1Recombinant DNA TechnologyStephen B. Gruber, MD, PhDDivision of Molecular Medicine and GeneticsNovember 4, 2002Learning Objectives• Know the basics of gene structure, function and regulation.• Be familiar with the basic methods of molecular genetics.• Understand the meaning of DNA sequence and amino acidpolymorphisms.• Know how DNA sequence analysis is performed and befamiliar with methods of screening for differences. • Have a general understanding of methods for gene transferinto tissue culture cells and the power of transgenictechnologies.2Learning Objectives (1)• Know the basics of gene structure, function and regulation.• Be familiar with the basic methods of molecular genetics.• Understand the meaning of DNA sequence and amino acidpolymorphisms.• Know how DNA sequence analysis is performed and befamiliar with methods of screening for differences. • Have a general understanding of methods for gene transferinto tissue culture cells and the power of transgenictechnologies.Chromosomes, DNA, and GenesCellNucleusChromosomesGeneProteinAdapted from Understanding Gene Testing, NIH, 19953Genetic CodeA codon is made of 3 base pairs64 codons total1 codon (AUG) encodesmethionine and startstranslation of all proteins3 codons stopproteintranslation61 codons encode 20amino acids(redundant code)U A A A U GMetGCAAlaDNA Transcription andTranslationmRNARibosomeGrowingchain ofamino acidsProteinNuclearmembraneCell membraneDNAAdapted from Understanding Gene Testing, NIH, 199545' endPromoterRNA transcriptionstart site3' endGene StructureStop siteIntron Exon 2 IntronExon 1 Exon 3Splice sitesExon 2Exon 1 Exon 3mRNARNA ProcessingTranslationProteinDNAPrimarymRNAMaturemRNAProcessingTranscriptionExon Intron Exon Intron ExonGUAG5Learning Objectives (2)• Know the basics of gene structure, function and regulation.• Be familiar with the basic methods of molecular genetics.– nucleic acid hybridization– Southern (DNA) and northern (RNA) blotting– PCR– DNA sequencing– basic steps involved in constructing & screening a cDNA library• Understand the meaning of DNA sequence and amino acidpolymorphisms.• DNA sequence analysis• Transgenic technologiesfrom Textbook: 5.41944DNA is thegenetic material1949Abnl Hemoglobinin sickle cell anemia1953Double helix1956Glu 6 Val insickle hemoglobin1966Completion of thegenetic code1970First restrictionenzyme1972Recombinantplasmids1975Southernblotting1981Transgenic mice1983HuntingtonDisease genemapped1985PCR1986Positional cloning(CGD, musculardystrophy,retinoblastoma1945 1950 19551960196519701975 19801985 1990199520001987Knockout mice1989Positional cloningwithout deletion (CF)1990First NIH-approvedgene therapyexperiment1996Complete yeastgenome sequence19951st completebacterialgenome sequence2001Draft humangenome sequence6Preparing DNA for AnalysisBlood sampleCentrifuge andextract DNA fromwhite blood cellsDNA for analysisSINGLE-STRANDEDDNA PROBESFOR GENE AMIXTURE OF SINGLE-STRANDEDDNA MOLECULES+BBBAACCCDDDEEEFFFONLY A FORMS A STABLEDOUBLE-STRANDED COMPLEXESA, C, E ALL FORMSTABLE COMPLEXESSTRINGENT HYBRIDIZATION REDUCED-STRINGENCY HYBRIDIZATIONATextbook: Figure 5.87Electrophoresis of DNAVoltage+DNA fragments loaded into wellsPath of migrationDNA fragmentsseparate by sizeand charge_ElectrophoresisRestriction enzymedigestionPrinciple of a Southern blothybridize labeled probe to fragment of DNAAdd radio-labelednormal DNAprobes8Polymerase Chain Reaction(PCR)Isolate anddenature DNAAnneal andextend primersRepeat asnecessaryAmplifiedsegmentsSequence to beamplifiedDNA Sequencing95'3'T G T TC T G A C T T C G A C A ASINGLE-STRANDED DNAOF UNKNOWN SEQUENCERADIOACTIVELY LABELEDPRIMERO CH O2HHH HHHDIDEOXYNUCLEOTIDE (ddNTP)DNA POLYMERASE IdATPdGTPdCTPdTTPddATPddCTP ddTTP ddGTPC T G A C T T C G A C A AddGREACTIONMIXTURESddATPREAD SEQUENCE OF ORIGINALSINGLE-STRANDED DNA(COMPLEMENT OF PRIMER-GENERATED SEQUENCE LADDER)GELELECTROPHORESISAUTORADIOGRAPHYTO DETECTRADIOACTIVE BANDS3'5'BASEddCTPddTTPddGTPLARGERFRAGMENTSSMALLERFRAGMENTSCTGACTTCGddGddGPRODUCTS IN ddGTP REACTIONPPPTextbook: Figure 5.17DNA SequencingATC TTA GAG TGT CCCATC TTA GTG TCC CStartA T C GNormalMutant (185delAG)AGA T C GdelAStartdelG10Learning Objectives (3)• Know the basics of gene structure, function and regulation.• Be familiar with the basic methods of molecular genetics.– nucleic acid hybridization– Southern (DNA) and northern (RNA) blotting– PCR and gel electrophoresis– DNA sequencing– basic steps involved in constructing & screening a cDNA library• Understand the meaning of DNA sequence and amino acidpolymorphisms.• DNA sequence analysis• Transgenic technologiesPolymorphisms and Mutations• Sequence variation-- differences among individuals(DNA, amino acid)– > 0.01 = polymorphism– < 0.01 = rare variant• Mutation-- any change in DNA sequence– Silent vs. amino acid substitution vs. other– neutral vs. disease-causing• Common but incorrect usage:“mutation vs. polymorphism”• balanced polymorphism= disease + polymorphism11Learning Objectives (3)(continued)• Understand the meaning and significance of DNAsequence and amino acid polymorphisms.• Understand the various types of DNA sequencepolymorphisms.– RFLPs (Restriction Fragment Length Polymorphism)– VNTRs (Variable Number Tandem Repeat)– SSRs (Simple Sequence Repeat; also STR [Short/SimpleTandem Repeat]))– SNPs (Single Nucleotide Polymorphism)Textbook: Figure 5.1912Learning Objectives (3)(continued)• Understand the meaning and significance of DNAsequence and amino acid polymorphisms.• Understand the various types of DNA sequencepolymorphisms.– RFLPs (Restriction Fragment Length Polymorphism)– VNTRs (Variable Number Tandem Repeat)– SSRs (Simple Sequence Repeat; also STR [Short/SimpleTandem Repeat]))– SNPs (Single Nucleotide Polymorphism)Disease-Associated Mutations Alter Protein FunctionFunctional proteinNonfunctional ormissing protein13P1 P2(TCTA)10(TCTA)11(TCTA)12(TCTA)13(TCTA)14(TCTA)15ABCDEFAB CD EF AF CE151413121110Textbook: Figure 5.22SNP (coding sequence)NormalmRNAProteinA U GMetAAGLysU UUPheG GCGlyGC AAlaU U GLeuA AGlnCSilent DNA sequence polymorphismSequencevariantmRNAProteinAU GMetA AGLysU UUPheG GUGlyGC AAlaUUGLeuAAGlnCG14Disease-Associated MutationsA mutation is a change in the normal base pair sequenceCommonly used to define DNA sequence changesthat alter