HC70A & SAS70A Winter 2011Genetic Engineering in Medicine,Agriculture, and LawProfessors Bob Goldberg & John HaradaLecture 4The Nuts & Bolts of GeneticEngineering:The Factor VIII Story -From Gene To DrugCourse AdministratorpTHEMES1. Continuation of How Do Genes Work - Parts One and Two2. What is Hemophilia?3. How Is Hemophilia Inherited?4. What is the Pedigree Pattern of a Sex-Linked Gene?5. How Find a Disease Gene When It is Not Known Wherethe Gene is Expressed?6. What Vectors Can Be Used For Cloning DNA?7. What Are the Advantage of Using a Virus Vector ForConstructing Genome Libraries?8. How Make a Library of the Human Genome?9. How Find a Gene With Only a Knowledge of the ProteinSequence?10. What is Chromosome Walking & What Role Did it Play inCloning the Factor VIII Gene?11. How Use DNA Testing to Detect Factor VIII DiseaseAlleles?12. How Isolate a Factor VIII cDNA Clone?13. How Produce Factor VIII Protein For Use as a Drug?How Do Genes Work (Lecture 3 Continued)! Replication2 Gene Activity toFunction &PhenotypeGene ActivityProteinFunctionPhenotype(Trait)A Gene is NOT Expressed Unless A FunctionalProtein Produced!Function ¢TraitDNA Replication Occurs Semi-Conservatively1. DNA Structure Allows DNA Sequence to Be Maintainedby Complementary Base Pairing2. Each Strand Serves as a Template for the Synthesis ofa Complementary Strand3. New DNA Molecules are Precise Copies of Parental DNA- Each Containing One Newly Synthesized ComplementaryStrandMeselson & Stahl, PNAS, 1957PCR is A Cyclical Process of DNA ReplicationRepeat Steps or CycleDNA Fragments All The Same SizePrimer-Sequence-PrimerDiagnostic For AmplifiedDNA Sequence2n Moleculesof DNAwhere n =Number ofCyclesRequires1. Template2. Primers3. Knowledgeof SpecificSequence4. Nucleotides5. Heat-StableDNAPolymerase6. CyclerUsing PCR in Crime ScenesDNA Doesn’t “Lie” !!Suspect VictimsCrimeScene“Match”What isProbabilityThat ThisWill Occurby Chance?OS = SuspectCS = Crime SceneRG & NB = VictimsHow Do Genes Work - Mutations!! ReplicationMistakes?2 Gene Activity toFunction &PhenotypeGene ActivityProteinFunctionPhenotype(Trait)A Gene is NOT Expressed Unless A FunctionalProtein Produced!Function ¢TraitNew Base PairC mispairs with ADNA Replication is PreciseBut Mistakes or Mutations Can Occur!!Gene AReplication1Replication2Gene A’Allelic VariantRare BaseMismatchChange DNA Sequence From A = T to G C∴ Change Protein Amino Acid Sequence ➩ Alter Function!Big Tomato to Small TomatoSeeMutation AsChange inPhenotypeMutation in Genes Are RareBut Are Inherited (1 out of 107 replications)One Gene PerGamete ♀+ ♂Two Genes perSomatic CellsMutation in theCystic Fibrosis GeneHow Follow Inheritance?What Allows Disease To Be Followed?DNA Marker or Fingerprint!Spontaneous Mutations Give Rise To Alleles, orDifferent Forms of the Same Gene, And result inSmall DNA Sequence Changes (e.g., SNPs or SingleNucleotide Polymorphisms)AllelesCan Follow TheseTraits With DNAMarkers As WellMutations Can Occur Different Ways1. Base-Pair Change2. Insert or Delete Base(Indel)3. Move Gene, or Partof Gene, to NewLocation (SwitchesChange)!Function ofProteinLost and/orChanged∴PhenotypeChangesHuman Genetic Disorders Occur As a Result of MutationsDominantRecessiveNeed One Allele Need Two AllelesNature, October 10, 2010• Sequenced Genomes of ~900 individuals• From Seven Different Global Populations• Found 250-300 Loss-Of-Function Mutations (KOs) Per Person• 10-8 bp Mutations per Generation (30 per Genome)Pedigrees Can Be Used To Follow Disease Genes in Human FamiliesFollowedByBleedingPhenotypeRecessive Sex LinkedPedigrees Can Be Used To Determine Ifa Trait is Dominant or RecessiveEach Type of Inheritance PredictsSpecific Results in Each GenerationMuscular DystrophyHuntington DiseaseSickle Cell AnemiaCystic FibrosisTay-Sachs DiseaseDominantRecessiveGenetic Diseases Can Be Followed in Families UsingMolecular Methods (e.g., DNA Blots or PCR)DNA FingerprintsPCR Can Be Used To Analyze Genes During PregnancyPCR Can Be Used To Analyze Mutant Gene in ASingle Embryo Cell Before PregnancyWhat is TheImplication ofThis ProcedureConsidering ThatThe HumanGenome Has BeenSequenced?PGDPre-ImplantationGeneticDiagnosisSex Determinationin 8-cell Embryo!“Mutations” Can Also Occur ByLarge Chromosomal ChangesThese changes affect many genes!e.g. Down’s Syndrome (3 Chromosome #21s)A Down’s Syndrome KaryotypeThree Chromosome# 21sIncreases With Ageof MotherHow Does A Gene Lead To A Phenotype?1 mRNA Synthesizedby Transcription2 mRNA Translatedinto Protein byTranslation of TheGenetic Code• Complementary toTranscribed, Non-SenseStrand• Same Sequence As SenseStrandGenetic Code on mRNATranslated to ProteinSequence∴ Sequence of Gene Sequence of mRNA Sequence of ProteinKnow SequenceKnow ProteinEngineer New Protein2The Genetic Code is Universal!How Know?Know Sequence of Gene-Know Sequence of ProteinUsing Genetic CodeBig Implication For Genetic Engineering! Can Make Genes,Genomes & Specify Proteins Wanted! Can Express GenesFrom One Organism in Another!Design An Experiment to Show Code is Universal!For RNA, The Ts are replaced by Us.1. Universal2. Triplet3. Punctuation4. DegenerateAn Elaborate Cellular Machinery RequiringThousands Of Genes is Required ToProduce Proteins Encoded By SpecificGenes!!It takes Genesto Express (and Replicate)A GENE!!!How Do Genes Work & What areGenes in Context of…1. What is a Gene?2. What is the Anatomy of a gene?3. How Does the Gene Replicate?4. How Does the Gene DirectSynthesis of a Protein?5. Does the Gene WorkIndependently of other Genes?6. What is the Sequence &Structure of the Protein?7. How does it work in cell?8. Does the Protein Structure implyany Potential “Harm”?9. Does the Gene Change theorganism? Fitness?Need Science-Based Questions &Science-BasedSolutions-NOTOPINIONS!There’s NO HOCUS POCUSall hypothesis are testable!!“Behind” All Traits!Same Processes!Thinking About The Consequences of GMOsThe Nuts & Bolts of GeneticEngineeringThe Factor VIII Story: From GeneTo Genetic Screening To DrugA Case Study of Cloning Genes and mRNAsReference: Lawn & Vehar, Sci. Amer., January, 1986 (Potentially Lethal Disease)Human Genetic Disorders Occur As A Resultof Mutations: Change Code-Alter ProteinSickle-Cell AnemiaHemophilia Has Been Known As AnInherited Disease For >2500 Years!Old Testament-CircumcisionsRoyal Family-Europea = activated formGenesis 17:10-14Abraham was circumcised at 93 and gave birth to Isaac at 99.His wife -