ODODOnononODO □ OnODODOnonOD ODODOnononono □ ODODODOnonon o □ o □ o " " " o □ o □ O □ O □ *-rlJ\f\. V ú O □ ODODOuv^uODOOO □ ODODODODODOD ODODODODODODO MASARYKOVA UNIVERZITA □ o o □ □ o o □ □ o o □ □ o o o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o Design sekvence PCR primerů Hana Konečná CEITEC - MU Centrální laboratoř - Proteomika Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. MINISTERSTVO ŠKOLSTVÍ, MLÁDEŽE A TĚLOVÝCHOVY EVROPSKÁ UNIE H INVESTICE DO ROZVOJE VZDĚLÁVÁNÍ OP Vzdiláviní pro konkurenceschopnost M m 1soudoi|3seoiiejn)|U(»| aid ]u?A9iepzA dO INVAVlSaZA 3ľOAZOy Oa 3DI1S3ANI AAOHOAA0131 v 3Z3aviH m ilNfl VXSdOUAl 'JAlSnO>H§ OA±SU31SINIIAl X>|!|qnd3j 9>|S3j aisyodzoj wju}Ets e lugpuoj wju|i3pos uiX>|sdojA3 uBAODireiHjn|ods sfipplojd o;usi B>)ZB>in B>10!l>lBJd Z OOIIO eJBMJios JUBAOl|JABU ÁpBSBZ 80U8A>|8S UßjSep BZ9JUÁS 90B>|!I!P0LU 90B>|!|dB Aaii03~i>inNOöno VlIZeGAINn VACWAWSVW onononononoDO □ ODODODODODOD OaOüOn^nODODO O, ODODÜu„uODODO dodododododod ododododododo □ OnononODODOD oaononononono ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ ODODODODODOD ODODODODODODO ODODODODODODO oligonukleotid orientace! polymeráza! )H 5' end HJ krátká jednořetězcová struktura DNA nebo RNA (event. PNA) hydroxyl na obou koncích (normálně na 5'- konci fosfát) oligonukleotid OODODODODODOD ODODODODODODO □ ononODODOnon ononooonODODO □ OnononODODOn (X. a _0-d = 0 (iB6ns) aßp|jq JS}saipogdsoL|d ©seq VNd j>|no ezeq 80Z8J8J 80U0>| eOB>1!l!PO|AJ ODODODODODODO ODODODODODODO zD-mnui-MMM VlIZ^BAINfl VAO>IAWSVW 111111SI i 1111 ODODODODODODO DODODODODODOD ODODODODODODO o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ 0 □ 0 □ 0 □ o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ 0 o □ □ o MASARYKOVA UNIVERZITA www.muni.cz □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ i i i i 1 1 g g g g g 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 o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o MASARYKOVA UNIVERZITA www.muni.cz □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o ododododododo Degenerované oligonukleotidy 2-deoxyinosin dodododododod ododododododo dodododododod ododododododo dodododododod 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 o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o MASARYKOVA UNIVERZITA www.muni.cz □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o Real-Time PCR • 2x značená sonda • REPORTER • QUENCHER nciHWARD pniMcn *'■ 2, Strand displacement: When the probe is intact, the reporter dye emission is quenched. r > B1 5_9, ľ ľ □ ODODODOnonon odododooododo □ ODODODOnonon ODODODODODODO □ odododododod 3, Cleavage: During each extension cycle, the DNA polymerase cleaves the reporter dye from the probe. r- i' B" ľ 9' 4, Polymerization completed: Once separated from the quencher, the reporter dye emits its characteristic fluorescence. ľ B' ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ ODODODODODOD ODODODODODODO Peptidonukleová kyselina pna N-terminal nenabitá molekula vazba k DNA/RNA N-(2-aminoethyl)-glycin DODODODODODOD ODODODODODODO DODODODODODOD ODODODODODODO DODODODODODOD C-terminal DNA 3'-end ODODODODODODO DODODODODODOD ODODODODODODO DODODODODODOD ODODODODODODO DODODODODODOD ODODODODODODO MASARYKOVA UNIVERZITA www.muni.cz ODODODODODODO OLIGONUKLEOTIDY • syntéza na pevné fázi • od 3'-konce k 5'-konci • bezvodé prostředí ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ ODODODODODOD ODODODODODODO ODODODODODODO DESIGN OLIGONUKLEOTIDU • manuální • počítačový www.protocol-online.org/prot/Research_Tools/Online_Tools/Oligo_Design/index.h Hlavní kritéria pro sekvenci PCR primem • vysoce specifické • netvoří dimery a vlásenky • stabilní duplexy s aktivní sekvencí • nepříliš stabilní 3'-konec ODODODODODODO □ ODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ ODODODODODOD ODODODODODODO ODODODODODODO OLIGO 6 PCR primery, hybridizační sondy sekvenační primery OLIGO 7 (od roku 2008) TaqMan sondy primery pro nested PCR molecular beacons siRNA ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ ODODODODODOD ODODODODODODO Terminologie PCR primem forward primer... část sekvence + vlákna reverse primer... část sekvence - vlákna pos: 350 tm: 57.1 ,,260, ,i27ů, , i319, . ií2ů, , i339, 1340 _ ľSO _ , 3E-Ü _ ,,370, CCTCCCTíTCACTACTCA »■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■»■ hhl-hhl-l-hhl-h TTAATGttTGGCTGTGACTACTCAC1111 IMGCATCCTATTCAGCCTATCTCCCAACATCACAAAAACAAACCCCCaCGACCATCCCTAATTACATI "GAACAAACAÍCAÍACACGAACTÍACCTC AATTAC G GAC C GACACTGATGAGTGAAAAATTC CTAC CATAACTC G GATACAC C CTTCTACTC11111GTTTG C C C CTC CTG CTAC C GATTAATGTAt KTTGTTTGTCGTCTCTGCTTCACTGGAG LMPGCDY5 L F K ] í IE P M i E DE KNKRGGRi ITL N K Q Q R R 5 DL UPPER - FORWARD - LEFT 5'-► + 5'-- 3'- DODODODOnonOö ononODODODODO DOnononononOö ODODODODODODO DODODODODODOD ■ 3' ■ 5' 5' LOWER - REVERSE - RIGHT ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ ODODODODODOD ODODODODODODO ODODODODODODO Terminologie forward primer... část sekvence + vlákna reverse primer... část sekvence - vlákna pos: 350 tm: 57.1 ,2E0 ?73 .......i.........i........ .2G0 ,290 ,300 5.3 ,320 ,330 ,340 • i.........i.........i.........i.........i.........i.........i......... 350 ,3EÜ 573 .........i.........i........ TTAATG C CTG G CTGTGACTACTCAC f 111IAAGGATGGTATTGAGCCTATCTGG GAAGATGAGAAAAACAAAC GGGGmGGACGA GG CTAATTACATTGAACAAACAGCACAGACCAACTCACt C AATTACG GAC C GACACTGATGAGTGAAAAATTC CTAC CATAACTCG GATACAC C CTTCTACTCTTTTTGTTTG CCCCTCCTGCTACC GATTAATGTAACTTGTTTGTCGTCTCTG CTTCACTG GAG 4 4 4 4 4 H < ACTC G GATACAC C CTTCTACTC LMPGCDY5L FKDCIEPHWEDEKNKRGGRW ITL NKQQRR5DL UPPER - FORWARD - LEFT 5' > polymeráza + 5] I- - 3'- 3' 5' polymeráza 4 5' LOWER - REVERSE - RIGHT DODODODODODOO ODODODODODODO DODODODODODOO ODODODODODODO DODODODODODOO o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o MASARYKOVA UNIVERZITA www.muni.cz □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o ODODODODODODO Nasedání PCR primem pos: 350 tm: 57.1 ?G3 ?73 i|23Üi ?33 100 ,310 _ 120 33Ü l'-O ISO 100 170 TTAATG C CTG G CTGTGACTACTCAC E 111IAAGGATGGTATTGAG C CTATGTG G GAAGATGACAAAAACAAAC G G G GAG GAC GA G G CTAATTACATTGAACAAACAGGACAGACGAAGTGAtt C AATTAC G GAC C GACACTGATGAGTGAAAAATTC CTAC CATAACTCG GATACAC C CTTCTACTCTTTTTGTTTG C C C CTC CTG CTAC C GATTAATGTAACTTGTTTGTCGTCTCTG CTTCACTG GAG 4 4 4 4 4 H < ACTC G GATACAC C CTTCTACTC L M P G ÍLVDV11W1D DDiYDDVDDVDDDD D WVD WVWD VDiYD WD D DiDIViD D DYDiiYiD DiYD1W.....nniTťilwnn DDiDD DIW11 VílíVlíVaiíHíaíHÍD i i i i i i i i i i i i i i i i i i i i i i i QIZ' QBZ' OSE i i i i.........i Oťť OEE1 ......mi i i i i i i i i i i i i i i i i i i i i i i i i i i 00E1 03S' i i i i.........i OÍZ1 oss' FZS I :uJl OSE I :sod ■ 1 ■■ ^ -^- _ rort1 púít1 imi1 post1 nou1 jůet noři1 non1 qmi1 mě1 oni;1 oni1 míí1 ms1 oni1 cme1 oa?1 mt ' [ 13npoJdUDd GJ 3o£6S :mi GJ zz vsz oBno jawo-] a tu 9SE — o LJ IS9"i sod Z st jauiMd 3l;.J3'V3íj u GJ 1+ :sijui:jj 6 m pusy OS8I"8I- 1 st jaui|jj pjímjoj u GJ iu S9ST * 06||0 p3333|D5 maiibag VMQ 909 ODODODODODODO ODODODODODODO zD-mnui-MMM VlIZ^BAlNfl VAO>IAWSVW IIIII IIIIIIII ODODODODODODO DODODODODODOD ODODODODODODO o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o MASARYKOVA UNIVERZITA www.muni.cz □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o o □ o □ c □ ono: o □ o □ c □ odo: o □ o □ OO Search for Primers & Probes — Search Options Subsearches Search in: iVf + Strand 5? - Strand Search Mode: @ Select Verify 5? Complex Substrate ® PCR Primers Compatible with the Forward Primer Q Reverse Primer 0 TaqMan Probes & PCR Pairs Compatible with the Upper Probe w Lower Probe 0 Molecular Beacons & PCR Pairs O Nested Primers Sequencing Primers C Hybridization Probes siRNA Probes After successfull search show: All Results Search ( Cancel ) ( Apply ) i Parameters ) ( Ranges ) ( Defaults ) \J U LJ W LJ V_/ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o MASARYKOVA UNIVERZITA www.muni.cz □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o c /: 6 ^ ^ Search for Primers & Probes Search Options Subsearches' Search method: Compatible Pairs 0 Eliminate Ambiguous Bases 0 Duplex-free Oligonucleotides •Vf Highly Specific Oligonucleotides (3'-end Stability) □ 5'-end Stability _ siRNA Internal Stability Mj Oligonucleotides with CC Clamp Oligonucleotides within Selected Tm Limits ^ Hairpin-free Oligonucleotides ^ Eliminate Mono- and Di-Nucleotide Repeats Mj Detect Sequence Repeats 8 Eliminate Frequent Oligonucleotides ^ Omit High Secondary Structure Regions in the Template Check Primers/Probe Sequence Constraints Mj Restrict the Number of C Bases B Eliminate False Priming Oligonucleotides and _ Continue Above Search in Other File(s) _ Consensus Primers Search ) ( Cancel ) ( Apply ) ( Parameters ) ( Ranges ) ( Defaults ) ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz DODODODODODOD ODODODODODODO File: I 0O PGR File: Human 4E.seq Optimal Annealing Temperature: 50.S °C (Max: 66.3 °Q Position and Length CC ßfl P.E# Score Product S62 7S.9 29.6 n/a 697 Forward Primer 9LS 56.9 45.5 471/47L S40 Reverse Primer L7S3 27 55.3 29.6 4S9 / 4S9 834 Upper ON go 979 24 56.5 33.3 479 / 479 9L7 Lower Oligo L694 23 55.4 39. L 457 / 457 84 L Product Tm - Reverse Primer Tm: 23.6 °C Primers Tm difference: L6 °C Comments: Concentration Forward Primer 200.0 nM Reverse Primer ZŮŮ.0 nM Upper Oligo 200.0 nM Lower Oligo ZŮŮ.0 nM Monovalent Cation 50.0 mM Free Mg[2+| 0.7 m M Total Na[+] Equivalent: L55.S rnM ODODODODODODO □ ODODODODODOD ISISIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ ODODODODODOD ODODODODODODO O □ O □ O d d O Selected Primers file: BRCA2 gene.seq AY436640:1543Sr22 5' CAATATATA C C CTA CTC C C CTA 3' Length: 22-mer 5core: 802 points 5' Position: 15438 Tm/tm: 53.4 52.5 °C AC/Ag (25 "Q: -30.5 -29.2 kcal/mol A5/As: -472.L -449.5 cal/ůK*mol AH/Ah: -1713 -163.2 kcal/mol 3'AC: -6.5 kcal/mol Degeneracy: ____1 P.E.#: (443/443) l/E: 4^63 nm ol yA2 B 0 31.1 M9/A2G0 AY436640:15917R20 y CACCTACATAT rACCCCACA 3' Length: 20-mer Score: 914 points 3' Position: 15917 Tm/tm: 53.1 53.8 °C AC/Ag (25 "C): -28.6 -28.5 kcal/mol A5/As: -430.5 -419.6 cal/ŕK * mol AH/Ah: -157.0 -153.6 kcal/mol 3'AC: -6.9 kcal/mol Degeneracy: ___1 P.E.#: (477/477) l/E: tt5 nm ol /A2 G0 31.0 pg/A2G0 Priming Efficiency PE Score ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ ODODODODODOD ODODODODODODO ODODODODODODO eee_ File: BftCAZ gene.seq Current Oligo ZL-rner [5042] Current Oligo Duplexes • HAIRPIN intramolekulärni DIMER intermolekulärni [Current + Oligo! - The most stable 3'-dimer: # of hydrogen bonds = LO: AG = -0.7 kcal/mol 5' GAATTAGArAAArrCAAATTA 3' III II II III V ATTAAACTTAAATAGATTAAG 5 r [Current- Oligo; - The most stable 3'-dinner: * of hydrogen bonds = 10; AG = -7.3 kcal/mol; Tm = 2.9°C 5 1 TAA TTTOAATT7TATC T AATTC 3 f I I I I I I I I I I 3r tTTAATCTATTTAAGTTTAAT Sr The most stable dimer overall: # of hydrogen bonds = LO: AG = -7.4 kcal/mol; Tm = 2.2X. 5' GAArrAGATAAATTCAAATTA 3" I I I I I I I I I I 3' ATTAAAC TTAAATAt] ATTAAu 5' Hairpin: loop = S nt: AG - -3.0 kcal/mol; Tm = S4.6 °C 5 1 UAATTAG-, I I I I I A 3'ATTAAAG TTAAAT ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz DODODODODODOD ODODODODODODO ooo Current Oligo Hairpin Stems File: BRCA2 gene.seq Current Oligo 21-rner (5042J L # of paired bases = 5; loop = 5 nt: AC = -3.0 kcal/niol: Tm = S4.6 °C 504 2 GAATT 5046 I I I 5056 CTTAA 5052 5 rCAAITAG-, 1 í 3 1 ATT AAA:: IT AAA T-' 2. ŕ of paired bases = 6; loop = 5 nt; AC = 0.2 kcal/niol: Tm = 2L7 °C 5043 AATTACA 5049 5 16AATTAfiAí I I I I II I I I I II 5061 TTAAAGT 5055 3 ' A IT AAA G IT A 3. # of paired bases = 4: loop = 2 nt: AC = 0.9 kcal/niol; Tm = 8.7 rC 5052 AATT 5055 I I 5061 TTAA 5053 5 1 GAATTAGATAAATTCn I I I I 3 ' AITAAAJ ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz □ odododododod ODODODODODODO ODODODODODODO ©06 Reverse Primer False Priming Sites File: M13MP18 Reverse Primer M13MP18 6310R19 (positive strand) Priming efficiency of the perfect match is 482 (above the threshold) Priming efficiency. 482 (above the threshold) 5'(6328) GGTTTTCCCAGTCACGACG (6310)3' I I I I I I I I I I I I I I I I I I I 3*(6328) ccaaaagggtcagtgctgc (6310)5' Priming efficiency 244 (above the threshold) 5*(6328) GGTTTTCCCAGTCACGACG (6310)3* III I I I I MINI 3'(626) agcaaatggtc—tgctgc (610)5' Priming efficiency 193 (above the threshold) 5'(6328) GGTTTTCCCAGTCACGACG (6310)3' I II MINIM III 3'(5125) tctaagtggtcagtg-tgc (5108)5' □ O □ O u u u u u o u o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o MASARYKOVA UNIVERZITA www.muni.cz □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o ODODODODODODO AHP2 cDNA (TAIR database)! Sequence: AT3G29350.1 Date last modified 2007-04-17 Name AT3G29350.1 Tail 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 ACT AAA GAGA 601 CTAGTCCATA AGAAGAAAAA AG AT GATGAC TTTCTTTCTT TAGTTTCTCT 651 TCTAAATTAT TTTGGATTTG GTGTTTGCTC AAAAACTCAA TAAAATATGT 701 GCAAAAAGAA ACAAAAACAA GTGATGGTTG TTTATAAATC AGTAGTATGT 751 ATTGTTTGAT CTCATCCGAG AAAATTGAAA CCATTGGACT AATGAATGTG 801 ATGATAATAT ATATTGGTTT GCTTCTG DODODODODODOD ODODODODODODO DODODODODODOD ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz DODODODODODOD ODODODODODODO 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'......GI AATTC......3 3"......CTTAA G......5 Design of primers AHP2 ex_up 5'- CCG GAA TTC ATG GAC GCT CTC ATT GCT C AG - 3' AHP2ex_low 5'- CCG GAA TTC TTA GTT A AT ATC C AC TTG AGG - 3' ODODODODODODO DODODODODODOD ISIÍIÍIÍISIÍI MASARYKOVA UNIVERZITA www.muni.cz DODODODODODOD ODODODODODODO ODODODODODODO 101 CCCAATCTCC GCTTCACTCT TCTC[4TGGAC GCTCTCATTG CTCÄČJCTTCA 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 AGTT CCTCAA GTGGATATTA ACTAA AGAGA EcoRI restriction site 5'......GI AATTC......3 3"......CTTAA G......5 Design of primers AHP2 ex_up 5'- CCG GAA TTC ATG GAC GCT CTC ATT GCT C AG - 3' AHP2ex_low 5'- CCG GAA TTC TTA GTT A AT ATC C AC TTG AGG - 3' ODODODODODODO DODODODODODOD ODODODODODODO MASARYKOVA UNIVERZITA www.muni.cz ODODODODODODO DODODODODODOD ODODODODODODO 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 DODODODODODOD ODODODODODODO DODODODODODOD ODODODODODODO DODODODODODOD o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o o □ □ o MASARYKOVA UNIVERZITA www.muni.cz □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o □ o 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 proteomikf (CZ. 1.07/2.3.00/09.0132) Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. INVESTICE DO ROZVOJE VZDĚLÁVÁNÍ