Week 3/4 Notes
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Chapter 6 DNA and RNA structure By yourself or with a partner WRITE the de nition of genetic engineering and give 2 general examples of the use of this technology o Endprocessing creating l 339 overhangs Helicases and nucleases 3 a First strand invasion 1 Recombinase g 9 Second strand invasion Recombinase Branched Intermediate 339 5i f1 0 Strand extension DNA polymerase 3 5 i F1 5 I 3 I Figure 1 32 Molecular Biology Principles and Practice 2012 W H Freeman and Company III quotConventiona Genetic Engineering of Eukaryotes tends to be Imprecise Homologous Recombination May be the Key Red strand is broken and blue strand is used as a template to engineer a new gene where the product is shown below It s a new DNA sequence using the blue strand Scheme for promoting homologous recombination TIIIlllIIjijllI ZFNs TALENs or CRISPRCasQ targeted DSB Ill 1111 p In I I I I I I I 1 WT donor Transgene donor vDNA RNA IIIIEIIIIII lilillllili 33111331 Ramalingam et al Genome Biology 2013 142107 Which method is best to introduce dsbreaks a ZFNs ZFNR lt3 A37 G I c T c c L 70 Q 9T T TIAAICClCIATCFqu TGAA 3 ldl VGleNAAT GGGTGAGMCACWTT C Three Technologies used 1 ZFNs Zinc Finger Nucleases Alpha helix of the zinc finger is binding on the nucleotides of the DNA and there is a residue on the zinc finger that can bind to the DNA 3 3 ZFNL If we string several together then they can specify a break in the DNA b TALENs by introducing Fok1 that cleaves but only when there is a dimer TALENL Type II Nucleases used 539 GCTTCTGACACAACTGTGT39I CACTAGCAACCTCAAACAGACACCATGGTGCATCTG 339 339 CGAAGACTGTGTTGACACAAGTGATCGTTGGAGTTTGTCTGTGGTACCACGTAGAC 539 2 TALENSI Transcrlptlon Actlvator Ilke NUCIGUS Modify transcription of the post protein TALEN R Using same Fok1 Type II nucleases and fusing it to the specific proteins RVDI Nll HID N39K N39G where it can change the nucleotides in the sequence Base A C G T Two amino acids that correspond to one base 0 3 CRISPRCASQ Clustered Regularly lnterspersed Short Palindromic Sequences c CR39SPRCAS9 gg CA89 Protein and Guide RNA that has a specific structure that has homology 8 to the DNA sequence and will bind to the DNA and CA89 will be able to a s Zn introduce a double strand break 0 quoti 339 UUUU 8 a a 80 Eag CasQ E23 Guide 0 RNA 3 3 D O 5 CNNNNNNNNNNNNNNNNNNN AAGGCUAGUCCGUUAUCAA llllll CC N N 339 NNNNNNNNNNN quot739quot NNNNNNNNNNNNNNNN 539 IlII 02L lIIII 5 NNNNNNNNNN NNNNNNNNNNNNNNN 3 NN GGNN Chromosome PAM l 23 bp genomic arget sequence Ramalingam et al Genome Biology 2013 142107 Most of Biotechnology is Applied Molecular Biology Nucleic Acid Structure and Chemical Properties are critical to Biotechnology and Molecular Biology Ch 6 NA Structure and Chemical Properties Ch 7 Biotechnology Methods Ch 8 Genomics Ch 9 DNA Topology Ch 10 Structure of chromosomes IChapter 6 DNA and RNA structure What do we need to know about nucleotides How do these characteristics affect nucleic acid structure How do structures vary based on sequence What are some of the key chemical and thermodynamic characteristics of nucleic acids Figure 61 Molecular Biology Principles and Practice 2012 W H Freeman and Company Nucleotide nomenclature Adenosine nucleoside A Nucleoside monophosphate AMP l Nucleoside diphosphate ADP Nucleoside triphosphate ATP Figure 61 2 Molecular Biology Principles and Practice 2012 W H Freeman and Company I Nucleotides I Pneumonic of Purines Pure As Gold also Ag is gold DNA Purines Adenine NHz Guanine 0 Phosphate Purine or pyrimidine S39CHZ o B base 439 139 H H mm H 339 239 H Pyrimidines OH H Cytosine Thymine Uracil H CH3 RNA H H RNA Phosphate Purine or I O pyrimidine 0 o S39CHZ base Figure62a 4 I 1 I Molecular Biology Principles and Practice H H 3 H 2012 W H Freeman and Company OH OH Figure6 2b Molecular Biology Principles and Practice 2012 W H Freeman and Company I Phosphodiester bonds and DNA properties 539 Terminus Write 5 to 3 Phosphodiester bond 3 Phosphodiester bond H H H H 3 H Phosphodiester bond s C H H H H 3 I H H 3 Terminus Figure 66 Molecular Biology Principles and Practice 2012 W H Freeman and Company I ISugar puckering OH OH BDFuranose Figu re 63 Molecular Biology Principles and Practice 4 2012 W H Freeman and Company Know that these are non planar molecules where the 2 or 3 carbon will be sticking out of the plane 3 3 Anitparallel Strands 5 Figure 611 Molecular Biology Principles and Practice 3911 2012 W H Freeman and Company IThe DNA helix Major Groove Adenine H 111 N H 110 N 0 A MmorGroove Thymine CH3 NH N O O kl r GC are closer than AT but not the same Guanine 108 A Major Groove H o N Cytosme oH H Minor Groove Repeats the alter helical structure Slight differences and shortens Beta form Helix Sugar phosphate 5 backbone quotr g v I P c ya One helical turn 34A Q r 39 I t 105 base pairs Major groove Axis Minor groove Base plane 20112 nm Figure 614 Molecular Biology Principles and Practice 2012 W H Freeman and Company I Different Helical Structures I B39DNA Z39DNA BDNA A DNA Z DNA Helix sense Righthanded Righthanded Leftlganded Diameter 20 A 26 A 18 A Base pairs per helical turn 105 11 o 12 o Helix rise per base pair 34 A 26 A 37 A Base tilt in relation to the helix axis 6 20 7 Sugar pucker conformation C2 endo C339 endo C239 endo for pyrimidines Major C3 endo for purines groove Glycosyl bond conformation Anti Anti AM for pyrimidines major syn for purines groove g Figure 61 7b Molecular Biology Principles and Practice Minor 2012W H Freeman and Company Minor groove groove A forms has more angled bonds Left handed helix Figure 617a Molecular Biology Principles and Practice 2012 W H Freeman and Company IConformations of sugars and bases in helices Figure 63 Molecular Biology Principles and Practice 9 2012 W H Freeman and Company a b syn position of guanine Deoxyguanosine C339 endo sugar pucker Deoxythymidine C339 endo sugar pucker Figure 61 6 Molecular Biology Principles and Practice 2012 W H Freeman and Company anti position of guanine Deoxyguanosine C2 endo sugar pucker Deoxythymidine C2 endo sugar pucker Z OH Has Considerable In uence on 2 and 3 Structure Stabilizes A form of RNA a b Figure 625 Molecular Biology Principles and Practice 2012 W H Freeman and Company B A and ZHelices BForm Helix Typical native form of DNA Has a shallow major groove that can bind a protein alphahelix AForm Helix Typical native form of RNA ZForm Helix Occurs with sequences that are GC rich or with salt Which helical form does DNA typically adopt A Aform B Bform C Zform Which helical form does RNA typically adopt A Aform B Bform C Zform Which helical form has a shallow major groove that can bind a protein alphahelix A Aform B Bform C Zform INA Structures Palindromes cruciforms gt2 strands a SS Single Stranded TGCGATACTCATCGCA 5 l 3 gt5I b dS Double Stranded with complimentary y hGCGATMCTchTCGCA y 339 I ACGCTATGAGTAGCGT Palindrome Mirror repeat c MEIAGCZACWZAC3Af i 1 I A Figure 620 Molecular Biology Principles and Practice 2012 W H Freeman and Company Illll TGGTG n m n q gt I A k r 5 m I gtmmngt gt m igtmnmI Guanosine tetraplex Fig u re 621 Molecular Biology Principle and Practice 1012 Wu H hemva and ompany Telomeric structure ends of chromosomes Repeats are special in nucleic acid because they change the structure I RNA Structure I a 1 ug quot1 2 Figure 622 Molecular Biology Principles and Practice 7 2012 W H Freeman and Company a Hairpin Single A c strands Internal loop A A Bulge U 9 A c C A A U A c AcC UU IIC39UACC CGUU GAG Loops 0 AG Stems G quotP A 6 C u G G A G G G c A 0 0 G A o O A G o bl Figure623 Predicted structure of HIV and affects rate of translation Molecular Biology Principles and Practice 0 2012 W H Freeman and Company a Sequence secondary form of RNA because the duplexes are shown for tRNA b comes from a crystal structures of tRNA tertiary form 3D shape mRNA structure may alter translation and folding RNA NonWatsonCrick base pairing and unpaired nucleotides I Figure 624 Molecular Biology Principles and Practice 3999 2012 W H Freeman and Company When there is a single stranded structure different base pairs may form Noncanonical base pair structure Noncanonical deviating from the normal structure Stabling base pairs c 39 C AGCCUGAUGCAG GI CGL GG CGGG A UAAJ U R A 2 c a G c 22 C A39uA A G CG G I AG C c I GG CU A c ACACGGUC A A GA Am amp39 G Guc iCGGlJ CAGGAAGAAG A o o A GUCAAGGUC CGG UIGACA GUM uuuucuuc G GG A A G k9 A AUG Gu UG GU Cu D IEIIIII ngt gtomocnc QC onnngtmgtc CAGA GCU G U U G G A G G c c G G c A G GU Au CG UA AU ES cGGG ccucuuG AGGGGG AACUACUGGA U II II I A ucc GGGGAG chcA cGAlLJIGGCA G A u UAA 1 A A C C CG AU G Ac A II A d Nucleic acids C deamination Chemical Reaction Cytosine Uracil Fig u re 63 3 Molecular Biology Principles and Practice wzoizwiHFveemananwmvaw Occurs 100xday in the human genome Because it is so common organisms have evolved a repair system that will replace the base Favored Unfavored Base methylation in DNA 5Methyldeoxycytidine N6Methyldeoxyadenosine Used by bacteria to identify self versus non self NHZ NH 5 of human C s are methylated and observed on CpG that N occurs in promoters that helps and marks promoters 6 gt K CpG cytosme phosphate guanine N N N NzMethyldeoxyguanosine 5Hydroxymethyldeoxycytidine 0 MHz N CHZOH N H gt 5 k N H3C N N o N H Figure 634a Molecular Biology Principles and Practice C s 2012 W H Freeman and Company Promoter Inhibitor Me CDS Me CpG Enhancing Promotes Z form DNA I Nucleic Acid Denaturation Unfolding of secondary structure Breaks hydrogen bonds Doublehelical DNA Denaturation occurs normally by increasing temperature but could also occur by adding certain chemicals Denaturation Annealing Partially denatured DNA Separated strands Assoaatlon of afrallili n c oils separation Strands by base of strands pairing Figu re 627 Molecular Biology Principles and Practice 2012 W H Freeman and Company I Denaturation T Varies by Sequence I Sections that unfold have fewer hydrogen bonds that hold them together duplex is unstable AT rich Repeatable locations of denaturation with slow heating Tm temperature at which half of DNA is denatured Adding salt will stabilize the duplex and increase the Tm A higher Tm means more energy is required to denature the DNA Singlestranded A260 Doublestranded l 40 60 Tan 100 Temperature C gure 629 Figure 628 Molecular Biology Principles and Practice 33 201 2 w H F reeman a nd Com pan Molecular Biology Principles and Practice 5 2012 W H Freeman and Company a Nucleic acid sample Runagarosegel I Using Physical Properties of NAs electrophoresis C9 More compact shape migrate faster More base pairs migrate slower Agarose gel Transfer to nitrocellulose Nitrocellulose membrane Hybridize with labeled nucleic acid probe b Complementary sequence base pairs PECAM1 Autoradiogram reveals mRNA 39 which fragments annealed 39 with probe GAPDH mRNA Example of a Northern Blot mRNA Figure 632 Molecular Biology Principles and Practice Southern Blot is with DNA 2012 W H Freeman and Company DNA Synthesis The Basis of Synthetic Biology om 5339 BM 0 at 39posntion CH2 DNA can be chemically synthesized Cyanoethyl a protecting group H 37 H 5 W39tft ii NC CH 2 2 NC CH 2 2 4053 DMT O H 93 CH 32CH N CHCH32 4 H DMT Diisopropylamino activating group 0 Protecting Next nucleotide I group removed added 393 CH32CH N CHCH32 R H a A Diisopropylamine byproduct Oxidation to form triester Repeat steps 0 to 6 until all residues are added I K Nc CH 2 0 Remove protecting groups from bases i 9 Remove cyanoethyl groups from phosphates a Cleave chain from silica support Mme e e We e 539 3 Oligonucleotide chain Figure 635 Molecular Biology Principles and Practice 2012 W H Freeman and Company Discuss with your neighbor how current understanding of DNA explains Chargraff and colleagues observations made in the 19405 Base composition often varies from one species to another DNA specimens isolated from different tissues and from different developmental stages of the same species have the same base composition In all cellular DNAs the ratio of A to T and G to C is always the same What model related to DNA metabolism did these rules inform A B C Beadle and Tatum s concept that one gene corresponds to one enzyme Avery s conclusion that DNA was the material from a virulent strain that can transform an avirulent strain Watson and Crick s model of DNA forming an antiparallel double helix with A pairing with T Chapter 7 Studying Genes Fig u re 71 9b Molecular Biology Principles and Practice 0 2012 W H Freeman and Company Cloning PCR Recombinant proteins Understanding protein localization and interactions Scheme for promoting homologous recombination ie genome editing Random insertion in a 1 1 1 1 1 1 1 1 1 1 1 I 1 1 transcriptionally active locanon ZFNs TALENs or CRISPRCasQ targeted DSB llllll 111 IE WT donor vDNA HEIDIEDIE IIIIEIIIIII liiillllili Need to first be able to clone genes to even begin gene editing gt I l 1 1 1 l I 1 1 Transgene donor DNA Ramalingam et al Genome Biology 2013 142107 What is superior about genome editing relative to conventional genetic engineering A Genome editing is faster B Genome editing targets a speci c site in the genome C Genome editing is new and cool General Cloning Scheme DNA gt Isolate gt Introduce it gt amplify make many copies K Amplifyj Repeat steps to move to eukaryote DNA cloning 11000 copies 5100 kb 10quot6 gt 4Mbp p 0 Gene inserted into plasmid Bacterial Plasmid chromosome it gene of Recombinant 39merest DNA of DNA Plasmld chromosome 9 Plasmid put into bacterial cell Recombinant bacterium httpbiol151nicerwebcomLockedmediach202002aGeneConingPreviewLjpg DNA cloning 5 generalfsteps Traditional Selecting Need DNA to clone and a vector plasmid Cutting Restriction Endonuclease enzyme that performs cutting that cuts in the middle and not at the end Joining Ligate using ligase Moving Transformation in vitro test tube gt in vivo bacteria typically Identifying Selection with antibiotics m 39xi Cloning m Eukaryotlc xzelx Vector 393 chromosome plasmid quotquotV V a DNA fragment of interest 0 Cloning vector is obtained by cleaving is cleaved with chromosome with a restriction restriction endonuclease endonuclease m g j 3 e Fragments are ligated to the prepared cloning vector 1 DNA ligase Recombinant vector DNA is introduced into the host cell e Propagation cloning of transformed cell produces many copies of recombinant DNA Figure 71 Molecular Biology Principles and Practice 2012 W H Freeman and Company I Restriction endonucleases I Endonucleases bind to and cleave DNA at specific sites Makes a cut leaving a 5 phosphate and 3 is turned into a OH group Endo cleaves in the middle exo cleaves at the end Arrows show where nucleases make a cut in the strands Table 72 Recognition Sequences for Some Type II Restriction Endonucleases l l BamHl 539GGATCC339 Hindlll 539AAGCTT 339 CCTAGG TTCGAA 1 3 l Named for speCIfIc Clal 539ATCGAT339 Notl 539GCGGCCGC339 site and organisms TAGCTA CGCCGGCG as T T l x l EcoRI 539GAATTC339 Pstl 539CTGCAG339 CTTAAG GACGTC T l Jr Jr EcoRV 539GATATC339 Pvull 539CAGCTG 339 CTATAG GTCGAC 1 l W l Haelll 539GGCC339 Tth111l 539GACNNNGTC339 CCGG CTGNNNCAG T T Note Arrows denote phosphodiester bonds cleaved by each restriction endonuclease Asterisks mark bases that are methylated by the corresponding methyltransferase where known N denotes any base Each enzyme name consists of a threeletter abbreviation of the bacterial species from which it is derived sometimes followed by a strain designation and a Roman numeral to distinguish restriction endonucleases isolated from the same bacterial species or strain Thus BamHl is the rst I restriction endonuclease characterized from Bacillus amyloliquefaciens strain H Table 72 Molecular Biology Principles and Practice 2012 W H Freeman and Company Restriction endonucleases sticky vs blunt 5 Blunt ends 3 GGTG GC TAG CCACTTAA TCG ATCGACATCG L Staggered sticky ends Figure 72 part 1 Molecular Biology Principles and Practice 2012 W H Freeman and Company Restriction endonucleases sticky vs blunt Chromosomal DNA Cleavage Recognition Cleavage site sequences site GG39I quotGC o o o o o o o EcoRl Pvull restriction restriction endonuclease endonuclease GGTG C39I39GTAGC o o f j o o o Sticky ends Blunt ends Figure 72 part 1 Molecular Biology Principles and Practice 201 2 W H Freeman and Company Cloning vectors plasmids EcoRl Pstl Sall 39 v Ampicillin Tetracycline r resistance resistance AmpR TetR pBR322 4361 bp Origin of replication ori Pvull Figure 74 Molecular Biology Principles and Practice 939 2012 W H Freeman and Company Cloning vectors arti cial chromosomes Cloning sites within IacZ F plasmid pargenes Figure 76 part 1 Molecular Biology Principles and Practice 0 2012 W H Freeman and Company ICloning vectors polylinkers I Synthetic polylinker EcoRl SmaI EcoRl stickyend PS I Hmdlquot I BaumquotW stickyend S IAATTCCTGCAGAAGCTTCCGGATCCCCGGG GGACGTCTTCGAAGGCCTAGGGGCCCTTAAI Plasmid cloning vector cleaved with EcoRI V J v E IZl E a DNA 6 O w 0 llgase V Figure 73 Molecular Biology Principles and Practice 2012 W H Freeman and Company I Cloning into vectors GG39139 C39I39GTAGC CCA GACATCG Sticky ends Blunt ends a b Plasmid cloning vector cleaved with EcoRI and Pvull Figure 72 part 2 Molecular Biology Principles and Practice 2012 W H Freeman and Company I Moving the recombinant DNA into cells Q DNA plasmid Bacterial chromosome C Uptake of plasmid CD Stable transformation Selecting for cells with the recombinant DNA EcoRl BamHl Pstl Ampicillin Tetracycline resistance resistance AmpR TetR pBR322 4361 bp Origin of replication ori Pvull Figure 74 Molecular Biology Principles and Practice a 2012 W H Freeman and Company httpwwwccpberinorgimg72bacteria on an Agarplatejpg Cloning into vectors producing many copies Which of the following is the correct order for traditional gene cloning Isolate DNA Ligate Restriction digest Transform Select Isolate DNA Restriction digest Transform Ligate Select Isolate DNA Select Transform Restriction Digest Ligate Ligate Selct Isolate DNA Transform Restriction Digest Isolate DNA Restriction digest Ligate Transform Select P190570 General Cloning Scheme DNA gt I introduce I gt PCR Targeted ampli cation of DNA segments Template strand 3 5 4 lt2 9 T C T C G A 9 9 PPP 5 3 Primer strand Incoming nucleotide Fig 524 from Tropp Molecular Biology Genes to Proteins PCR The three stages D8 339 W DNA 5 DENATURATION I 3 T39ITTI3939l39T39IT3939ITTI39TI39TI39l3939IT39ITTI39TI39TI39I39TT39ITTI39TI39TI39I39TT W ANNEALING P1 P2 mm Illlllllllllllllllllllllllllllllllllllllll I vL mm 5 50 C gt EXTENSION 72 C httpwww mnhu educowriesPCRgif I PCR How does it work Region of target DNA to be ampli ed I I 3 I I 5 I I 339I 39 I 5 I I 539 539I I 339I I 539 I I 539 539I I 339I l I l I I 4 539I l Figure 79a part 1 Molecular Biology Principles and Practice 2012 W H Freeman and Company I PCR How does it work 1 quot1 i quot1 HMIIIH WEE N N H U After 25 cycles the target sequence has been ampli ed about 105fold Figure 79a part 2 Molecular Biology Principles and Practice 2012 W H Freeman and Company Polymerases in PCR E coli DNA polymerase NH COO Domain 1 Domain 2 5 gt 3 3 gt5 Domain 3 exonuolease exonuolease Polymerase Figs 526 amp 527 From Tropp Molecular Biology Genes to Proteins IPolymerases in PCRI How We Know 7 Figure 2 Molecular Blolog y Principles and Practice 0 2012 W H Freeman and Company General Cloning Scheme DNA gt I introduce I gt ICloning Expressing proteins in bacteria Bacterial promoter P and operator 0 sequences Gene encoding polylinker repressor that binds O and regulates P Ribosome Transcription binding site termination sequence ori Selectable genetic marker eg antibiotic resistance Figure 71 3 Molecular Biology Principles and Practice 0 2012 W H Freeman and Company 0 Q 39 b 2 6 3 z Mr 0 s 97000 39 r 66000 39 31000 p Figure 714 Molecular Biology Principles and Practice 393quot 2012 W H Freeman and Company Cloning Expressing proteins in other organisms Figaro 71 6 quhrmologyhinnles39 WWII 101 w n Drunun mu Lumunn httpstaticddmcdncomgiflightvirus1jpg ICloning Expressing proteins that are altered Gene for target protein 2 Gene for GST Transcription Gene for fusion protein 1 OC OC Express fusion protein in cell cell protein mixture ITagged proteins in protein puri cation I Prepare cell extract containing fusion protein as part of the 335 Elute fusion protein c Glutathione V anchored to Add protein medium mixture binds to column GSTtag I gt V Other 3 proteins 7 ow through column 0 O Q o 00 O o O o 0 Figure 718b Molecular Biology Principles and Practice 2012 W H Freeman and Company Western Blot Run agarose gel electrophoresis Agarose gel 751 Transfer to membrane Membrane Wash with rst antibody 38394 Wash with second antibody l Visualization Autoradiogram Figure 722a Molecular Biology Principles and Practice 2012 W H Freeman and Company a Gal4p DNA binding domain Protein X Gal4p binding site Protein Y Gal4p activation domain Increased transcription polymerase b Yeast strain 1 with Gal4pbinding domain fusions Yeast strain 2 with Gal4pactivation domain fusions Mate to produce diploid cells Plate on medium requiring interaction of the binding and activation domains for cell V survival Survivors form colonies Sequence fusion proteins to identify which proteins are interacting Figure 725 Molecular Biology Principles and Practice 2012 W H Freeman and Company Protein fusions vvuv vv39 v Transcription 2 Gene for fusion protein Protein fusions GFP tvvvivv c H d T l ea al a w Ventral Figure 719 Molecular Biology Principles and Practice 0 2012 W H Freeman and Company The importance of recombinant DNA