essential Tools for Gene Exploration • Restriction Enzymes : Precise Sequence-Specific Cleavage of DNA • Blotting Techniques : Southern and Northern Blotting • DNA Sequencing : Determination Precise Nucleotide Sequence of DNA (human 3 billiion base-pairs) • Solid-Phase Synthesis of Nucleic Acids : Oligo(deoxy)nucleotide • Polymerase Chain Reaction (PCR) : Amplification of a segment of DNA (Nobel Prize in Chemistry 1993 Dr Kary B. Mullis, U.S.A., for his invention of the polymerase chain reaction (PCR) method) • Computerized Analysis : Bioinformatics

3. Palindromic Inverted repeat, Usually 4-8 base pairs Recognition Site for Sac I (Streptomyces achromogenes) Restriction Endonuclease (Restriction Enzyme) • Found in a variety of prokaryotes. • Foreign DNA cleavage. • Self DNA protection by methylation • Sequence-specific cleavage • Hydrolysis of a phosphodiester bond • Palindromic sequence recognition • Staggered or even cutting • 1978 Hamilton Smith, Daniel Nathans • Rule of Naming Restriction Enzymes • First three letters for the host organism (e.g. Hin for Haemophilus influenza) • Next one letter for strain designation, if necessary (e.g. HinD for Haemophilus influenza D) • Last one letter for a number, if more than one restriction enzyme have been identified from the same species. (e.g. HinD III for the third RE found in Haemophilus influenza D)

4. Non-Denaturing Gel Electrophoresis Systems For < 1 k bps : Polyacrylamide Gel Electrophoresis For < 20 k bps : Agarose Gel Electrophoresis Pulsed-Field Gel Electrophoresis (PFEG) can separate DNA molecules in the range of million base pairs. Denaturing Gel Electrophoresis Systems Urea Polyacrylamide Gel can distinguish DNA fragments differing in length just by one nucleotide out of several hundred base pairs EtBr (ethidium bromide) Staining of Nucleic Acids; intercalation into base stacks; fluorescence upon UV light; 50 ng of DNA is detection limit Visualization of DNA Fragments by Gel Electrophoresis

5. Ethidium bromide intercalating agent commonly used for nucleic acidstaining may be a mutagen, carcinogen

6. Denaturation By formaldehyde Hybridization b-rays from radioisotope Southern Blotting for DNA & Northern Blotting for RNA Hybridization Probes for Southern Blots : single-stranded DNA for Northern Blots : single-stranded mRNA

7. Partial termination of DNA polymerase reaction by including dideoxy nucleotides • Urea Polyacrylamide Gel Electrophoresis • Visualized by autoradiography or fluorescence Sanger Di-Deoxy Method for DNA Sequencing

8. Automated Solid-Phase DNA Synthesis Deoxyribonucleoside 3’-phosphoramidite : Incoming Nucleotide DMT : Dimethoxytrityl Group ; Protect 5’ hydroxyl group of incoming nucleotide bCE : b-Cyanoethyl Group ; Protect 3’ phosphoryl oxygen All bases are protected during synthesis. Phosphotriester is oxidized by iodide to make phosphodiester. Dichloroacetic acid removes DMT to generate a free 5’ hydroxyl group. Each cycle can be completed within 10 min and the efficiency is better than 98%. At the end of the synthesis, NH3 is added to remove all the protecting groups.

9. Required Components • Pair of primers that hybridize the target • All four deoxyribonucleotides (dNTPs) • A heat stable DNA polymerase • Target sequence (template) • Cycling Reactions • Strand Separation (Denaturation): 95 • Hybridization of Primers (Annealing): 54 • DNA Synthesis (Extension): 72 • Repeats the above three steps Polymerase Chain Reaction (PCR) : Amplification of Specific DNA Sequences

10. Parent DNA annealing?

11. Theoretical PCR Amplification Fold = 2n (n : cycle number) Thus, Million Fold Amplification after 20 Cycles, and Billion Fold Amplification after 30 Cycles

12. PCR machine PCR Application 1. Medical Diagnostics - Detection of pathogen (bacteria and virus) - Detection of cancers (mutations of ras genes) 2. Forensics - Some genes are highly variable within a population (human leukocyte antigen type, HLA) 3. Molecular Evolution - DNA is very stable and remain intact for thousands of years or longer, particularly when shield from air, light and water

13. Vector • Autonomously replicable DNA in an appropriate host. • Delivering vehicle for the gene of interest into a host • Insert • DNA fragment of the gene of interest • Restriction Enzyme • To generate joinable DNA fragments • Ligase • To join the cut DNA fragments • Linker • Small DNA fragment containing restriction enzyme sites • Can be attached to any DNA fragment by a ligase and cut by a particular restriction enzyme to generate specifically desired cohesive ends

14. Useful as a cloning site • Plasmids • Naturally occurring circular DNAs acting as accessory chromosomes (small extra chromosomes) found in bacteria; episome, disposable • Plasmids can be autonomously replicated in bacteria independently of the host chromosome. • Plasmids utilized in molecular cloning in the laboratory usually contain cloning sites and selection markers (e.g. antibiotics resistance genes).

15. Plasmids • Evolved… • Polylinker Region •  More Cloning Sites • Further Selection for Recombinants •  X-gal Color Selection • Stronger Replication Origin •  More DNA Synthesis

16. Bacteriophage l Inserts up to 10 kb • Cosmid : A Circular Vector with Bacteriophage Backbone  Inserts up to 45 kb (hybrid of plasmid/ l phase) • BAC : Bacterial Artificial Chromosome •  inserts up to 300 kb • YAC : Yeast Artificial Chromosome •  inserts up to 1000 kb arms Bacteriophage l

17. YAC Chromosome Walking Efficient Analysis of Long Stretches of DNA

18. Construction of l-Phage Genomic Library Cloning of a Gene from Genomic Library Genomic Library Screening with a Radioactive Probe Containing the Sequence of the Gene of Interest 32P-labeled probe

19. Deduce the Possible Nucleotide Sequences from Amino Acid Sequence  Synthesize Oligonucleotides Containing Corresponding Sequences Make cDNA from mRNA by using Reverse Transcriptase  PCR using cDNA Templates and Specific Primers for the Gene of Interest How to Make a Specific Probe for Library Screening ?

20. cDNA Library Screening Poly(A) mRNA Isolation using Oligo(dT) Column Synthesis of Double-Stranded cDNA (Total cDNA) Linker Ligation Cloning of Total cDNA into Plasmid (or Bacteriophage Vectors) Transform (or Infect) Host Bacteria Obtain Pool of Bacteria (or Bacteriophage) cDNA Library cDNA Library Construction

21. Deletions : Single Cleavage by RE •  Exonuclease Digestion  Ligation • Substitutions : Oligonucleotide-Directed Mutagenesis • Insertions : Cassette Mutagenesis • Designer Genes : Chimeric Proteins • (cf. Immuno-Toxin Therapy for Cancer) Mutation of DNA  Generation of New Proteins Digestion of template strand by Dpn1 enzyme

22. Classical Random Mutant Screening (Forward Genetics) vs. Generation of a Specific Mutant by Design (Reverse Genetics)

24. Complete Genome Sequencing Gene # ? Efficiency ? Comparative Genomics Evolution ?

25. Labeling Total cDNAs from Sample 1 with Green Fluorescence • Labeling Total cDNAs from Sample 2 with Red Fluorescence • Mix Two Pools of cDNAs • Hybridize against the Gene Chip • Variations in Gene Expression Profile Can Be Visualized. Breast Cancer Specific Changes in Gene Expression Comprehensive Gene Expression Analysis Using Microarray Figure out!

26. Animation! http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

27. Expression of Foreign Genes in Host Cells • +oligo T primer • High pH • Terminal transferase, add dG to 3’ +synthetic linker Expression of a Gene of Interest from Genomic DNA ??? Introns… Splicing Apparatus Is Absent in Prokaryotes… Introns Are Already Removed in cDNAs Made from Mature mRNAs

28. Necessity : Post-translational Modification • Deficiency in Bacteria • Transfection • Calcium Phosphate Precipitation Liposome-Mediated Vesicle Fusion • (cf. Stable vs. Transient Transfection) • Micro-Injection into the Nucleus • Viral Infection • DNA Virus : Adenovirus, etc. (Transient Expression) • Retro-Virus : Moloney Murine Leukemia Virus (MMLV), • Lenti-Virus, etc. (Stable Expression) • Vaccinia Virus : Viral DNA Replication in the Cytoplasm • Shut Down Host Protein Synthesis • Baculovirus : Infection into Insect Cells (Sf9), • Efficient Protein Production Factory Expression of Foreign Genes in Mammalian Cells

29. Growth Hormone Deficiency  Dwarfism Growth Hormone Excess  Gigantism Transgenic Animals Can Express Exogenous Genes of Interest • Metallothionein: heavy metal binding protein with many cysteins • Growth hormone induction (500x) by adding cadmium into drinking water • Microinjection of several hundred copies of GH expressing plasmid into the male pronucleus of a fertilized mouse egg  insertion into the foster female uterus • Check by southern blotting

30. (or Markers) Myogenin(+/+) Mouse Muscle Targeted Gene Disruption by Homologous Recombination Myogenin(-/-) Mouse Muscle Gene Disruption in Animal (Knock-Out Animal) Gene Knock-Out (or Knock-In) Technology Has Enabled Us to Address In Vivo Functions of a Gene of Interest at the Level of a Whole Organism.

31. Gene Expression Knockdown by RNA Interference • Double stranded RNA cleavage by Dicer • 21 nucleotide double stranded RNA (siRNA : short interfering RNA) • Recognition by RISC • (RNA Induced Silencing Complex) • Hybridization with mRNA • mRNA cleavage • C.elegans, E.coli

32. Ti (Tumor Inducing) Plasmids Can Deliver Foreign Genes into Plants Dicot, monocot A small synthetic plasmid carrying the gene of interest in T-DNA fragment can be added to Agrobacterium colonies harboring naturally occurring Ti plasmids, then, by recombination, recombinant Ti plasmid can be generated. Crown Gal Plant Tumor Caused by Agrobacterium tumafaciens

33. More Methods for Gene Transfer into Plants • Electroporation • Make protoplast by using cellulase • High electric pulse makes membranes transiently permeable. • Plasmid DNA can enter the cells. • Marker selection • Gene Guns • DNA is coated onto 1-mm-diameter tungsten pellets • These microinjectiles are fired at the target cells with a velocity greater than 400 m s-1. GMOs in wheat, soybean, corn, and rice; most efficient way

34. Mutual Re-Enforcement of Protein and Nucleic Acid Chemistry