Class Note for ECOL 320 at UA
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Date Created: 02/06/15
echnology lquot Ww i Asilomar 1973 Singer Zinder Brenner Berg Foreign DNA Remidicn Er me Fragment Reslridion cut Junmng al cuheswe ends i Hnmm 39 3 Eec anBnl 39 5 human DNA Technology The following are some of the most important molecular methods we Will be using in this course They Will be used among other things for 0 Sequencing genes and genomes 0 PCR 0 Making recombinant DNA 0 Detecting mutations at the molecular level 0 Studying the control of gene action DNA Sequencing Reminder DNA polymerase adds nucleotides one at a time to a single nucleotide strand in the 539 to 339 direction using a DNA template and RNA or DNA primers 0 Polymerase DNA template pair of DNA primers gt copy DNA sequence between the primers Interrupt reaction by dideoxynucloside triphosphates which Cambridge biochemist Fred can have labels attached to them You don t need to know Sang W011 N0b 1 PfiZ S fOf the method sequencing is done for you at facilities at UA 56911161 ng prOteinS and DNA and elsewhere Sequence both strands Proofread sequences i i f if m 7 i if i i i i if 1397 F39i i i in a A AEAE m 4311 iii Tip r TATAEA TEE it fan1 139 mm Relative light absorbance at 260 nm 140 130 120 110 100 DNA Denaturation and Renaturation Melting and Annealing EDenatured i Strand separation Kgsingle strands rther Temperature at which enaturat39on half the base pairs are a denatured and half remain intact 7 Denaturation begins Double stranded DNA muIIllmllllmnlmlml 50 70 Tm 90 100110 Temperature C 30 DNA Denaturation and Renaturation Melting and Annealing Molecule A has 30 GC Molecule B has 45 GC Which one Will melt at a lower temperature Polymerase Chain Reaction PCR1 A prede ned DNA sequence in the genome can be greatly ampli ed by repeated Polymerization cycles using 2 primers which hybridize to the ends of the target DNA In each cycle the amount of target DNA is doubled After 10 20 and 30 cycles there is a 1000 million and billionfold ampli cation respectively A Original duplex DNA DNA IIIllI g 2 gg IIIIIIIlllIllIIIgX I gz IIIlII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII B First cycle primers attached IIIIIIKER39I39GKCG39I39IIIIIIIIIIIIIIIIBTK39I39GEK39I39GIIIIII I GATACGTAC Primer Region to be amplified Primel GATACGTAC quotIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII C Elongation DNA synthesis Illlll g zg g zIIIIIIIIIIIIIIIlgzgzgggzgllllll I39llIIIIIIIIIIIIIIIIIIIIIIIIIllllllll 39iggxgigggiillIIIIIIIIIIligzgzgggzgllllll IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII D Second cycle after elongation gggzg g filllllllllIlllligxgzggizgllllll lullIIIIIIIIIIIIIIIIIIIIIIIIIIrlrg E Third cycle after elongation xsiiaii iilllllllllllIlllaiiiaEXia39 iii iiii llIIIllllllllllgi i i i i Polymerase Chain Reaction PCR2 A First cycle 39 DNA se uence to I gtbe amplcllfied 1VDenaiuration annealing Primer oligonucleotides quot has 3 steps h I replicati a Melting of DNA B Secondcvcle e g 94 2 L b Hybridization 0f 74 primer e g 40 03 20 bP 10mg c 5333 J JA39 iL ge c39Z A l J 0 DNA synthesis eg 68 PCR Requires Thermostable Polymerase Thermus aquaticus bacterium found in hot springs of Yellowstone National Park in 1969 Survives at up to 80 C DNA polymerase from Thermus aquaticus Taq DNA Polymerase has optimum temperature 80 Monetary value of natural habitats and organisms If we don t conserve them we won t be able to eXploit them Size separation of DNA fragments by electrophoresis in agarose gels Slots for Bands visible after samples suitable treatment A Electrode r W i ESIfJ2n t Gel it it Direction of it movement gt G A 9 DNA is negatively charged due to phosphates on its surface As a result it moves towards the positive pole Distance migrated by a DNA fragment in a gel is related to log10 of its size Heaviest fragment Direction of movement Lightest fragment Gel Electrophoresis to Verify Amplification PCR Very powerful amplifies tiny amounts of contaminating DNA Should have negative control all reagents but no added DNA If we amplified the correct DNA segment we should get a product of the size of that segment Tiqu 1 Positive Tumsz TnsIIES 39 Control 39 Size Standard QampA Question A pair of PCR primers are designed to be complementary to sites 120 135 and 440 465 of the human B globin gene What is the expected size of the amplification product if these primers are used With a human DNA template QampA Question A pair of PCR primers are designed to be complementary to sites 120 135 and 440 465 of the human B globin gene What is the expected size of the amplification product if these primers are used With a human DNA template Answer 120 465 1 346 bp Question If the template is DNA from a baboon do we expect to get the same size QampA Question A pair of PCR primers are designed to be complementary to sites 120 135 and 440 465 of the human B globin gene What is the expected size of the amplification product if these primers are used with a human DNA template Answer 120 465 345 bp Question If the template is DNA from a baboon what size product do we expect Answer Similar but not necessarily identical because the genes of these closely related animals are similar but not identical and can differ in length as well as in sequence Memorize and understand 21 2 224 23 8 24 16 2532 26 64 27 128 28 256 29 512 210 1024 z103 211x 2 X103 212z4 X103 220 z 106 Powers of 2 Useful in thinking about 0 Anything that doubles in number PCR increase in cell number during binary fission Dilution series in the lab Genetic ratios 11 31 9331 etc etc Engineering DNA The same enzymes that are used in DNA replication can be used to rearrange DNA molecules at Will Endonuclease breaks 539 339 phosphodiester bonds in backbone of DNA quotnicksquot DNA Restriction endonucleases are enzymes made by bacteria They recognize and cleave specific sequences These sequences are usually palindromes Eg EcoRI 539 GIA A T T C 339 339 C T T A AIG 539 6 cutter staggered Avall 539GIGWCC339 WonrT 339 C C W GIG 539 5 cutter staggered degenerate Sau3A 539IG A T C 339 339 C T A Gl539 4 cutter staggered Alul 539 A GIC T 339 339 T CIG A 539 4 cutter blunt end SVVVSVJiVi BLUSEUULQVJUW3333VJLLVV SVVVJUSLLLJVULE iii i vi V i i i i W ii iiL i vi 1 HQBJU EDUUUH Q BVVVSVUiVi B1399 j i V3 3V344TSVVVJBSLLLJVJLE iii i ivBSUWB i v i i iSWVLLSLLLSJDWVVBiSWE UWVWBVJLVi 51339UBLSVD j V iLV SVVVUQSLLLJVJLE iii i vL S i i i iBWWiL iii vvv i i smo 13033 AAOH VNG Buueeug ug C 39E ii ii i i L WBSWBLJVBBLBJVJJvLIL P iB V 39555i i VB W i i V i Bi l L S 13 3596 I in EILLDVJCILLCJEIZIUZIWCWELi J 393ULUWBEIWBLZIVBEILEIIJ VCICIVL Ii JWVEILBEWVZIEIEEISLEILZIVEI39 EIEIWEILCIZILZWEILCICWZIEILEIEILV if E 301112811 pus1un1g 39VNCl 3919311 3VNC JO 9u0qgt1312q u spuoq 191s91p0qu0qd gg s9gt112u1 ase n Jepugwea mm 1UQLILE1UJCICIEIJ E W10 I I 1 W VHO 395 LBSBLDUi iv l VWV L iW l i W iJJvBBLBjV WiI EV333VSBVBVLLWJLLLSVJJWLSWBV iiVW L j lS iW i Vi VWv i i i i i vSVii iii V33vi liv i i i g NIJ31UE LUEEJ 21 mm m 1JEILJJEELU g ELVVLBVVJLBBLVULJLB VVLLUWVBVLJLVBJUI EVLLVJLLLSVJUVLQVSVJiiV QLLULVBVLUBB IEI39JC39EI LinquotA D313LJ133J El VNICI I p 393 1385133 vanavaa E i V 395 ii jiL V W ijL i VS V L vvUSSSBLDLJWBVSSVSLJUii V C Si V Si iV IHDDE HILM Dal lJls J v VNC 39S LS i ji lvVLBWWWDLBBLWDLDLBWWLJJVVBWLJLVBJUI HVUUJVSSWBWLLVULLLBW l vSiiWWSiL iv i fr Q VHO m SiliV ii DVJVSLJLJDLJVSBVVLi iSJV iI JVVQLBBVVJSSBBLSLUVSWSSVBLJUliVv i i iv V VHO VNCI lueulqwooea VVLLJVSSLBJVJJVL39 VVLLJVSBLSJ33VLI f 391 Cloning DNA Making Cells Amplify DNA for US Plasmid small DNA molecule that can replicate inside bacterial cell independently of cell chromosome to achieve high copy number 3 cut with Wm ex plesmid veetdr with my gate quot 0 unique 9er site Vectm mam t enRI fragment f to he eldned uquot l bf 0 74 0 J ado O J 0 OJ transformation plateml O O medium to amp select sells 393 euleng x with vector ff eldne E m insert r nest I isolateur39ff g 139 139Jr E39Ni39i39i asquot re ll I 3955 g A Feleetrepherese if 1 isdlate 5 J fragment I h Cloning DNA Clomng Vector needs 55mm Repllcatlon onger Selectable genes e g anublotlc reslstance Resmcuon sltes ln genes F5 um um may he sumewhere else Em lrcle Selecting cells wmn vector insert Join DNA em with restriction enzyme 10 plasmid em wmn same enzyme eg Banl Transform bacteria wmn DNA egt cells cells plasmid cells plasmid insert Antibiotic sensitiveresistant phenotypes Cloning DNA Clomng Vector needs 55mm Repllcatlon onger Selectable genes e g anublotlc reslstance Resmcuon sltes ln genes Fm um um may he sumewhere else Em lrcle Selecting cells wmn vector lnsen Join DNA cm with restriction enzyme 10 plasmid cm wmn same enzyme eg Banl Transform bacteria wmn DNA egt cells amps lets cells plasmid ampR beLR cells plasmid lnsen ampR bets IHow would I select cells plasmid insert
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