onononononono nonononononon onononononono □onononononon MASARYKOVA UNIVERZITA ononOu^uOnono nonononononon onononononono onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono □onononononon onononononono □ onononononon onononononono □ onononononon onononononono □ onononononon onononononono □ onononononon onononononono □ onononononon onononononono □ onononononon onononononono □ onononononon Design sekvence PCR primerů Hana Konečná CEITEC - MU Centrální laboratoř - Proteomika Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. 17^ /jp. WBm I ministerstvo školství, OPVzděBvínt EVROPSKÁ UNIE ^0 ■ mládeže a tělovýchovy pro konkurenceschopnost □ onononononon onononononono INVESTICE DO ROZVOJE VZDĚLÁVÁNÍ □ onononononon onononononono □onononononon onononononono □ onononononon onononononono □ onononononon onononononono nonononononon onononononono □onononononon O n o n o CL'1 o □ o n o o n □ O IF^NJ^Vao □ ononOuwuOnono nonononononon onononononono MASARYKOVA UNIVERZITA definice aplikace modifikace syntéza purifikace kontrola kvality i OLIGONUKLEOTIDY design sekvence zásady navrhování software OLIGO 7 praktická ukázka Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. ministerstvo školství, mládeže a tělovýchovy EVROPSKÁ UNIE ■ INVESTICE DO ROZVOJE VZDĚLÁVÁN OP Vzdělávání pro konkurenceschopnost Vi onononononono nonononononon onononononono □onononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.mum.cz oligonukleotid krátká jed no řetězcová struktura DNA nebo RNA (event. PNA) hydroxyl na obou koncích (normálně na 5'- konci fosfát) oligonukleotid i syntetický oligonukleotid i primer orientace! polymeráza! 5' end H 3' and nonononononon onononononono □onononononon onononononono □onononononon O H 'O—P=0 H2Cs- jol 3*1 O H O—P=0 i CH H onononononono nonononononon onononononono □onononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz Aplikace syntetických oligonukleotidů • syntéza genů • primery pro syntézu komplementární DNA PCR, Real-Time PC R • hybridizační sondy pro klonování • místně cílená mutageneza • sekvenování • diagnostika - testy a biosensory • gene arrays • blokace genové exprese antisense oligo • potenciální léčiva • NMR studia interakcí DNA-protein • strukturální rentgenová analýza NA nonononononon onononononono □onononononon onononononono □onononononon onononononono nonononononon onononononono □onononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz Modifikace degenerace konce řetězce báze fosfát cukr PNA phosphodiester hrldge O - P S-o-deoxyrlbose í»ugar} onononononono nonononononon onononononono □onononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz Degenerované oligonukleotidy Příklady: ACG TAC GTA CGT ACG TAC nedegenerovaný ACG TAM GTA CGT ACG TAC M = A/C ACG TAC GTA CDT ACG TAC D = A/G/T ACG TAC GTA CGT ACG NAC N = A/C/G/T onononononono nonononononon onononononono □onononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA Degenerované oligonukleotidy 2- deoxyinosin univerzální báze: 3- nitropyrrol 5-nitroindol nonononononon onononononono □onononononon onononononono □onononononon www.mum.cz M A or C R A or G W A or T S C or G Y C or T K G or T V A or C or G H A or C or T D A or G or T B C or G or T N G or A or T or C X G or A or T or C onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA □onononononon onononononono www.muni.cz 5' Modifikace na 5'- konci fosforylace postsyntetické modifikace —- aminoskupina -► thioskupina digoxigenin -► biotin enzymy psoralen akridin cholesterol sekvenování -► fluoresc. barviva fragmentační analýza zhášedla gene arrays 2,4-dinitrofenyl Real-Time PCR TBR-chelát spacer větvení □onononononon onononononono □onononononon onononononono □onononononon blokáda onononononono □onononononon onononononono □ODonoDOnonoD onononononono MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono Modifikace na 3'- konci ^^^^b 3' derivatizovaná matrice fosfát thioskupina —► aminoskupina spacer akridin —> biotin —► fluorescbarviva —* zhášedla cholesterol 2?4-dinitrofenyl □onononononon onononononono □onononononon onononononono nonononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono □onononononon Real-Time PCR 2x značená sonda REPORTER QUENCHER HHWURDFRMEH V 2, Strand displacement: When the probe is intact, the reporter dye emission is quenched. r i a1 r 5_9. □onononononon onononononono □onononononon onononononono □onononononon 3, Cleavage: During each extension cycler the DNA polymerase cleaves the reporter dye- from the probe. r_2ZV_9, ľ 9' 4, Polymerization completed: Once separated from the quencher, the reporter dye emits its characteristic fluorescence. I". r s1. ľ i" onononononono nonononononon onononononono □onononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono Další modifikace fosforothioáty «-páteř fosforodithioáty H-fosfonáty metylfosfonáty cukr modifikace v 2'pozici modifikace ribózové jednotky □onononononon onononononono □onononononon onononononono □onononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono o □ o □ o □ MASARYKOVA UNIVERZITA www.mum.cz Terapeutika nedegradována nukleázami! modifikace fosfodiesterové vazby 0 1 0 = P I o -s I 0=P-CK i O. 0 1 O-P-NH-alkyl I O. phosphorothioate methylphosphonate phosphoramidate l i"2 ? CH2 H3C-N 0 = P-BHľ l 3 I 1 3 3'-thioforniacetal methylene(methylimlnio) boranophosphate onononononono nonononononon onononononono nonononononon ododododododo dodododododod iiliiiiiiiiil MASARYKOVA UNIVERZITA www.muni.cz dodododododod ododododododo ANTISENSE oligonukleotid • oligonukleotid nebo analog • komplementární k segmentu RNA nebo DNA • vazbou inhibuje jejich normální funkci Antigene (triplex) Rlbozyme Antisense Sense/Aptamer □ODODOPODOnon ododododododo □ odododododod OnonODODOnono □ODODOPODOnon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono Peptidonukleová kyselina pna N^ťermiiiäľ- nenabitá molekula vazba k DNA/RNA N-(2-aminoethyl)-glycin □onononononon onononononono □onononononon onononononono □onononononon C-terminal 0=P-0" DNA 3'-end onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz nonononononon Peptidonukleová kyselina • nenabitá molekula • vazba k DNA/RNA N-(2-aminoethyl)-glycin nonononononon onononononono □onononononon onononononono nonononononon PNA « ... *. vr.l.. HN ř v B DNA Onononononono nonononononon onononononono nonononononon Onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz Vlastnosti PNA • vysoká termostabilita • Tm nezávisí na obsahu solí • vyšší specifická • vyšší afinita • rezistentní k enzymům... nonononononon onononononono □onononononon onononononono nonononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono Locked Nucleic Acid 2'-0, 4'-C methylenový můstek potlačená flexibilita ribofuranózového kruhu struktura je zamčena do rigidní C3-endo konformace zlepšená hybridizace výjimečná biostabilita onononononono ^^^^^^^^^ji o n o □ o n H> □ o □ o □ o H] o n o n o n ip9J}sojd epoAzeq. puo>|-,g >| 90U0>|-x po . jZBJ 9UA9d BU ez9juAs • npno8|>|nuo6!|o e ZD'jUnUJ'MMM o n o n o VllZ^3AINn VAOIAWSVW I ill I o □ o □ o □ o □ o □ o n o n o ononono □ O □ O □ 0 □ o □ o □ o □ o n o n o n 0 n ononono n o n o n o n ononono n o n o n o n onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz OLIGONUKLEOTIDY design syntéza purifikace * -i Ji f I I T/M M EXPEDITE 8909 onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz EXPEDITE 8909 • rychlost • nízká spotřeba reagencií • několik koncentračních rozsahů • dvě paralelní syntézy • protokoly pro TNA, RNA, PNA, fosforothioáty • Workstation: možnost editace základních protokolů -syntéza modifikací (máčení biotinem, fluroscenčními značkami, degenerované ol.gonukleotidy, užití inosinu, aminoderiváty aj.) □onononononon onononononono nonononononon onononononono □onononononon onononononono □onononononon onononononono □onononononon MASARYKOVA UNIVERZITA www.mum.cz Kontrola kvality HPLC Perfúzní chromatografie anex RP 190.bio - 20.Cul i M j r "Sk í -J onononononono □onononononon onononononono □onononononon OnODODODODODO nonononononon iiiiiilliiiii MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono 1341ong.bio - 20.0jil Perfúzní chromatografie POROS klasický sorbent □ onononononon onononononono noDOnonononon onononononono nonononononon POROS 0.15- 0.10- 0.05- 0.00 100 134.bio - 2 0.0ul 2 6 0 n m 0.15- 0.10- 0.05- 0.00- forot. ŕrd( Ml. .0 2.5 Min 100 -50 onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono Maldi-TOF MS ce 7103 . Mass/charge D ■ ° CapíEary efcctaophoirais trace onononononono □onononononon onononononono nonononononon onononononono □onononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz u u u u page g$ >r *r □onononononon onononononono nonononononon onononononono □onononononon 85 85 7£ 75" 65 6£ cntde purffarf crude purged cwde purif&t onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon PURIFIKACE Sephadex RP cartridge HPLC MASARYKOVA UNIVERZITA www.mum.cz l?Q,bl9 - 20.Opi nonononononon onononononono □onononononon onononononono □onononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz DESIGN OLIGONUKLEOTIDU • manuální • počítačový www.protocol-online.org/prot/Research_Tools/Online_Tools/Oligo_Design/index.html Hlavní kritéria pro sekvenci PCR primem • vysoce specifické • netvoří dimery a vlásenky • stabilní duplexy s aktivní sekvencí • nepříliš stabilní 3'-konec onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz ligo OLIGO 6 • PCR primery, • hybridizační sondy • sekvenační primery OLIGO 7 (od roku 2008) • TaqMan sondy • primery pro nested PCR • molecular beacons siRNA onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono Terminologie PCR primerů forward primer... část sekvence + vlákna reverse primer... část sekvence - vlákna pos: I 350 tm: | 57.1 2G3 273 283 233 333 3.3 323 333 30 .......i.........i.........i.........i.........i.........i.........i.........i.........i......... 350 3C3 373 .........i.........i........ TTAATGCCTGGCTGTWTACTCAa 111 lAAíGATCíTATTíACCCTATíTCíGAACATíACAAAAACAAACíGCíAííACGATGíCTAATTACATl "GAACAAACAGCAGAGA«AAGTWt_C AATTAC GGACC G AC ACTG ATGAGTG AAAAATTC CTAC C ATAACTC G GATACAC C CTTCTACTCTTTTTGTTTG CCCCTCCTG CTAC C G ATTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG ......... ACTCÍÍATACACCCTTCTACTC .............................. CCTCCTCCTACCGATTAA LHPGCDY5LFKDCIEPHWEDEKHKRGGRW ITLHKQQRR5 PL UPPER - FORWARD 5'- + 5'- - 3'- LEFT —► ■ 3' ■5' 5' LOWER - REVERSE - RIGHT □onononononon onononononono □onononononon onononononono □onononononon onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono Terminologie forward primer... část sekvence + vlákna reverse primer... část sekvence - vlákna pos: tm: I 5ŤX ?G3 ,270 283 233 333 3.3 323 333 3<^3 .......i.........i.........i.........i.........i.........i.........i.........i.........i......... 3Í3 3G3 373 .........i.........i........ TTAATGCCTCGCTGTGACTACKAt 1111 lAAGíATíGTATTíAíCCTATGTGíGMCATíAíAAAMCAAACíGííAíGACíATGíCTAATTACATl "GAACAAACAGCAGAGACGAAGTGACCTC AATTAC G G AC C G AC ACTGATG AGTG AAAAATTC CTAC CATAACTC G GATACAC C CTTCTACTC11111GTTTG C C C CTC CTG CTAC C G ATTAATGTAACTTGTTTGTC GTCTCTG CTTC ACTG GAG ......... ACTtíGATACACtCTTCTACTC L.MPGCDY5L.FKDGIĚPMHĚDĚKNKRGGRWLITL NKQQRR5DL UPPER - FORWARD - LEFT 5' » polymeráza-- + 5' — -y y-1 iv polymeráza * 5' LOWER - REVERSE - RIGHT □onononononon onononononono □onononononon onononononono □onononononon onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono Nasedání PCR primerů pos: f ' tm: | Šfl 2G3 .270 283 230 333 l. 3 323 3<^3 .......i.........i.........i.........i.........i.........i.........i.........i.........i......... ÍÍ3 3C3 i?3 .........1.........1........ TTAATGttTCGtTGTGACTAtKAt 1111 lAACtATtCTATTCAtCCTATCTCtCAACAKACAAAAACAAACCCttAÍCACCATCtCTAATTACATl "GAACAAACACCAGAGACCAACTCACCTC AATTAC G G AC C G AC ACTGATG AGTG AAAAATTC CTAC CATAACTC G GATACAC C CTTCTACTC11111GTTTG C C C CTC CTG CTAC C G ATTAATGTAACTTGTTTGTC GTCTCTG CTTC ACTGrGAG .........ACKÍGATACACÍCTTCTACTC ■l-l-IHHH-l-l-l-IHHHH-l-l-l-IHHH-l-l-l-l-IHHH-l £ C T C CTC C TAC C G ATTAA LMPGCDY5LFKDGIEPMÍEDEKNKRGGRWLITL NKQQRR5 DL UPPER - FORWARD - LEFT + 5'-- 3'- 5' □onononononon onononononono □onononononon onononononono □onononononon 5' 3' 5' LOWER- REVERSE - RIGHT onononononono nonononononon onononononono □onononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono 5'CTTCTG CTCAATCTTTCTAC 3' FORWARD + R 1 ATGG "TTCTG CTCAATCTTT CTAC AACCAA AGCTCTGTCT TGAAAATGAA w 51 TGTGATGGTT GTGGACGATG ATGATGTTTT CCTTGATATC ATGTGAGGGA 101 TGCTTGAAGA CTCCAAATAC AGAGGTAATT AAATATTATT ATCATATTAT 151 ATATAATATG TTATTGATTT TTTGTTTGTG ATTTGATTTA GATTTTTATT 201 TCTATGATTT CTTAGGATGA AATAGAATTT TTGGAGAAAC AACTAGGAGT 251 TTTAAAAAGA AAACTTGAAT TTTGAGAAAT TGAAAGATGT TATATATATA 301 TGTGAAAATT TAAGAATTAT TCTTCTAAAT GATCGGGATT CGGTTTAGAT 351 GTAGAGATCT AGAATTTTGA ATTGAGGTAT TCTTGTTTTG ATGGCTTTGA 401 GACGAATAGT TTGATTGATA AAAAAAATTC TAACGAATAT GATATATAAA 451 GTTTATTTTC TTTTTGTGAA ACGATACTTT ATACTATGTA ACTTTTTTAA 501 GAGATTATTG AAAATAGTTT ATTTATAAAA TAGTAAGCTA TTGTTGAATT 551 AAAAAAAAAA aaaaaatt^t AAATrcTnTTTnrAAAmAr. ATfrrf^ATTTA 601 TCTTAGTTT/IAAACTAGCTG ATATTCTTCA AATCGACTGT TCTTATAAGT 651 AATGAAGCA/ITTAQCATCAA TmCAATAAA^TGTAAAGAC TTGAATGAAA 701 ATGGTGATTT TAAAGAATAT GTTTTACTTA TGTTATGAAC TATCTGAAAT 751 TTGTGAAATA TTTCATAACT AATGTGGAAA ACTATATAAC CCCTCCATAC 801 AAAACGTAAG TAAAATTTAT GAAATGCTAT GATTTTTAAA GGTTAAACCA 851 ATGAAAAAGT AATAATTCTT GGTACTTGGA ATATTTTTGT GATTATATTT 901 TAGTTTATTA ATTTTATTTT GATTAAATGG TTTTAGATGC ATGAGTTATG 951 GAGATCGCAG TTATAGCTGT AGACGATCCG AAGAAAGGAT TATCTACTCT 1001 AAAAATTCAA CGAGACAATA TAGATCTCAT AATGAGAGAT TATTATATGC 1051 CTGGTATGAA CGGTTTACAA CTCAAAAAAC AAATCACTCA GGAATTTGGA 1101 AATTTACGGG TCTTAGGTAA GATTTTTTGT TCTTTAGAAC TTAAATTAAA 3' d£:T£AAGAATATCAGCTAGTTT 3' REVERSE onononononono □onononononon onononononono □onononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono File: Human 4E.seq Sequence DIN A 5e que nee 5elected Oligo Position Length # Feature Location Sequence Length: 1868 nt XD □ Forward Primer 259 18 1 source -18..1850 Reading Frame: + L □ Reverse Primer 32S 18 2 CDS 1..651 Current Oligo Length: 21 nt B Upper Oligo --- — Position: 356 □ Lower Oligo 294 H> tm: 593°C PCR Product 87 nt pos: | 350 tm: | 57,1 .260 ,270 ,230 ,290 .300 ,310 .320 ,330 .340 .......1.........1.........1.........1.........1.........1.........1.........1.........1......... 350 ,360 ,370 .........1.........1........ cctgg:tgtga:ta:tca hthhi-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i-i hthi-i-i-i-i-i-i-i ITAATGaTatTCTCAtTAtKAtTTmAAaATCG™^ aatta: ggacc gacactgatgagtg aaaaattc ctac t ataactc g gatacac c :tt:ta:tctttttgtttg c c t ctc ctg ctac t gattaatgtaacttgtttgtc gtctctg cttcactg gag ACTCGGATACACCCTTCTACTC CCTCCTGCTACCGATTAA H H 1 H H 1 1 H H LMPGCDY5LFKDCIEPMWEDEKHKRGGRW ITLHKQQRR5PL ===========. —^ onononononono □onononononon onononononono □onononononon onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono □ o n o : o n o n < □ o n o : 0 n o n Search for Pnmers & Probes ' Search Options Subsearches 1 Search in: 0 4- Strand 0 - Strand Search Mode: © Select Q Verify 0 Complex Substrate © PGR Primers Compatible with the 0 Forward Primer C Reverse Primer 0 TaqMan Probes & PGR Pairs Compatible with the Q Upper Probe O Lower Probe O Molecular Beacons & PGR Pairs O Nested Primers O Sequencing Primers O Hybridization Probes O siRNA Probes After successful! search show: . All Results ( Search ) ^ Cancel ^ ( Apply ) Parameters ) ^ Ranges ^) V Defaults ) KJ l_l W l_l KJ l_l W onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono Search for Primers &. Probes Search Options Subsearches ^ Search method: Compatible Pairs ^ Eliminate Ambiguous Bases 0 Duplex-free Oligonucleotides 0 Highly Specific Oligonucleotides (3'-end Stability) □ 5-end Stability □ siRNA Internal Stability 0 Oligonucleotides with CC Clamp 0 Oligonucleotides within Selected Tm Limits 0 Hairpin-free Oligonucleotides & Eliminate Mono- and Di-Nucleotide Repeats 0 Detect Sequence Repeats 0 Eliminate Frequent Oligonucleotides 0 Omit High Secondary Structure Regions in the Template 23 Check PrimersyProbe Sequence Constraints 0 Restrict the Number of C Bases 0 Eliminate False Priming Oligonucleotides and Q Continue Above Search in Other File(s) £J Consensus Primers ( Search ^ Cancel ^) ( Apply ) Parameters ^ Ranges ^) Q Defaults □onononononon onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono 88Q_PCR File: Human 4E.seq —C- Optimal Annealing Temperature: 50.8 *C (Max: 66.3 Position and Length Tm ra GCpfil P.E# Score Product 862 78.9 29.6~ n/a 697 Forward Primer 91S 22 56.9 45.5 471 / 471 840 Reverse Primer 1753 21 55.3 29.6 4S9 / 4S9 S34 Upper Dligo 979 2A 56.5 33.3 479 / 479 917 Lower Dligo 1694 23 55.4 39.1 457 / 457 841 Product Tm - Reverse Primer Tm: 23.6 *C Pri m e rs Tm di Ffe re nee: 1.6 "C Com m e nts: Concentration Forward Primer 200.0 nM Reverse Primer 200.0 nM Upper Dligo 200.0 nM Lower Dligo 200.0 nM Monovalent Cation 50.0 mM Free Mg[2+J 0.7 mM Total Na[+| Equivalent: 155.8 mM onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono e o Selected Primers File: HRCA2 gene.seq AY436640:LS43GF22 AY436640:L59L7R20 5' C AATATATA C C CTA CTCCCCTA 31 5' C ACCTA CATATTA C C C CA CA. 3' Length: 22-mer Length: 20-mer Score: S02 points Score: 9L4 points 5' Position: L543S 3" Position: L59L7 TmAm: 53.4 52.6 BC Tm/tm: 53.L 53.S "C AC/Ag (25 ÜC): -30.5 -29.2 kcal/mol AC/Ag (25 ÜC): -2S.6 -2S.5 kcal/mol A5/As: -472.1 -449.5 cal/°K*mol A5/As: -430.5 -419.6 cal/*K*inol AH/Ah: -171.3 -L63.2 kcal/mol AH/Ah: -157.0 -153.6 kcal/mol 3 AG: -6.5 kcal/mol 3'AC: -6.9 kcal/mol Degeneracy: ___1 Degeneracy: ___1 P.E.#: (443/443) P.E.#: (477/477) 1/E: 4^63 nmol/A260 1/E: 5T05 nmol/A2so 3L.1 M9/A2S0 31.0 pg/A2G0 Priming Efficiency PE Score onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono HAIRPIN intramolekulärni 0OO Current Oligo Duplexes . DIMER intermolekulärni File: BRCA2 gene.seq Current Oligo 2L-mer [5042) [Current t Oligo| - The most stable 3-dim er: $ of hydrogen bonds = LO; AC = -0.7 kcal/mol 5 h (MTTAGATAAATI^AAATTA 3 h III II II III 3' ATTAAACTTAAATAGATTAaG 5' [Current- OligoJ - The most stable 3"-dim er: * of hydrogen bonds = LO; AC = -7.3 kcal/mol; Tm = 2.9°C 5 r TAATITCAATTOATCTAATTC 3 r I I I I I I I I I I 3r CTTAATCTArrTAAGTTTAAT 5' The most stable dinier overall: $ of hydrogen bonds = LO; AC = -7.4 kcal/mol; Tm = 2.2°C 5h GAATTAGATAAATTCAAATTA 3' I I I I I I I I I I 3' ATTAAACTrAAATAUATTAAÜ 51 Hairpin: loop = 5 nt; AC = -3.0 kcal/mol; Tm = 54.6 *C 5 1 ÜAATTAG-, I I I I I A 3 1 ATTAAAGTTAAAT-1 O n o □onononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono 8QO File: BRCAZ gene.seq Current Oligo Hairpin Stems Current Oligo ZL-rmer (5042J L#of paired bases = 5: loop = 5 nt; AC = -3.0 kcal/mol; Tm = 54.6 °C 5042 GAATT 1 1 1 1 1 5046 5 'GAATTAG-, 1 1 1 1 1 n 5056 1 1 1 1 1 CTTAA 5052 1 1 1 1 1 A 3 1 ňTTAflACTTAMT-1 2. # of paired bases = 6; loop = 5 nt; AC = HZ kcal/mol; Tm = 2L7 °C 5043 AAITACA 1 1 1 1 II 5049 5 1GAATTAGATA-, 1 1 1 1 II n 5061 1 1 1 1 II TTAAACT 5055 3 1 1 1 1 II A r MTAAACTTA-1 3. # of paired bases = 4; loop = 2 nt; AG = 0.9 kcal/mol; Tm = S.7 °C 5052 AATT 5055 I I I 5061 TTAA 5059 5 1GAATTA GAT AAA T' rc -, I I I I 3 1ATTAAA-1 onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono □onononononon onononononono □onononononon onononononono □onononononon t ©OO Reverse Primer False Priming Sites File: M13MP1S Reverse Primer ML3MPLS:6310RL9 (positive strand) Priming efficiency of the perfect match is 4S2 (above the threshold Priming efficiency: 482 (above the threshold) 5' (6323 J GGTTTTCCCAGTCACCACG (631.0)3 ' I I I I I I I I I I I I I I I I I I I 3 h (6323) ccaaaagggtcagtgctgc {631.0)5' Priming efficiency: 244 (above the threshold) 5 (6328) tol'l'l'lCCCAGTCACGaOe (6310)3' I I I I I I I I I I I I I 3' (626 ) ac Priming efficiency: L93 (above the thresh 5' (6323) CCrTTTCCCACTCACCACC (631.0)3 ' III 11111111 111 Priming efficiency: L91 (above the threshold) 5 (6328) Gtm^TTCCCAGTCACGACG (6310)3' II I I I I I I I I I I I I 3 ' (3 C 3 ; Priming efficiency: L79 5'(6323) GGTTTTCCCAGTCACCACC 531.0)3' 111111 3'(63L5) tgctgcaacai. (6297)5' Reverse Primer ML3MPLS:G310RL9 (negative strand) Priming efficiency of the perfect match is 4S2 (above the threshold) Priming efficiency: 105 5,l CCTTTTCCCACTCACGACG 1631.0)3' I I I I I I I I I I I 3'(5744) ccaaaaagcgggaaactgc (5762)5' onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon O ^ ^ Forward Primer Composition File: BRCA2 gene.seq Forward Primer AY436640:6275F19 64.2" [nearest neighbor method) Tm 56.5° [nearest neighbor method) Tm 70.S1 [MCC method) Tm 56° [Z(A+"n" + 4(G+aB method) Tm(RNA)[lM Na| SI" [MCC method) Tm(DNA:RNA)[lM Na| 74.7" [%GC method] 1.59 [single strand] Molecular Weight 5.SK [one strand) Molecular Weight 11.7K [two strands) ug/OD_ 47.4 IdsDNA! Base Number &% A 2 [10.5%) C 5 [263S6) C 4 [21.1%) T S [42.1%) A + T 10 [52.6%) G+C 9 [47.4%] DNA Melting Temperature in Various Salt and Form amide Concentrations fC) (mM| x55C OK 10% 50% 1 0.006 24.S 1S.3 -7.7 10 0.06 41.4 34.9 S.9 50 0.3 52.S 46.3 20.3 165 1 60.S 54.3 2S.3 330 2 65.1 5S.6 32.6 soo 3 67» 60.9 34.9 1000 6 70.S 64.3 3S.3 Approximate tm of the mismatched oligo Mismatch tm = 7^ - 1.206 mismatch)" mism. # tm mism. # tm 0 64.2 3 45.3 1 57.9 4 39.0 2 51.6 5 32.7 onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono GOO Oligonucleotide Database File: New/Database .odb 9. 9L □ #of Records: 29 Date ID Number Sequence 3'-Dim.AC P.E/p.e. Tm /tn 21 12/02/06 22 12/02/06 12/02/06 12/02/06 12/02/06 12/02/06 12/02/06 12/02/06 23 24 25 26 27 2S AY436640: AY436640: AY436640: AY436640: AY436640: AY436640: AY436640: AY436640: 5916R19 5916R20 5937R21 5937R22 4695U22 5325U22 57S6L23 5S60L19 A ATC C CTC C CTTTA CTCTC CAATCC CTC C CTCTA CTCTC TCAATTTCTTTACCTTCCCAT TTC AATTTCTTTA C CTTC CC AT TC C CTTAA CAAAA CTAATC CAT AATTAC CTCTTTCTTATC C C A A CTCTCCCTACAA CATTATCA CTC A A C AA C C A AA C C C AA C CTC 03 n ^ SC 5C 5r Selected oligo 03 0.3 -0.3 -0.9 5C SC SC 5C 430 366 .449 I5S 432 4S3 451 430 450 449 45S 432 453 4SL 54.1 54.7 55.9 54.5 53.3 54.S 55.3 54.5 57.2 53.1 53.S 53.0 53.0 55.0 55.9 OI igo n uc leotide 5e.ts (64) T Forward Primer Reverse Primer Upper Oligo Lower Oligo 2S 2S Checked Set of nested primers u 39 9 15 25 27 u 33 9 16 25 27 u 61 9 17 25 27 u 48 9 IS 25 27 This database is linked to HRCA2 gene.seq nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono Restriction Enzvme Sites in Protein File: GRCA2 gene.seq Enzyme |iä4iD ^itno ,210x1 ,237110 ,is+0d ^asao ^jjdd ,2.^000 ,míw ,24*011 ,m*w j24*00 ,0000 ^noa ,28*00 ,25*00 psaoo ^hjdd ,211700 ^wdd ^důdd ,2wqo 33 I EcoRI 34 EcoRV 35 E&P3I 36 F&el 37 Fspl 3S Csul 39 Hindlll -0 Hpal 41 Kpnl 42 N'lul 43 N'unl 44 Nael 45 Narl 46 Ncol 47 Ndel # Enzyme Site #Cuts Positions & Fragment 5izes 41 Kpnl CT2VpzY6 8 -21253 236S4 63 23722 52 23774 237 24011 585 24596 162 24758 629 253S7 1219 26606 22S51 42 Mlul TRlRVyA7 5 -22233 22674 2S24 2S49S 576 26074 106 26180 244 26424 23033 43 Munl QL3Nawl5 L0 -21287 23620 355 23975 351 24326 282 24608 242 24850 72 24922 351 25273 714 25987 187 26174 420 26594 22863 44 Nael AG2PAxR6 7 -21823 230S4 597 236S1 1286 24967 86 25053 573 25626 149 25775 623 2639S 23059 45 Narl CA2APZR6 1 -20043 24S64 24593 46 Ncol PW3HGwM5 4 -22361 22546 336 22882 887 23769 531 24300 25157 47 Ndel HM2lawY5 2 -20366 24541 1211 25752 23705 4S Nhel A52l_Ax-6 L6 -22276 22631 322 22953 185 23138 S3 23226 27 23253 461 23714 369 240S3 312 2439S 288 24683 151 24834 273 25107 536 2S643 402 26045 30 26075 210 26285 372 26657 22800 f n 1 n 1--J. 1_ _i_3_ - i ■-■ _i_o_ -.-1 - - ■-■ Search: 22454 to 27004 End Cut Type: Blunt, Odd, 3'-overhjng, S'-overhang onononononono nonononononon onononononono nonononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono 0O Hybridization Time I File: M13MP1& DNA Length: Concentration: 21 nt. 200.0 nM L298 Mg/mL = 45.4 sec T =3 rnin 47 sec □onononononon onononononono □onononononon onononononono □onononononon Onononononono □onononononon liillillliili MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono o □ o □ o 60A Concentrations File: BRCA2 gene.seq @ Constant Concentration Q Constant Volume e Current + Dligo: 5.0S nmol/ODf 32.5 ug/OD o Current -Oligo: 4.67 nmol/ODf 30.9 ug/OD o Entire Sequence (ds): 0.001 nmol/DDf 4S.1 ug/OD 0 Forward Primer: 5.98 nmol/ODh35.0 ug/OD o Reverse Primer: S.3L nmol/ODr34.0 ug/OD o PCR Product (ds): 0.146 nmol/QD, 4S.L ug/OD o Upper Oligo: 4.S3 nmol/ODf31.2 ug/OD 0 Lower Oligo: 4.67 nmol/ODr 30.9 ug/OD or or in yields 32.5 1.0 OD(260) 5.0S4 nmol 50S.4 uL 10.0 uNJ □onononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono AHP2 cDNA (TAIR database)! Sequence: AT3G29350.1 Date last modified 2007-04-17 Name AT3G29350.1 Tair Accession Sequence:4010737427 Sequence Length (bp) 827 1 ACAATTCGCG AGAAAGACAA AACACAAGTT TCTTCTTCTT GGGATTGGCT 51 ATTTCCAGAA ATCCAAGTCA ATAATCAAAG TCCAAACAAA AAAATCCTCT 101 CCCAATCTCC GCTTCACTCT TCTCATGGAC GCTCTCATTG CTCAGCTTCA 151 GAGACAATTT CGTGATTACA CCATTTCTCT CTACCAACAG GGGTTTTTGG 201 ATGATCAATT TACTGAGTTG AAAAAGCTAC AAGATGATGG AAGTCCTGAT 251 TTTGTGTCTG AAGTGCTTTC ACTTTTCTTT GAAGATTGTG TGAAGCTTAT 301 CAGTAACATG GCTAGAGCTT TGGACACGAC AGGAACTGTA GATTTTAGTC 351 AGGTAGGTGC TAGTGTGCAT CAATTGAAGG GTAGTAGCTC AAGTGTTGGT 401 GCCAAGAGGG TCAAAACTTT GTGTGTTAGC TTCAAGGAAT GTTGTGAAGC 451 TAAGAACTAC GAAGGGTGTG TGAGATGTTT GCAGCAAGTG GATATTGAGT 501 ACAAGGCGTT AAAGACAAAG CTTCAAGATA TGTTCAATCT TGAGAAACAG 551 ATCATTCAAG CTGGTGGTAT AGTTCCTCAA GTGGATATTA ACTAAAGAGA 601 CTAGTCCATA AGAAGAAAAA AGATGATGAC TTTCTTTCTT TAGTTTCTCT 651 TCTAAATTAT TTTGGATTTG GTGTTTGCTC AAAAACTCAA TAAAATATGT 701 GCAAAAAGAA ACAAAAACAA GTGATGGTTG TTTATAAATC AGTAGTATGT 751 ATTGTTTGAT CTCATCCGAG AAAATTGAAA CCATTGGACT AATGAATGTG 801 ATGATAATAT ATATTGGTTT GCTTCTG □onononononon onononononono □onononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono 101 CCCAATCTCC GCTTCACTCT TCTCATGGAC GCTCTCATTG CTCAGCTTCA 151 GAGACAATTT CGTGATTACA CCATTTCTCT CTACCAACAG GGGTTTTTGG 201 ATGATCAATT TACTGAGTTG AAAAAGCTAC AAGATGATGG AAGTCCTGAT 251 TTTGTGTCTG AAGTGCTTTC ACTTTTCTTT GAAGATTGTG TGAAGCTTAT 301 CAGTAACATG GCTAGAGCTT TGGACACGAC AGGAACTGTA GATTTTAGTC 351 AGGTAGGTGC TAGTGTGCAT CAATTGAAGG GTAGTAGCTC AAGTGTTGGT 401 GCCAAGAGGG TCAAAACTTT GTGTGTTAGC TTCAAGGAAT GTTGTGAAGC 451 TAAGAACTAC GAAGGGTGTG TGAGATGTTT GCAGCAAGTG GATATTGAGT 501 ACAAGGCGTT AAAGACAAAG CTTCAAGATA TGTTCAATCT TGAGAAACAG 551 ATCATTCAAG CTGGTGGTAT AGTTCCTCAA GTGGATATTA ACTAAAGAGA EcoRI restriction site 5'......G| AATTC......3 3"......CTTAAl G......5 Design of primers AHP2ex_up 5'- CCG GAA TTC ATG GAG GCT CTC ATT GCT CAG - 33 AHP2ex_low 53- CCG GAA TTC TTA GTT A AT ATC CAC TTG AGG - 3' onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono 101 CCCAATCTCC GCTTCACTCT TCTQITGGAC GCTCTCATTG CTCACJCTTCA 151 GAGACAATTT CGTGATTACA CCATTTCTCT CTACCAACAG GGGTTTTTGG 201 ATGATCAATT TACTGAGTTG AAAAAGCTAC AAGATGATGG AAGTCCTGAT 251 TTTGTGTCTG AAGTGCTTTC ACTTTTCTTT GAAGATTGTG TGAAGCTTAT 301 CAGTAACATG GCTAGAGCTT TGGACACGAC AGGAACTGTA GATTTTAGTC 351 AGGTAGGTGC TAGTGTGCAT CAATTGAAGG GTAGTAGCTC AAGTGTTGGT 401 GCCAAGAGGG TCAAAACTTT GTGTGTTAGC TTCAAGGAAT GTTGTGAAGC 451 TAAGAACTAC GAAGGGTGTG TGAGATGTTT GCAGCAAGTG GATATTGAGT 501 ACAAGGCGTT AAAGACAAAG CTTCAAGATA TGTTCAATCT TGAGAAACAG 551 ATCATTCAAG CTGGTGGTAT AGTTpCTCAA GTGGATATTA ACTA4AGAGA EcoRI restriction site 5'......G| AATTC......3 3"......CTTAAl G......5 Design of primers AHP2ex_up 5'- CCG GAA TTC ATG GAG GCT CTC ATT GCT CAG - 3' AHP2ex_low 53- CCG GAA TTC TTA GTT A AT ATC CAC TTG AGG - 3' onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz LITERATURA - PCR Primer: A Laboratory Manual (2003) ■ Artificial DNA: Methods and applications (2003) ■ OLIGO Primer analysis software, Version 7 □onononononon onononononono □onononononon onononononono □onononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.mum.cz Tato prezentace vznikla s podporou projektu OP VK „Rozvoj týmu pro výuku, výzkum a aplikace v oblasti funkční genomiky a proteomiký' (CZ.1.07/2.3.00/09.0132) Discovery is not in seeking new landscapes, but in having new eyes... Marcel Proust Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. EVROPSKÁ UNIE MINISTERSTVO ŠKOLSTVÍ, MLÁDEŽE A TĚLOVÝCHOVY i OP Vzdělávání pro konkurenceschopnost INVESTICE DO ROZVOJE VZDĚLÁVÁNÍ