Class Note for BIOC 461 at UA
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Date Created: 02/06/15
Umom A o mm 0322 a 203m 0003m3mlt 3 mm Moom OIgtum m ECU 0220 Omzmm rmOHcmm Ou0m 3 mmijOjOZ m2Nltltmm NV Omr mrmOjNOUIOmem OH 02gt 8 02gt mmOcmZQZO 22gt mmOcmZQZO 02gt mlt2Immm Av uOltltmmgtmm OIgt2 mmgt002 coo NV mv mmOOltw2gt2 02gt OOZmdNCOjOZ gt20 Omzm 90220 9 02gt Or0220 ltmOOmm d Omzm wmgtmmmn ltgtAZO gt20 mommm220 Imlt mv OImOltOmOltm ltgtuu20 9 mXUmmeOZ OH Or02m0 Omzmm 40v m202mmm20 20ltm UmOj zm Page 2 of 25 Recombinant DNA technoloqv started in late 197039s 0 An incredibly powerful set oftool for gene manipulation 0 Methods associated with this quottechnologyquot make genetic engineering a reality 0 DNA genes RNA and protein structure and function can be altered by design for beneficial or detrimental bioogica warfareterrorism results KEY TOOLS and METHODS OF GENE EXPLORATION O ENZYMES to cut join and replicate DNA in test tubes in vitro a restriction enzymes are DNA cutters b DNA ligases are DNA joiners 0 DNA replication requires DNA polymerases 0 GEL ELECTROPHORESIS to separate and isolate specific DNAs 0 BLOTTING METHODS based on hybridization BASEPAIRING of complementary DNA andor RNA 0 SOLID PHASE methods to seguence and synthesize DNA 0 POLYMERASE CHAIN REACTION for gene detection and amplification Page 3 of 25 RESTRICTION ENZYMES ENDONUCLEASES DNA scissors hundreds of restriction enzymes are known Recognition sequences different lengths often 48bp palindromic 2 fold rotational axis of symmetry speci c cleavage sites Fig 61 They can leave overhanging ends or blunt ends Named ex Hindlll for source bacterial Cleavage Site strain l 5 C C G C G G 3 t l l 1 H Haemophilus in influenzae d strain d I third one identified l Cleavage site 3 6 6 6 KG C C 5 Symmetry axis Number of cuts in a specific DNA ranges from few if long recognition site to many short or ambiguous recognition site patterns of fragments are diagnostic of a given DNA species and physical maps of whole chromosomes can be made Fig62 l 5 3 GGAITCC BumHl 3 CCTAGG 5 5 GAATTC 3 I EcoRl 5 CTTAAG 5 l GGuCC 5 Haelll 339 CCGG 5 l l 539 GCGC 3 D Hhal 339 CGCG 5 l l 5 T A 3 C CG G W 339 GAGCTC 5 Page 4 of 25 GEL ELECTROPHORESIS OF DNA 0 Agarose gels separate DNA restriction fragments o Asualize DNA by staining or autoradiography 0 even differences ofm base pair can be detected on gels o Hybridization Basepairing of DNADNA DNARNA Complementary single stranded DNA and RNA molecules form basepaired structures even if only 2 or 3 bases can pair like at ends ofrestriction fragments IMPORTANT Hybridization DNADNA DNARNA is almostalwazs used in one or more ways to to detect particular DNA or RNA sequences and to construct new combinations of DNA fragments NUCLEIC ACID BLOT39I1NG AND HYBRIDIZATION DNA bands and patterns on gels Page 5 of 25 can be transferred to nitrocellulose filters Southern blotting and identi ed by hybridization with a speci c gene probe Fig63 0 Southern DNA Northern RNA and Western protein blotting methods are aH powerful probes of gene function DNA fragments Transfer 7 Add quot DNA a of DNA Prlabeled probe E by blotting DNA probe Auloradiugraphy revealed gt gt gt B E 7 w Agarnse Mirrocerrurnse Amaradiogram gel sheet 0 Rest riction fragment length polymorphism RFLP gel analysis is a powerful diagnostic tool ex sicklecell anemia genetic typing Mstl RFLP for Sickle Cell Mutation Detection Fig 752 Normal Sickle cell I moovQ rrqmnrr 4 5386 bases 155844 bases 18 million bases 3 million bases bases Page 6 of 25 LANDMARK DNA SE UENCES COMPLETED tRNA 1964 X174 DNA 1977 tobacco chloroplast DNA 1986 H influenzae 1995 E coli 1997 human 2000 g complicated method DNA SEQUENCING ALL METHODS REQUIRE THE FOLLOWING reactions specific for each base controlled random reactions equimolar collection of reaction products same frequency of stopping each time the same base occurs DNA SEQUENCING BY CONTROLLED RANDOM CHAIN TERMINATION OF DNA SYNTHESIS METHOD SANGER DIDEOXY METHOD Fig64 Use a templateprimer complex a DNA polymerase dNTPs and 239339 dideoxynucleoside triphosphate one for each base in each ofm reactions DNA synthesis occurs specific for each base until a dideoxy nucleoside phosphate is inserted into the nascent DNA then the reaction stops no free 339 OH group to attack the incoming dNTP substrate Reaction is carried out under conditions adjust ratio of dNTP ddNTP to give equal representation and distribution of products Run reaction products on gel 500600 bases can be easily read on one gel Page 7 of 25 Stratey for Chain termination DNA sequencin Fig64 2 3 Dideoxy analog DNA to be sequenced 3 GAATTEGCTAATGC 539 CTJAA I Primer DNA polymerase I Labeled dATP dTTP dCTP dGTP Dideoxy analog oi dATP 3 GAATTCG CTAATG C 539 CTTAAGCCATTA 3 GAATTCGCTAATGC 539 CTTAAGCGA New DNA strands are separated and electrophoresed Page a c 25 Cunem memnqs m DNARNA sequence qenerminminn 0 Use nunrescenuzheueq nuclenuqes makes eacn base veacuun mw uuvesce a dwevem cu uv m 3H 4 veacuun pvuducts anq detect wmh amuma ed numememsm de ect DNA veactmn pvudums anq name by Dmpmev mmemate y an E 5 Can sequence Wha e qencnnes ex m E E r banenau 0 Can a su sequence DNA by de ectmg hybndwzauun swgna s un anays cvsncn DNAsequences ucunq m mwcvuchwps RNA SEQUENCING VR NA can be sequenceq dwec y um nuw u s qcne by mdeuxy methud usmg vevevse nanscnmase Wun a syntheuc DNA pnmev E E E w u c c a 2 a a E SequenceCArAcCTGTTTCCTGTGTGAAA onganucleonde lengm Page 9 of 25 CHEMICAL SYNTHESIS OF DNA SOLID PHASE AUTOMATED METHODS mutagenesis oligodeoxynucleotide chain short DNA parts of genes for probes and primers aid DNARNA sequencing cloning and gene probing by hybridization easy to make DNA 100 nucleotides long 1820 used most often Chemically synthesized DNAs are key to protein engineering by sitedirected 10013 Dimethoxytrityl DMT group Start with blocked nucleotide linked to a solid support glass bead b ELd protected nucleotides added Eplg e e stepwise but 339 to 539 reverse of DNA polymerase p StepWIse lInkIng ofactlvated I I Wmemv N monomers deoxyrIbonucleosIde 3 uca group 0 phosphoramidites that are blocked NC at 539P and have protected amino Adeoxyribonuclenside3 phosphuramidite with DMT and BCE attad led groups Chemical synthesis of DNA Fig67 Activated monomer 1 p S t 339 Q O a w JMl G 3 0 u 3 in r Louplmg 5 Phosphite triester intermediate Growing chain Repoat Sf ahon iml lm til ex U wr n r 39 5 D e5 DEpl UlELiIUI I with dichloroacezic acid g 5 Elongated chain Fhusphohiester intermediate Page 10 of 25 POLYMERASE CHAIN REACTION PCR Discovered in 1984 0 KB MullisScientific American1990 2625665 0 Incredibly powerful method to amplify synthesize DNA starting with as little as one copy of a DNA molecule Figs68 and 69 0 PCR CONCEPT Fig68 Two oligonucleotides 5233 Targe sequme spanning a gene of interestm on opposite strands are used to prime multiple cycles of DNA synthesis catalyzed by a heat stable DNA Diggg fgggrggmms polymerase ex Taq polymerase from a thermophilic bacterium 7E7 H icwm anneal primers J l riim s E lSynthesim new DNA ExPCR startin with one of 2 strands of DNA avquot 39Kh 2W Thard t s quot 131 Frimo rmquot i ii FoLymuA sq um l 395 Dunurt 4 I l n 37quot quot1 See Fig 69 for 333 drawing of three if M I cycles of PCR r a 1 I i i p14 3 Fame Nymmse 4 svm fquot 1 was a 1 I quot dw39 Q Fuquot an NM 6 Page Df 25 0 PCR RESULT In 2540 replicakion cycles enough DNA is symhesized to clone orto sequencedirecuy Pag212u125 mm Fuvensms magnum Mgeneu dams m uzero sequencmg DNA mm uss s detemmg smaH amuums Mbac ena ex ammax 2h FORENSICS EXAMPLE E uud am ana ysws ulwcum v and dummy u Manamaappayenuygumyw Fwa m nuns Hun quotII 0 ans 4 3 smn g lt III Mkb Page 13 of 25 CONSTRUCTION CLONING AND EXPRESSION OF DNA Novel combinations of genes can be constructed cloned amplified and expressed in foreign environments KEY STEPSPROCEDURESITOOLS Fig611 4 quot Ed Construct recombinant molecule link DNA insert with a vector Amplify and clone DNA Introduce DNA into host cells as naked DNA or as DNA incorporated into virus particle DNA must be replicated autonomously in a host cell Selection by antibiotic resistance gene probing antibody reaction Law L 5 bn IInu I QIHDNA 0 Pllsmid victor Joinm mwuc I wally52 immuucm we host ceII by lmndmmzvmn N W A A mm m a vecomblnant DNA molecule I Page 14 of 25 CONSTRUCT RECOMBINANT MOLECULE link a DNA insert with a vector Figs61112 CUT AND JOIN DIFFERENT DNA MOLECULES Restriction enzymes and DNA ligase 0 Restriction enz mes that ive cohesive ends short com lementa ends that can base pair cut unique cloning sites in vectorsEX Eco RI Fig611 GAATI39C CTI39AAG Cleave with EcaRI restriction enzyme WWW G A mime Anneal DNA fragments and rejoin with DNA ligase G CTTA The vector and insert are covalently linked with DNA ligase 0 Restriction enzymes that give blunt ends need linkers added by T4 DNA ligase to get cohesive ends for cloning Ex Eco RI linker Fig612 39 7 70H 339 5 339 Ho 539 DNA fragment or vector M I Me 539 CGGAA I39I39CGG gtOH 339 3 339 HO GGCTTAAGCC7 539 Decameric linker 5 CGGAATTCGG CGGAATICGG OH 3 3 HO GGCITAAGCC GGCTTAAGCC 7 5 ECORI restriction enzyme 539 7 AATI39CGG CCiCrOH 339 339 H0 GCC GGCrTAA 7 539 Page 15 of 25 Amplify and clone DNA Introduce DNA into host cells as naked DNA See illustration page 13 or as DNA incorporated into virus particle DNA must be replicated autonomously in a host cell Selection of recombinant DNAcontaininq host cells antibiotic resistance gene probing antibody reactions etc 9 Platle V9059quot SoleM 39 5 amquot aij uHoil39Cells Tetracycline resistance m Amplclllln resistance A procaryd ic we 6quot SOME CLONING VECTORS autonomously replicating Plasmids lnsertional inactivation of antibiotic resistance often used Use for small DNAs Lambda phages Larger DNA pieces tha plasmids Especially useful for libraries of cDNA or eucaryotic genomic DNA YACs and BACs yeast and bacterial artificial chromosomes for long DNA especially big pieces of chromosomes Page 16 of 25 CLONING VECTORS O Plasmids accessory chromosomes which replicate autonomously are excellent vectors for cloning DNA of 26 kb in E coli Antibiotic AmPicillin resistance resrstances are used for selection of recombinant plasmids lnsertional inactivation signals the presence of a DNA insert in an antibiotic resistance gene See page 15 and Origin of replication Plasmid pBR322 0 Special lambda A phages they work a lot like T2 bateriophage used to clone large pieces 1020kb of DNA between each end of lambda DNA Especially suitable for cloning of libraries of cDNA or eucaryotic genomic DNA Fig61415 V A DNA r A I m lambda A phage L If g W K lifecycle 39 pathway f n Entry of Progeny I A DNA r 9 DNA Lysed bacterium Activatinn with released i ii phage E coil Bacterial Lysogen DNA cell pathway 39A DNA integrated in E coir genome Cloning DNA 1 between each end of A DNA lambda A DNA l Removal of middle section by restriction digestion l l Splicing with foreign DNA 1rr Too small to be packaged In vitro packaging of recombinant molecule infective X virion harboring foreign DNA m n n Page 17 of 25 0 Yeast arti cial chromosomes YACs Can clone large pieces of DNA 100000 to a million base pairs Fig621 Telomere Autonomous replication 5e uence ARS q l Centromere DNA insert 100 to 1000 kb Telomere GENE LIBRARIES What are they Genomic libram Made from all restriction fragments of a cell39s DNA A collection of cloned sequences which represents a whole genome O Genomic library vectors Bacteriophage lambda YACs and BACs cDNA libram Made from mix of all of a cell s mRNAs Use reverse transcriptase to get DNAA cDNA library is a mix ofDNAs complementary to ALL genes that are being expressed as mRNA s CDNA library vectors lambda phage plasmids Page 18 of 25 Constructinq a qenomic librarv in bacteriophaqe A Made from all restriction fragments of a cell39s DNA A collection of cloned DNA sequences which represents a whole genome a b c d Gennllm DNA Fragmen39auun by shearing er enzymatic digestion Joining is 7 DNA pieces EjcjEj in vim packaging i iregmenns ni lureign DNA Ampiiiicd on by iniecnen DiE on Genomic library in phage Constructin a cDNA libr from a cell39s mRNAs Use reverse transc ptase to get DNA A cDNA library is a mix of DNAs that regresents all of the cell s mRNAs man eigenien er mRNA tempiait REVEGE W 233 Wm transmplase W BN WT T H Anath niigemc a H dNYPs i i mm m enumom s Himi on 3 H AI V L A on V mRNA PnMA la ii mRNA migomq primer 7 7 DNA puiymeiese 3 HO 99015 1177 i dNWS V LtqitiAAnuAi H 5 a Hoib r GGiYTTnT a Doublmsuanded DNA Page 19 of 25 SCREENING GENE LIBRARIES searching for a needle in phagestack 0 screen 500000 clones for a specific sequence in a genomic library 0 Easier for abundant mRNA molecules in a cDNA library 0 Hybridization screening with gene probe 0 Synthetic DNA probes predict sequence by reverse translation of protein sequence to a DNA sequence 0 Immunochemical antibody screening of an expression library 0 Chromosome walking to connect long pieces fo chromosomes 0 Can map whole chromosomes use lambda or YACs for cloning Screening for a specific gene with a radioactive gene probe Or antibodies or fluorescence quot l Clone containing gene a y Plaquesnn Auloradingram master plate of replica plate Screening a cDNA libram 7 Bacterial promoter site 7 Eukaryutic DNA insert Expression vector plasmid Translorm E all Colony produting protein of interest Bacterial colonies on agar plate Transler olonies to a replica plate Lyse barterl a to expose proteins Transler proteins to I39IIUOUZHUIOSE sheet Add radiolaheled antibody specillc for protein at interest Dark spot on film 39 quot identities the batterial olony K expressing the V 7 X gene of interest Auloradiogram Page 20 or 25 W Make a gene probe from a known protein sequence o Predrct DNA base sequence rrorn ammo acrd sequencer usmg Geneuc Code 0 syntnesrze DNA probes rrorn tne predrcted gene sequences 0 ans examp e ng e 20 reqmres a rnN or 256 drrrerent ohgonudeoudes to guarantee a penect rnatcn Tnese mrxtures rearWarm Amino acid sequence V Cys Pru Asn Lys Trp Thr His A A Potential oligonucleotide T6 62 AA AA mu Acg CA sequences T T CHRDMDSDME WALKING Use to mapexp ore ong regrons or cnrornosornes by rteratne nybndwzauon subcxonrng and rescreemng o DNA fragments near ends of one done are used to rdentny onger domes wnrcn contam tnerr sequence and adjacent sequences extendrng past tne ongrna DNAs ends ng 6 22 Detected by A39 e f Prepare 5 Fvagment Detected by B r Prepare C Fragment Detected by C r Prepare D Fragment L a 0 3 mm s c d L2 chromosome 7 r Page 21 or 25 EXPRESSION OF CLONED GENES DNA vector delivem to cells I calcium phosphate precipitated DNA a microiriiectiori o virus vectorsSv40 vacciriia retroviruses ex Maloriey murine leukemia virus a GENE GUNquot microproiectiles coated with DNA a liposomes o electroporatiori Expression vectors o Designed to give erricient transcription andtranslation by cloning a gene near a strong promoter o The gene must be cloned in the correct readingrrameWith a properly spaced riposome binding site o lmmunochemical ScreeninngH identirv clones it an antibody to the cloned protein is available SOME EXAMPLES o Proinsulin cDNA was cloned in a plasmid and the proinsulin was made by wcella This is a standard method to express cloded genes as proteins and is the basisror much or the biotechnology industry Fig 6 23 Gene stir Pioinsulin proinsiiim i Reverse 10mm 3 x mnscvlpms plasmid lilletlE mli quot l m Ah Pancreas Mammalian Pmilliulln Recamlzinaul Tcanslnrmed pioinsulm mm plasmid ha elium mRNA Page 22 of 25 0 Genetically engineered giant mice result from injection of somatotropin gene into male pronucleus of a fertilized mouse egg Cd controls expression ofthis gene by its placement under control of the metallothionein gene Fig 632 Mouse metallothionein Nterminal exon promoter Intron Activated by Cd2 Rat growth hormone Plant Genetic Engineering Genes are cloned into the Tiplasmid of Agrobacterium tumefaciens Part ofthis plasmid the TDNA is incorporated into plant chromosomes after Agrobacterium infection DNA which is cloned between the right and left ends of the TDNA can be expressed by quottransformedquot plant cells after integration in a chromosome Figs63334 Page 23 of 25 Tiplasmid of Agrobacterium tumefaciens Tumor morphology and octopine synthesis Octo ine I P I breakdown Agropine breakdown Octopine Ti plasmid Gene disrugtionlreglacement Genes can be inactivated knockout mutant or replaced by a modified or completely new gene by homologous recombination A Targeted gene r A a J I o Mutated gene B E Homologous recombination C l l Mutakion in the targeted gene Page 24 of 25 ENGINEERING NOVEL PROTEINS 0 Modify DNA coding information to get protein with different amino acid sequence 0 These are really novel combinations which would not occur in nature 0 Solid phase synthesis of Whole genes of any type is now possible Sitespecific mutagenesis Hybridize synthetic oligonucleotide with a mismatched base Fig636 Mismatched nucleotide c PrlmErSACACCTT TCCCCGA OH rs39to T chAG Ad c c cts39 Template strand Cassette Mutagenesis Fig637 Cleavage sites 0 Use restriction fragments to combine parts of genes coding for I different domains of different I 2 I proteins ills j 0 introduce a DNA fragment with one or more changes from normal Plasmid gene with original gene Cutwitli endonudeases I and 2 Purify the large fragment II Add new cassette Ligate Purin the large circular DNA C Plasmid with new gene Page 25 of 25 We 0 Can start wrtn a DNA sequence and uttrrnatety rsotate an Unknown protern Atso can start wrtn a known protern and rsotate rts gene th 6738 Wquot quotM nanrmrm 5 mquot pram t Neda quot me WW Enmdndprmmn manta Prepare WEme aman and ynment dvmbudy peum Sequwue peptnte my me annulled pmlew Cuncorcnkn A Don tva Syntnem Screen DNAltbraly WW DNAr vahes hvsmuhem Mum39ng 4 4397 e PmnamDNAhhvaWr Freya sveullL ammuuy mm and sum mun by Wexrnrn Mumng lt5 3 N am CURRENT AND FUTURE APPLICATIONS OF RECDMBINANT DNA TECHNOLOGY 0 cnrornosorne rnapprng and sequencmg o Dtscovery or rnotecutar bases at devetopmem evomuonary retatronsnrps 0 New proterns wrtn newfuncuons or otd proternswrtn newmncuons o nurnan norrnone syntnesrs rn bactena o antNrrat agents 0 ADS vaccme devetoprnent 0 new pnarrnacotogrca agents protetns RNA DNA 0 antrsense RNA therapy 0 rnedrca dragnostrc reagents gene probes tor detectron or genetrc drseases rnrectrons and cancers 0 gene dehvery wrtn drsarrned Wuses to aHevtate drseases caused by known gene detects o agncutura revotutron wrtn anrrnats havtng attered trarts rnore nutntrous ptants neatdrougnt resrstant crops etc o forenstcs r motecutar detectwes
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