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 n o n o o n 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 IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono oligonukleotid • krátká jednořetězcová struktura • DNA nebo RNA (event. PNA) • hydroxyl na obou koncích (normálně na 5'- konci fosfát) oligonukleotid syntetický oligonukleotid orientace! polymeráza! i" ®ntd 3' and onononononono nonononononon onononononono □onononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz Aplikace syntetických oligonukleotidů • primery pro syntézu komplementární DNA PCR, Real-Time PC R • syntéza genů a rekombinantní proteiny • hybridizační sondy pro klonování • místně cílená mutageneza • sekvenování a genetické profilování • diagnostika - testy a biosensory • gene arrays • blokace genové exprese antisense oligo • potenciální léčiva a DNA vakcíny • 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 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 nononociononon 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 nonononononon onononononono □onononononon onononononono nonononononon onononononono nonononononon 11 llllll 11 III MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono □onononononon Real-Time PCR 2x značená sonda REPORTER QUENCHER HHWURDFRMEH 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 □ MASARYKOVA UNIVERZITA www.mum.cz Terapeutika nedegradována nukleázami! modifikace fosfodiesterové vazby i 0 = P I o 0 = P-f CH O 0 1 0=P- I o. phosphorothioate methylphosphonate phosphoramidate l i"2 ? CH2 H3C-N 0 = P-BHľ i 3 I 1 3 3'-thiofornriacetal methylene(mothyliminlo) boranophosphate onononononono nonononononon onononononono □onononononon onononononono □onononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz ANTISENSE oligonukleotid • oligonukleotid nebo analog • komplementární k segmentu RNA nebo DNA • vazbou inhibuje jejich normální funkci _ Antigene (triplex) □onononononon onononononono □onononononon Ribozyme RNA s katalytickou aktivitou Antisense Sense/Aptamer 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 nonononononon onononononono □onononononon onononononono □onononononon C-terminal 0=P-0" DNA 3'-end onononononono nonononononon onononononono □onononononon onononononono □onononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz □onononononon Peptidonukleová kyselina • nenabitá molekula • vazba k DNA/RNA N-(2-aminoethyl)-glycin □onononononon onononononono □onononononon onononononono □onononononon pna « ... *. vr.l.. HN ř v B DNA onononononono nonononononon onononononono nonononononon onononononono □onononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz o □ o □ o □ □ o □ o □ o Vlastnosti PNA • vysoká termostabilita • Tm nezávisí na obsahu solí • vyšší specifická • vyšší afinita • rezistentní k enzymům... nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono □onononononon onononononono □onononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz LNA 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 nonononononon onononononono □onononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz OLIGONUKLEOTIDY design syntéza purifikace * -i f I 9 I T/M pi EXPEDITE 8909 onononononono □onononononon ilIIIIilIIIil MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono ononononon Syntéza oligonukleotidu syntéza na pevné fázi od 3'-konce k 5'-konci bezvodé prostředí OMT- O - O — p _ N(iPf), on + Tetrszole onononononono nonononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono nonononononon Kontrola kvality □onODOnononon onononononono nonononononon HPLC Perfúzní chromatografie • anex • RP onononononono □onononononon onononononono □ onononocionon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz 1341ong.bio - 20.0jil Perfúzní chromatografie klasický sorbent POROS 0.15- 0.10- 0.05- 0.00' Verve HQ - -N-1—t—;- 100 50 5 10 15 20 25 30 35 Min 134.bio - 20.0111 2 6 0 n T. 0.15- 0.10- 0.05- 0.00 □onononononon onononononono □onononononon onononononono □onononononon forot ftC( \__ .0 2.5 Min 100 50 onononononono nonononononon IIllllllIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono Maldi-TOF MS CE 7103 . Mass/charge D ■ ° CapíEary efcctaophoirais trace onononononono □onononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono VYTEZEK 1.000 0.900 0.800 0.700 0.600 Yield 0.500 0.400 0.300 0.200 0.100 0.000 II □ "D" □ □ TJ 10 i i i r 20 30 40 50 i i i r 60 70 80 90 100 Oligonucleotide Length nonononononon onononononono Efficiency ■ 0.995 ■ 0.990 □ 0.980 PAGE 85 85 75 75 65 65 crude purified crude purified crude purified onononononono □onononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.mum.cz PURIFIKACE • Sephadex • RP cartridge • HPLC □onononononon 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.htm 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 LMPťiCDYSLFKDťilEPMWEDEKHKRGGRW 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 nonononononon 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 + 51 - -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 3G3 i?3 .........1.........1........ TTAATGttTCGtTGTGACTAtKAt 1111 lAACtATtCTATTCAtCCTATCTCtCAACAKACAAAAACAAACCCttAtCACCATCtCTAATTACATl "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 ACTGGAG .........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 IIllllllIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono 5' CTTCTG CTC AAT CTT TCT AC 3' forward + 5 1 ATGOfclTCTG CTCAATCTTT CTA(IaACCAA AGCTCTGTCT TGAAAATCAA W 51 TGTCATäüTT gtgcWjgatc aTcatgtttt ccttgatatc atgtcacgca 101 TGCTTCAACA CTCCAAATAC AGAGGTAATT AAATATTATT ATCATATTAT 151 ATATAATATG TTATTGATTT TTTGTTTGTG ATTTCATTTA GATTTTTATT 201 TCTATGATTT CTTAGCATGA AATACAATTT TTGGAGAAAC AACTAGCAGT 251 TTTAAAAACA AAACTTGAAT TTTGAGAAAT TCAAAGATGT TATATATATA 301 TGTCAAAATT TAACAATTAT TCTTCTAAAT CATCCGGATT CCGTTTACAT 351 GTACACATCT ACAATTTTCA ATTGAGGTAT TCTTGTTTTG ATGCCTTTGA 401 GACGAATAGT TTGATTGATA AAAAAAATTC TAACCAATAT GATATATAAA 451 GTTTATTTTC TTTTTGTCAA ACCATACTTT ATACTATGTA ACTTTTTTAA 501 GAGATTATTG AAAATAGTTT ATTTATAAAA TAGTAACCTA TTGTTGAATT 551 AAAAAAAAAA aaaaaattct AAATrnTnTTTnrAAArnAr ATfTTf^ATTTA 601 TCTTAGTTT/IAAACTAGCTG ATATTCTTCAlAATCGACTGT TCTTATAAGT 651 AATCAACCA/T TTAQCATCAA TmCAATAAA^TGTAAACAC TTCAATGAAA 701 ATGGTGATTT TAAAGAATAT GTTTTACTTA TGTTATGAAC TATCTCAAAT 751 TTGTGAAATA TTTCATAACT AATGTGGAAA ACTATATAAC CCCTCCATAC 801 AAAACGTAAG TAAAATTTAT GAAATCCTAT CATTTTTAAA GGTTAAACCA 851 ATCAAAAAGT AATAATTCTT GGTACTTGCA ATATTTTTGT CATTATATTT 901 TAGTTTATTA ATTTTATTTT GATTAAATGG TTTTAGATCC ATCAGTTATG 951 GAGATCGCAG TTATAGCTGT AGACGATCCG AAGAAAGCAT TATCTACTCT 1001 AAAAATTCAA CGAGACAATA TAGATCTCAT AATCACAGAT TATTATATGC 1051 CTGGTATGAA CGGTTTACAA CTCAAAAAAC AAATCACTCA GGAATTTGGA 1101 AATTTACCGG TCTTAGGTAA CATTTTTTGT TCTTTACAAC TTAAATTAAA 3' D£:JJ3AAGAATATCAGCTAGTTT 3' reverse onononononono □onononononon onononononono nonononononon 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: LS6S nt XD □ Forward Primer 259 18 1 source -L8..1850 Reading Frame: + L □ Reverse Primer 328 18 2 CDS 1..651 Current Oligo Length: 21 nt B Upper Oligo --- — Position: 356 □ Lower Oligo 294 H> tm: 593°C PGR Product 87 nt pos I 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........ cctggctgtgactactca 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 TTAATG«TatTCTCAtTAtKACTTmAAaATCGTATT^ AATTAt GGACC GACACTGATGAGTG AAAAATTC CTAC tATAACTC G GATACAC C CTTCTACTCTTTTTGTTTG C C C CTC CTG CTAC t GATTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG ACTCC^ATACACCCTTCTACTC 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 nonononononon onononononono nonononononon II II II II II III MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono 88Q_PCR File: Human 4E.seq - Optimal Annealing Temperature: 50.8 *C (Max: 66.3 *Q 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 GTA CTCCCCTA 31 5' C ACCTA CATATTA C C C CA CA. 3' Length: 22-mer Length: 20-rner 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 ÜCJ: -2S.6 -2S.5 kcal/rnol A5/A&: -472.1 -449.5 cal/üK*mol A5/A&: -430.5 -419.6 cal/*K*mol 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/rnol Degeneracy: ___1 Degeneracy: ___1 P.E.#: (443/443) P.E.#: (477/477) 1/E: 4^63 nrnol/A260 1/E: 5T05 nrnol/A^so 3L.1 ug/A2S0 31.0 ug/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 (MTTAGATAAATTCAAATTA 3 h III II II III 3' ATTAAAC TTAAATAG-ATFAAG 5' [Current- ON go) - The most stable 3"-dim er: * of hydrogen bonds = LO; AC = -7.3 kcal/mol; Tm = 2.9°C 5 r TAATTTGAATTTATCTAATTC 3 r I I I I I I I I I I 3r CTTAATCTArrTAAGTTTAAT 5' The most stable dinier overall: $ of hyidrogen bonds = LO; AC = -7.4 kcal/mol; Tm = 2.2°C 5h (jAATTAjGATAAATTCAAATTA 3' 11111 11111 3' ATTAAAC TTAAATAGATTAAG 51 Hairpin: loop = 5 nt; AC = -3.0 kcal/mol; Tm = 54.6 *C 51GAATTAG- I I I I I A 31ATTAAAG TTAAAT o n o nonononononon onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz □onononononon onononononono 8QO File: BRCAZ gene.seq Current Oligo Hairpin Sterns 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 ňTTMACTTAMT-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 ©O0 Reverse Primer False Priming Sites Flte: M13MP18 Reverse Primer M13MP18:6310R19 (positive strand) Priming efficiency of the perfect match is 482 (above the threshold) Priming efficiency: 48Z (above the threshold) 5,(632BJ GGTTTTCCC AG TC AC GAC G (6310)3* Ml I I I I I I I I I I I I I Ml 3'(6328) ccaaaagggtcagtgctgc (6310)5' Priming efficiency: 244 (above the threshold) 5H(6328) GGTTTTCCCAGTC ACCACC (6310)3' III MM llllll 3'(626) agcaaatggtc—tgctgc (610)5' Priming efficiency: 193 (above the threshold) 5' (6328) GCTTTTCCCAGTCACGACG (6310)3" III MINIM III 3' (5125) tcLaasjLggLcagtg-tgc [5100)5 ■ r □ o n o u " V U U U V u onononononono nonononononon IIIIIIIIIIIII MASARYKOVA UNIVERZITA www.muni.cz nonononononon onononononono © f> f) Forward Primer Composition File: BRC.A2 gerc.s-:; Forward Primer AY43G640:G275rt9 nonononononj ° u u 64.2* [nearest neighbor method] Tm 56,5* [nearest neighbor method] Tm 70.8* [%CC method) Tm 56* [21A+T]* + 4[C+C)* method] Tm[RNA>[lMNa] ar [%GC method] Tm(DNA;RNAJ[lMNal 747* [%CC method] I.S9 (single strand] Molecular Weight 5SK [one strand] Molecular Weight 11.7K [two strands) ug/OD 47.4 [dsDNA] Base Number & ft A 2 110.5«: C 5 [26.3ft] c 4 [21.1ft] T £ [42.1ft] A + T 10 [52.6ft] C + C 9 [47,4ft] 1 o 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.AG 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 CTTTACTCTC 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 0.3 n ^ 5C 5C Selected oligo 0.3 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 Sets (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 □onononononon onononononono □onononononon onononononono □onononononon 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 |ia4ÍD ^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 23654 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 16 -22276 22631 322 22953 185 23138 S3 23226 27 23253 461 23714 369 240S3 312 24395 288 24683 151 24834 273 25107 536 25643 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, 5'-overhüng onononononono □onononononon 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 |jg/rnL = 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.S pg/OD o Current -Oligo: 4.67 nmol/ODf 30.9 pg/OD o Entire Sequence (ds): 0.001 nmol/DDf 4S.1 pg/OD 0 Forward Primer: 5.9S nmol/ODh35.0 pg/OD o Reverse Primer: S.31 nmol/ODr34.0 pg/OD o PCR Product (ds): 0.146 nmol/QD, 4S.1 pg/OD o Upper Oligo: 4.S3 nmol/ODf31.2 pg/OD 0 Lower Oligo: 4.67 nmol/ODr 30.9 pg/OD or or in yields 32.5 ug 1.0 OD(260) 5.0S4 nmol 50S.4 pL 10.0 pNJ □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 ononononODOno n o □ O n o □ o n o MASARYKOVA UNIVERZITA www.mum.cz I LITERATURA I Artificial DNA: Methods and Applications; Khudyakov, Y.E., Fields, W.A., Ed. (2003) PCR Primer: A Laboratory Manual (2003) OLIGO Primer analysis software, Version 7 nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono nonononononon onononononono MASARYKOVA UNIVERZITA www.muni.cz Discovery is not in seeking new landscapes, but in having new eyes... Marcel Proust 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) Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. INVESTICE DO ROZVOJE VZDĚLÁVÁNÍ