1. DNA Sequencing 2. DNA Cloning 3. Studying Gene .

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Chapter 20:Biotechnology1. DNA Sequencing2. DNA Cloning3. Studying Gene Expression4. Manipulating Genomes5. Therapeutic & DiagnosticTechniques

1. DNA SequencingChapter Reading – pp. 409-412

DNA Sequencing TECHNIQUEDNA(template s(fluorescently tagged)dATPddATPdCTPddCTPdTTPddTTPdGTPddGTPP P PGOHP P PGH DNA replication in vitro using 1 of 4 different “chainterminating” dideoxynucleotides (ddNTPs) results in a set of DNA fragments ending in all positionswith A, C, G, or T that can be resolve by length on a gel

DNA Sequencing usesChain TerminatorsNormal nucleotide(dNTP)Dideoxynucleotide(ddNTP)*

DNA synthesis is carried out in reactionscontaining the following: DNA template to be sequenced dNTP’s DNA primer DNA polymerase ddATP, ddCTP, ddGTP or ddTTP each ddNTP is labeled with a differentfluorescent tagThe color reveals the identity of the ddNTP thatterminated DNA synthesis at each position,thus revealing the sequence!

DNA Sequencing TECHNIQUE (continued)5′3′DNA (templateC strand)TGACTTCGACAAddCTGTTddGCTGTTLabeled dCTGAAGCTGTTShortestDirectionof movementof strands3′5′LongestLongest labeled ortest labeled strand

DNA SequencingDirectionof movementof strandsLongest labeled strandDetectorLaserShortest labeled strandRESULTSLast nucleotideof longestlabeled strandLast nucleotideof shortestlabeled strandGACTGAAGC

“Next Generation”Sequencing

2. DNA CloningChapter Reading – pp. 409-417

What is “Recombinant DNA”?The joining of DNA from different sources.This can happen in nature (in vivo) the transfer of DNA involving bacteria or viruses or in the laboratory (in vitro) the cutting & splicing of DNA fragments bymolecular biologistsThe term “recombinant DNA” generally refers to the invitro kind which is commonly called “gene cloning”

Gene Cloning Bacterium1 Gene inserted A (plasmid)Gene ofinterest2 Plasmid put intobacterial cellCell containinggene of interestDNA ofchromosome(“foreign” DNA)Recombinantbacterium gene of interest is inserted into a plasmid vector bacterial host transformed with recombinant plasmid bacterial clone with recombinant plasmid is identified

Gene Cloning3 Host cell grown inculture to form a cloneof cells containing the“cloned” gene of interestCloned gene canthen be used in avariety of ways.Protein expressed fromgene of interestGene ofinterestProtein harvestedCopies of geneBasicresearchon gene4 Basic researchand variousapplicationsBasicresearchon proteinHuman growthGene for pestGene used to alter Protein dissolvesresistance inserted bacteria for cleaning blood clots in heart hormone treatsinto plantsup toxic wasteattack therapystunted growth

Restriction ′1 Restriction enzymecuts sugar-phosphatebackbones.5′3′5′3′5′ Sticky 3′3′5′end5′2 DNA fragment added3′3′5′from another moleculecut by same enzyme.Base pairing occurs.5′3′3 DNA ligasee.g. EcoRI cuts at:3′ 5′3′ 5′3′G AATT CC TTAA GG AATT CC TTAA G5′3′5′ 3′.GAATTC.CTTAAG.5′One possible combinationseals strands5′3′Gene cloning usuallyinvolves the use ofrestriction enzymesthat cut DNA at veryspecific sequences:3′Recombinant DNA molecule5′There are many differentrestriction enzymes,each cutting a differentsequence

The Gene Cloning TechniqueHummingbird cellTECHNIQUEBacterial plasmidampR genelacZ geneRestrictionsite cut plasmid &DNA to becloned withsame RE ligate vector &DNA insert withDNA ligase transform bacteriawith ligated DNAStickyendsGene ofinterestHummingbird DNAfragmentsRecombinant plasmidsNonrecombinantplasmidBacteria carryingplasmids

Selection for Bacterial ClonesAntibiotic resistance allows selection for bacterialclones containing the plasmid vector.Bacteria carryingplasmidsRESULTSColony carrying nonrecombinant plasmidwith intact lacZ geneColony carryingrecombinantplasmidwith disruptedlacZ geneOne of manybacterialclonesBlue/whiteselection vialacZ genedisruptionallowsidentificationof clonescontaining aDNA insert.

TECHNIQUE1Mixture ofDNA molecules ofdifferentsizesPowersource CathodeAnode WellsGel2 Powersource oresisSeparation of DNAfragments through aporous gel matrix: gel is either agarose orpolyacrylamide electric current pullsnegatively charged DNAtoward the positive pole rate of movement isinversely proportional toDNA fragment size

DNA Libraries A collection of cloned genes from anorganism is called a DNA library.Genomic DNA Library a collection of chromosomal DNA fragments clonedinto a particular vector essentially cloned pieces of the organism’s genomecDNA Library a collection of DNA fragments produced frommessenger RNA (mRNA) cloned into a vector produced from mRNA by reverse transcriptase a collection of expressed genes with NO intron DNA

DNA LibrariesForeign genomeCut with restriction enzymes into eithersmalllargeorfragmentsfragmentsBacterial artificialchromosome ) BAC clonePlasmidclone(a) Plasmid library(c) Storing genome libraries

DNA innucleusmRNAs incytoplasmmRNA5′ReversetranscriptasePoly-A tailA A A A A A 3′T T T T T 5′3′DNA PrimerstrandA A A A A A 3′T T T T T ′3′cDNAProducing cDNAcDNA (DNA complementary tomRNA) is produced as follows: purify mRNA fr. desired cell type treat with reverse transcriptaseand oligo-dT primer results in double-stranded DNAcopies of all mRNA from the cell clone cDNA fragments intovector such as a plasmid transform bacterial hostsResults in a collection of clonedcDNA corresponding to codingsequences of all expressed proteins

Screening DNA LibrariesTECHNIQUERadioactivelylabeled probemolecules5′3′ CTCATCACCGGC GAGTAGTGGCCG5′3′Gene ofinterestProbeDNASinglestrandedDNA fromcellMultiwell platesholding libraryclonesNylon membraneLocation ofDNA with thecomplementarysequenceFilmNylonmembrane DNA from library clones immobilized on a membrane radioactively labeled DNA similar in sequence to desiredclone is added as a probe hybridization of probe to complementary DNA IDs clone

PCR5′TECHNIQUETargetsequenceGenomic DNAThe Polymerase ChainReaction is a techniqueto selectively amplify adesired DNA sequence:3′1 Denaturation3′5′5′3′3′5′2 AnnealingCycle 1yields2molecules essentially DNA replicationin vitroPrimers3 ExtensionNewnucleotides results in exponentialamplification of target DNAsequence artificial primers specificfor target DNA sequencelimit replication to DNAcomplementary to primersCycle 2yields4moleculesCycle 3yields 8molecules;2 molecules(in white boxes)match targetsequence

Overview of PCREvery PCR reaction requires the following:1) source of target DNA template2) artificial primers “flanking” DNA of interest3) heat-stable DNA polymerase (from hyperthermophile)4) dNTP’s5) automated thermocycler to facilitate repeated: denaturation of DNA (separating the 2 strands) hybridization of primers to template DNA synthesisResulting PCR products can then beanalyzed by gel electrophoresis.

“PCR Cloning” involvesdesigning restrictionsites into the primers tofacilitate cloning

3. Studying Gene ExpressionChapter Reading – pp. 417-419, 421-422

TECHNIQUE1 cDNA synthesismRNAscDNAs2 PCR amplificationPrimersβ-globingene3 Gel electrophoresisRESULTSEmbryonic stages213 4 5 6RT-PCR produce cDNAwith reversetranscriptase carry out PCRusing specificprimers fordesired gene gel electrophoresisof resulting PCRfragmentsAmount of DNAreflects amount oforiginal mRNAsequence.

in situ Hybridization

in situ HybridizationHow is in situ hybridization carried out? fluorescently labeled anti-sense RNA probesadded to tissue sample hybridization with complementary mRNAsequences location of specific gene expression within thetissue (i.e., in situ) is revealed

DNA MicroarraysA DNA microarray is a solid surface containing aprecise array of single-stranded DNA sequencesfrom 1000s of different genes in an organism. labeled cDNA is produced from test cells andallowed to hybridize with sequences in thearray intensity of signals reveal expression ofspecific genes within the test cells.When cDNA from different sources is labeled differently,gene expression from each source can be compared in asingle microarray (as shown on the slide after next).

TECHNIQUE1 Isolate mRNA.2 Make cDNA by reversetranscription, usingfluorescently labelednucleotides.Tissue samplemRNA moleculesAnalyzinggeneexpressionusing a DNAmicroarray.Labeled cDNA molecules(single strands)3 Apply the cDNA mixture to amicroarray, a different genein each spot. The cDNA hybridizeswith any complementary DNA onthe microarray.DNA fragmentsrepresenting aspecific geneDNA microarray4 Rinse off excess cDNA; scan microarrayfor fluorescence. Each fluorescent spot(yellow) represents a gene expressedin the tissue sample.DNA microarraywith 2,400human genes

Comparing Gene Expression

Single NucleotidePolymorphisms (SNPs)SNPs are single nucleotide differences that correspond withspecific disease-causing alleles.PCR and hybridization techniques (e.g., microarrays) canreveal the presence of such alleles (genetic testing).DNATNormal alleleSNPCDisease-causingallele

4. Manipulating GenomesChapter Reading – pp. 422-425, 429-430

Cloning PlantsSome plants can be cloned from a single adult plant cell.Crosssection ofcarrot root2-mgfragmentsFragments werecultured in nutrient medium;stirring causedsingle cells toshear off intothe liquid.Single cellsfree insuspensionbegan todivide.Embryonicplant developedfrom a culturedsingle cell.Plantlet wascultured onagar medium.Later it wasplanted in soil.Adultplant

EXPERIMENTThisexperimentproducedthe firstartificiallyclonedanimal.Frog embryoFrog egg cellFrog tadpoleUVLess differentiated cellFully differentiated(intestinal) edEnucleatedegg cellEgg with donor nucleusactivated to begindevelopmentRESULTSCloningAnimalsMost developinto tadpoles.Most stop developingbefore tadpole stage.

TECHNIQUEMammarycell donorEgg celldonor1Culturedmammarycells2Eggcell fromovary3 Cells fused4 Grown in cultureNucleusremovedNucleus frommammary cellEarly embryo5 Implanted in uterusof a third sheepSurrogatemother6 EmbryonicdevelopmentRESULTSThe first clonedmammal, “Dollythe sheep”, wascloned by thismethod.Other mammalssuch as catshave since beencloned andpresumablyhumans could becloned this wayas well.Lamb (“Dolly”) geneticallyidentical to mammary cell donor

Transgenic OrganismsTransgenic organisms have a foreign gene (e.g. fromanother species) inserted into their genome. also known as “genetically modified organisms” (GMOs)

CRISPR-Cas9Cas9 active sitesGuide RNAcomplementarysequence a new method to modify genomesCas9 proteinGuide RNA engineered to“guide” the Cas9 proteinto a target gene5′Active sites thatcan cut DNANUCLEUS25′3′3′5′5′Part of thetarget gene3′Complementarysequence that canbind to a target geneResulting cut intarget gene byCas9Cas9–guide RNA complex13CYTOPLASMNormal(functional)gene for use asa template byrepair enzymes(a) Scientists can disable(“knock out”) the target geneto study its normal function.(b) If the target gene hasa mutation, it can berepaired.NUCLEUSRandom nucleotidesNormal nucleotides

5. Therapeutic & DiagnosticTechniquesChapter Reading – pp. 425-431

Stem CellsAdultstem cellsEmbryonicstem cellsCells generatingsome cell typesCells generatingall embryoniccell typesCulturedstem types ofdifferentiatedcellsNervecellsBloodcellsStem cells areundifferentiated cellsthat can give rise tomultiple differentiatedcell types.Embryonic stem cellsare considered morepluripotent than adultstem cells, thoughtechniques are beingdeveloped to expandthe potential of adultstem cell.

Stem Cell TherapyTechniques arebeing developed totreat a person’sown cells in vitro toproduce a neededcell type which isthen reintroduceinto the patient torepair damagedtissue.1 Remove skin cellsfrom patient.2 Reprogram skin cellsso the cells becomeinduced pluripotentstem (iPS) cells.Patient withdamaged hearttissue or otherdisease3 Treat iPS cells sothat they differentiateinto a specificcell type.4 Return cells topatient, wherethey can repairdamaged tissue.

Gene TherapyCloned gene1 Insert RNA version of normal alleleinto retrovirus.Viral RNARetroviruscapsid2 Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.3 Viral DNA carrying the normalallele inserts into chromosome.Bonemarrowcell frompatient4 Inject engineeredcells into patient.BonemarrowBone marrowstem cells canbe geneticallymodified in vitroto fix a geneticdefect and thenreintroduced backinto the patient.

Restriction Fragment LengthPolymorphisms (RFLPs)Normal β-globin allele175 bpDdeISickle-cellalleleLarge fragment201 bpDdeINormalalleleDdeIDdeILargefragmentSickle-cell mutant β-globin allele376 bp376 bpDdeI201 bp175 bpLarge fragmentDdeIDdeI(a) DdeI restriction sites in normal andsickle-cell alleles of the β-globin gene(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles DNA from different sources cut with the same RE willresult in different size DNA fragments (RFLPs)

Short Tandem Repeat AnalysisMany parts of thehuman genomecontain short DNAsequences repeatedmany times in a row(in tandem) the number of repeatsin these regions ishighly variable fromperson to person RFLP analysis canproduce a DNAfingerprint that isunique for eachindividual(a) This photo showsWashington just beforehis release in 2001,after 17 years in prison.Source ofsampleSTRmarker 1STRmarker 2STRmarker 3Semen on victim17,1913,1612,12Earl Washington16,1814,1511,12Kenneth Tinsley17,1913,1612,12(b) These and other STR data exonerated Washingtonand led Tinsley to plead guilty to the murder.

Southern BlottingTECHNIQUEDNA restriction enzymeRestrictionfragmentsIII IIIHeavyweightNitrocellulosemembrane (blot)GelSpongeI Normal II Sickle-cell III Heterozygoteβ-globin alleleallele1 Preparation ofrestriction fragmentsIII IIIRadioactively labeledprobe for β-globingeneNitrocellulose blot4 Hybridization with labeled probeAlkalinesolution2 Gel electrophoresisPapertowels3 DNA transfer (blotting)Probe base-pairswith fragmentsFragment fromsickle-cellβ-globin alleleFragment fromnormal β- globinalleleIII IIIFilmoverblot5 Probe detection

Key Terms for Chapter 20 recombinant DNA, gene cloning, plasmid vector transformation, ligation, restriction enzymes genomic & cDNA libraries, hybridization, probe gel electrophoresis, PCR, RT-PCR dideoxynucleotide, RFLP, STR, SNP in situ hybridization, DNA microarrays transgenic, stem cell, southern blot, CRISPR-Cas9 in vivo, in vitro, in situRelevant ChapterQuestions1-10, 12

The term “recombinant DNA” generally refers to the in vitro kind which is commonly called “gene cloning” Bacterium. Bacterial chromosome. Plasmid. 2. 1. Gene inserted into plasmid. Cell containing gene of interest. Recombinant DNA (plasmid) Gene of interest. Plasmid put into bacterial cell. DNA

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