MASARYK UNIVERSITY Czech Republic Design of PCR Primers ľGC- CTTCTG CTCAATCTTT C "ACAACCAA AGCTCTGTCT TGAA> GTCAI I I U I CJLiAOOA i U ATCATGI I I I CCTTGATATC ATGT 'GCTTCAACA CTCCAAATAC AGAGGTAATT AAATATTATT ATCA ^TATAATATG TTATTGATTT TTTGTTTGTG ATTTCATTTA GATTT "CTATGATTT CTTAGCATGA AAT A C AATTT TTGGAGAAAC AACT TTAAAAACA AAACTTGAAT TTTGAGAAAT TCAAAGATGT TATA" "GTCAAAATT TAACAATTAT TCTTCTAAAT CATCCGGATT CCGT 3TACACATCT ACAATTTTCA ATTGAGGTAT TCTTGTTTTG ATGC 3ACGAATAGT TTGATTGATA AAAAAAATTC T AAC C AAT AT GATA 3TTTA I I I I C I I I I TGTCAA AC C AT ACTTT ATACTATGTA ACTTT 3AGATTATTG AAAATAGTTT ATTTATAAAA TAGTAACCTA TTGT1 ^TGGTGATTT TAAAGAATAT Gill I ACTTA TGTTATGAAC TATC" TGTGAAATA TTTCATAACT AATGTGGAAA ACTATATAAC CCCT VTCAAAAAGT AATAATTCTT GGTACTTGCA ATA I I I I I GT CATT/ "AGTTTATTA AI I I IAI I I I G ATT AAATG G TTTTAGATCC ATCAG 3AGATCGCAG TT AT AG CTGT AGACGATCCG AAGAAAGCAT TA1 AAAAATTCAA CGAGACAATA TAGATCTCAT AATCACAGAT T ATI CTGGTATGAA CGGTTTACAA CTCAAAAAAC AAATCACTCA GG; AATTTACCGG TCTTAGGTAA CAT I I I I I GT TCTTTACAAC TTAA 5TT TGCAAACGAC ATG1 CA AATCGACTGT TCTT> \AA TTGTAAACAC TTCA Hana Konečná Proteomics Core Facility CEITEC Central European Institute of Technology NCBR National Centre for Biomolecular Research MASARYK UNIVERSITY Czech Republic definition aplications modifications synthesis purification quality control design of sequence rules software OLIGO 7 example MASARYK UNIVERSITY Czech Republic oligonucleotide • short single stranded structure • DNA or RNA (also PNA) • hydroxyl on both ends (no phosphate on 5-end as usual) oligonucleotide orientation! polymerase! 5' end 3' end CH H ITfTl MASARYK l[uj UNIVERSITY B^l Czech Republic Aplications of synthetic oligonucleotides • primers for synthesis of complementary DNA PCR, Real-Time PCR • gene synthesis and recombinant proteins • hybridisation probes for cloning • site directed mutagenesis • sequencing ang genetic profiling • diagnostics - tests and biosensors • gene arrays • blocation of gene expression antisense oligo • prospective therapeutics and DNA vaccines • NMR monitoring of DNA - protein interactions • structural X-ray analysis of NA MASARYK UNIVERSITY Czech Republic Modifications • degeneration • end of sequence • bases • phosphate • carbohydrate • PNA S'-conJugate^}^ B heterocyclic base 3-conjugate MASARYK UNIVERSITY Czech Republic Degenerated oligonucleotides Examples: ACG TAC GTA CGT ACG TAC non-degenerated 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 MASARYK UNIVERSITY Czech Republic Degenerated oligonucleotides 2-deoxyinosin M AorC R AorG W AorT S C orG Y CorT K G orT V A or C or G H A or C orT D A or G orT B C orG orT N G or A or Tor C X G or A or Tor C MASARYK UNIVERSITY Czech Republic postsynthetic modifications sequencing fragmenation analysis gene arrays Real-Time PCR Phosphorylation -► Amino group -► Thio group Digoxigenin -► Biotin Enzymes Psoralen Acridine Cholesterole -► Fluorescent dyes Quenchers 2,4- dinitrophenyl Spacer Branching Blocation MASARYK UNIVERSITY Czech Republic derivatized matrix Phosphate Thio group Amino group Spacer Acridine Biotin Fluorescent dyes Quenchers Cholesterole 2,4 dinitrophenyl MASARYK UNIVERSITY Czech Republic nOHWARD PPIMEn • 2x labeled probe • REPORTER • QUENCHER 2, Strand displacement: When the probe is intact, the reporter dye emission is quenched. r. a1 5_9, r I' 3, Cleavage: During each extension cycle, the DNA polymerase cleaves the reporter dye from the probe. SV J" —^ B' 9' 4, Polymerization completed: Once separated from the quencher, the reporter dye emits its characteristic fluorescence. MASARYK UNIVERSITY Czech Republic Other modifications Phosphorothioates *-backbone Phosphorodithioates H-phosphonates Methylphosphonates ■ . . . Modifications in 2'- position carbohydrate-- Ribose modificationM MASARYK UNIVERSITY Czech Republic Therapeutics + Nondegradable by nucleases! Modification of phosphodiester i 0 = P I o o = 0 1 p I o CH 0 = phosphorothioate methylphosphonate phosphoramidate CH2 H3C-N 0 = P-BHa" 1 I 1 3 -thioformacetal methylene(methyliminio) boranophosphate MASARYK UNIVERSITY Czech Republic antisense oligonucleotide oligonucleotide or analogue complementary to segment of RNA or DNA inhibition of normal function due to coupling Nucleus \ mRNA Translation Ribosome Protein Cytoplasm Antigene (triplex) Ribozyme RNA with catalytic activity Cell membrane Antisense Sense/Aptamer MASARYK UNIVERSITY Czech Republic Peptidonucleic acid PNA DNA • uncharged molecule • binding with DNA/RNA N-(2-aminoethyl)-glycine-► i—NH 0 H2N— N-terminal , v_ 5'-end HN V i O B 17 u j ~p o=( o=p-o~ t 0=( 0=P-0_ i O 3 -end o C-terminal 0=\ 0=P^-0" PNA DNA MASARYK UNIVERSITY Czech Republic Peptidonucleic acid noncharged molecule binding with DNA/RNA N-(2-aminoethyl)-glycine- •NH O PH R,N- PNA DNA B 0=P-0" O 0=P-0 0= DNA MASARYK UNIVERSITY Czech Republic Why PNA • thermostable • Tm not depending on salts • high specificity • high affinity • resistant towards enzymes Gambari R. Expert Ooin Ther Pat. 2014, 24(3):267-94. Peptide nucleic acids: a review on recent patents and technology transfer. MASARYK UNIVERSITY Czech Republic I Locked Nucleic Acid • 2'-0, 4'-C methylene bridge • supressed flexibility of ribofuranose ring • structure is locked into rigid C3-endo conformation • enhanced hybridisation • outstanding biostability Molecular Therapy (2012); 20 8,1590-1598. LNA-based Oligonucleotide Electrotransfer for miRNA Inhibition MASARYK UNIVERSITY Czech Republic design synthesis purification EXPEDITE 8909 MASARYK UNIVERSITY Czech Republic I j.' i /™n li • synthesis on solid matrix Oligonucleotide Synthesis . fryom 3-_end t0 5^.end • anhydrous environment HO- O II CH— C - O O II B, o II -0— p -o II - 0— p -q _ Cleevaga/Deprotection DMT— O I OR Step 4. Oxidation B. OMT— O —v - O — • "M B, -O - Stepi. Deblocking Bj O II CHj- C - O Step 3. Capping - O DMT- O N B, O — p -Q i A OR Step 2. Coupling o-m Activation DMT- O "\ - O — p _ N(iPr)j OR ♦ Tetrezole MASARYK UNIVERSITY Czech Republic Quality Control HPLC perfusion chromatography anex RP MASARYK UNIVERSITY Czech Republic classical sorbent POROS 1341ong.bio - 20.Opi 0.15H 0.10 0.05H 2 6 0 n 0.0C 0.1SÍ o.ioH 0.05H rlOO h50 oi-^ 134.bio - 20.0ul 0.00 100 0.0 MASARYK UNIVERSITY Czech Republic MM Mass/charge Capillary cfccteopboraia trace MASARYK UNIVERSITY Czech Republic 85 85 75 75 65 65 crude purified crude purified crude purified MASARYK UNIVERSITY Czech Republic PURIFICATION • Sephadex • RP cartridge • HPLC ,.0 MASARYK UNIVERSITY Czech Republic • manual • computer assisted www.protocol-online.org/prot/Research_Tools/Online_Tools/Oligo_Design/index.html Main features of good PCR primer sequence • highly specific • no dimers and hairpins • stable duplexes with active sequence • lightly unstable 3'-end MASARYK UNIVERSITY Czech Republic OLIGO 6 PCR primers hybridisation probes sequencing primers OLIGO 7 (from 2008) TaqMan probes primers for nested PCR molecular beacons siRNA MASARYK UNIVERSITY Czech Republic Terminology of PCR primers forward primer... part of the + string reverse primer... part of the - string pos: 350 tm: 57.1 3S0 , CCTGGCTiTGACTACTCA ►>■►►*■......►......►......► ► ► ► i hhhhhhthhth TTAATG C CTG G CTGTG ACTACTC AC WW EAAGGATG GTATTGAG C CTATGTG G G AAG A GAGAAAAACAAAC GGGGAG g AC G ATG g CTAATTAC ATTG aacaaacwsc AÍ AC ACG AACTÍ acc C m™cggaccgacactgatgagtgaaaaattcctaccataactcggatacacccttctactc i i i i igtttgcccctcctgctaccgattaatgta; tCTTGTTTGTC GTCTCTG CTTC ACTG G ag LHPGCQY5 LFKO-G IE P M i E DE KNKRGGRi I t L NKQQRR5DL UPPER - FORWARD - LEFT 5'-► + 5'-- 3'- ■ 3' ■ 5' 5' LOWER - REVERSE - RIGHT MASARYK UNIVERSITY Czech Republic Terminology forward primer., reverse primer.. part of the + string part of the - string pos: 350 tm: 57.1 , 2G0 _ ,,290, ^33 1-3 111 3 IM 170 TFAATG C CTG G CTGTGACTACTCAC1 111IAAGGATGGTATTGAGCCTATCTGGGAAGATGACAAAAACAAACGGGGmGGACGA GGCTAATTACATTGAACAAACACCACACACCAACTCACC C AATTAC GGACC G AC ACTG ATG AGTG AAAAATTC CTAC C ATAACTC G G ATAC AC C CTTCTACTCTTTTTGTTTG CCCCTCCTGCTACC GATTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG 4 4 4 1 4 H * ACTC G GATACAC CCTTCTACTC LMPGCDY5L FKDCIEPHWEDEKNKRGGRW I T L NKQQRR5DL UPPER - FORWARD - LEFT 5'-► polymerase f 5] ^^^^=1 3' - v i J v polymerase * 5' LOWER - REVERSE - RIGHT MASARYK UNIVERSITY Czech Republic Annealing of PCR primers pos: 350 tm: 57.1 , 2G0 _ _ 1290 _ Ö33 3?0 33Ü 3^0 110 ÖG3 170 TTAATG C CTG G CTGTGACTACTCACE 1111AAGGATGGTATTGAGCCTATCTGGGAAGATGACAAAAACAAACGGGGAGGACGA GGCTAATTACATTGAACAAACACCAGAGACCAAGTGACC C AATTAC GGACCGACACTGATGAGTGAAAAATTC CTAC CATAACTC G GATACAC C CTTCTACTCTTTTTGTTTG C C C CTC CTG CTAC C GATTAATGTAACTTGTTTGTCGTCTCTG CTTCACTG GAG 4 4 4 1 4 H < ACTC G GATACAC CCTTCTACTC LMPGCDY5 L FKDG I E P M W E DE KNKRGGRW I T L NKQQRR5DL UPPER - FORWARD - LEFT +■ 5'-- 3'- 3' 5' 5' LOWER - REVERSE - RIGHT MASARYK UNIVERSITY Czech Republic 5' CTT CTG CTC AAT CTT TCT AC 3' FORWARD ' 1 ATGdCTTCTG CTCAATCTTT Cl"ACAACCAA AGCTCTGTCT TGAAAATCAA 51 TGTCAI (JMJ I I U IüüAUÜAI U ÄTCATGTTTT 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 TTG ATTG ATA AAAAAAATTC TAACCAATAT G ATATAT AAA 451 GTTTATTTTC TTTTTGTCAA ACCATACTTT ATACTATGTA ACTTTTTTAA 501 GAG ATT ATTG AAAATAGTTT ATTTATAAAA TAGTAACCTA TTGTTGAATT 551 AAAAAAAAAA AAAAAATTGT A AAT CG TG TT TGCAAACGAC ATGTGATTTA 601 TCTTAGTTm AAACTAGCTG ATATTCTfCA AATCGACTGT TCTTATAAGT 651 AATCAACCÄA TTAQOATOAA TGAGAAiAAA TTGTAAACAC 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' 5' TGAAGAATATCAGCT AGTTT 3' REVERSE MASARYK UNIVERSITY Czech Republic .BOO File: Human 4E.seq Sequence =1 DNA Sequence Sequence Length: Reading Frame: Current Oligo Length: Position: Selected Oligo Position Lcngtn v # Feature Location B Forward Primer 259 IS source -IE..1S5Ö- JJ JLeierse Primer 32S CD5 1..651 JJ B Upper Oligo — JJ S Lower Oligo 294 22 JJ PGR Product S7nt ÍUOU 11 LOU ,1J0U ,1401 .15011 .lLOU pos: I 350 tm: | 57,1 ......... ,270 ,200 i i.........i i i i ,290 ,300 ,310 i.........ii~.......i i i i ,320 ,330 ,340 i i i i.........i....... 350 ,3E0 ,370 i.........i...... CC~CGC~C~CAC~AC~C^ TTAATG C CTG G CTGTGACTACTCAtTTTTTAAGGATGGTATTGAG C CTATGTG G GAAGATGAGAAAAACAAACG G G GAG GAC GATG G CTJ^TTACATTGAAC AATTAC GGACC G AC ACTG ATG AGTG AAAAATTC CTAC CATAACTC GGATACACC CTTCTACTCTTTTTGTTTG C C C CTC CTG CTA^rfTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG .........ACTC G GATACACCCTTCTACTC ..............................CCTCCTGCTACCGATTAA "^ďfTAAl \ LMPGCDYSLFKDGIEPMWEDEKHKRGGRW ITL MKQQRR5DL _ MASARYK UNIVERSITY Czech Republic O n r> Search for Primers & Probes Search Options Subsearches Search in: 5tf + Strand §j - Strand Search Mode: @ Select Verify B Complex Substrate ©PCR Primers Compatible wi ith the Forward Primer Q Reverse Primer 0 TaqMan Probes & PCR Pairs Compatible with the Upper Probe w Lower Probe \ Molecular Beacons & PCR Pairs Nested Primers Sequencing Primers C Hybridization Probes siRNA Probes After successfull search show: All Results ' Search i ( Cancel ) ( Apply ) \ Parameters"^ ( Ranges ( Defaults ) MASARYK UNIVERSITY Czech Republic ©r>r> Search for Primers & Probes r Search Options Subsearches * Search method: Compatible Pairs 0 Eliminate Ambiguous Bases ft Duplex-free Oligonucleotides B Highly Specific Oligonucleotides (3'-end Stability) _ 5'-end Stability _ siRNA Internal Stability 0 Oligonucleotides with CC Clamp M Oligonucleotides within Selected Tm Limits 0 Hairpin-free Oligonucleotides iminate Mono- and Di-Nucleotide Repeats 0 Detect Sequence Repeats fl Eliminate Frequent Oligonucleotides 0 Omit High Secondary Structure Regions in the Template B Check Primers/Probe Sequence Constraints 3 Restrict the Number of C Bases 0 Eliminate False Priming Oligonucleotides and _ Continue Above Search in Other File(s) _ Consensus Primers Search ^ ( Cancel ) ( Apply ) ( Parameters ) ( Ranges ) ( Defaults ) MASARYK UNIVERSITY Czech Republic s o ^ File: Human 4E.seq PCR Optimal Annealing Temperature: 50.8 °C (Max: 66.3 ÜQ Position and CC [%} Length P.F_# Score 78.9 29.6 n/a 697 Forward Primer 9LS 22 N 56.9 45.5 47L/47L 840 Reverse Primer L753 27^ S 55.3 29.6 489 / 489 834 --rřT 24 56.5 33.3 479 / 479 9L7 Lower Oligo 1694 23 55.4 39. L 457 / 457 841 Product Tm - Reverse Primer Tm Primers Tm difference: L6ŮC 23.6 °C Comments: Concentration Forward Primer 200.0 nM Reverse Primer 200.0 nM Upper Oligo 200.0 nM Lower Oligo 200.0 nM Monovalent Cation 50.0 mM Free Mg(2+) 0.7 mM Total Na|>] Equivalent: L55.S mM MASARYK UNIVERSITY Czech Republic Selected Primers file: GRCA2 gene.seq AY436640:1S438F22 AY436640:15917R20 5' CAATATATACCCTACTCCCCTA 31 5' CACCTACATATTACCCCACA 3" Length: 22-mer Length: 20-mer Score: 802 points 5core: 914 points 5' Position: 15438 3' Position: 15917 "WW 53.4 52.6 °C Tm/tm: 53.1 53.8 *C AC/Ag (25 *€): -30.5 -29.2 kcal/rnol AC/Ag (25 *C): -28.6 -28.5 kcal/rnol AS/As: -472.1 -449.5 cal/*K*mol A5/As: -430.5 -419.6 cal/úK * mol AH/Ah: -171.3 -163.2 kcal/rnol AH/Ah: -157.0 -153.6 kcal/rnol 3 AC: -6.5 kcal/niol 3'AC: -6.9 kcal/rnol Degeneracy: ___1 Degeneracy: ___1 P.LJ: (443/443) P.E.#: (477/477) 1/E: 4-^3 nm ol /A2 go 1/E: 5^T5 nm ol /A2 G0 31.1 ug/A260 31.0 ug/A26o Priming Efficiency PE Score MASARYK UNIVERSITY Czech Republic Secondary structures \ ^JSZSST eoe Current Oligo Duplexes file: BRCA2 gene.acq Current Oligo 2L-mer [5042] [Currentt Oligo! - The most stable 3-dimer: # of hydrogen bonds = 10: AC = -0t7 kcal/mol 51 GAATrAGATAAArrCAAArrA 31 III II II III 3 r ATTAAACTTAAATAGjU7TAA.G 5 r [Current- Oligo] - The most stable 3-dinier: * of hydrogen bonds = L0: AC = -7.3 kcal/mol; Tm = 2.9°C 5 1 TAArPTGAATTTATCTAATTC 3 f I I I I I I I I I I 3' G TTAATG TATTTAAU TTTAAT 5" The most stable dimer overall: & of hydrogen bonds = L0: AC = -7.4 kcal/mol: Tm = 2.2^ S1 UAATTAGArAAATTCAAATTA 3' I I I I I I I I I I 3 ' ATTAAAC TTAAATAt]ATTAAt] 5 1 Hairpin: loop = 5 nt: AC = -3.0 kcal/mol; Tm = 54.6 °C z>' ^AArrAC- I I I I I A 3 'ATTAAaCTTAAAT-1 MASARYK UNIVERSITY Czech Republic ooo Current Oligo Hairpin Stems File: BRCA2 gene.seq Current Oligo 2L-nner [5042j L # of paired bases = 5; loop = 5 nt; AG = -3.0 kcal/mol; Tm = 54.6 °C 5042 GAA1 1 1 1 5056 CTTJ 5046 \A 5052 5 r GAATTAG-, III A 3 'ATTAAACTTAAflT-1 2. # of paired bases = 6; loop = 5 nt; AC = 0.2 kcal/mol; Tm = 2L7 *C 5043 AATTi 1 1 5061 TTAAJ \ZA 5049 5 1GAATTAG ATA-, II II 1 1 A LCT 5055 | 3 r ATTAAACTTA-1 3. # of paired bases = 4: loop = 2 nt; AC = 0.9 kcal/mol; Tm = 8.7 T 5052 AAT 1 1 5061 TTA r 5055 A 5058 5 1 GAATTAGATAAATTC-. 1 1 1 1 3 r AÍT AAA- MASARYK UNIVERSITY Czech Republic O ^ Reverse Primer False Priming Sites lie: M13MP18 Reverse Primer M13MP18 63L0R19 (positive strand) l± Priming efficiency of the perfect match is 482 (above the threshold) [▼ Priming e 5 (6328 3'(6328 Priming e 5 (6328 3'(626 Priming e 5 (6328 3'(5125 ficiency 482 (above the threshold) GGTTTTCCCAGTCACGACG (6310)3' I I I I I I I I I I I I I I I I I I I (6310)5 fkiency: 244 (above the threshold) GGTTTTCCCAGTCACGACG (6310)3' III I I I I I I I I I I (610)5' ficiency. 193 (above the threshold) GGTTTTCCCAGTCACGACG (6310)3' I II MINIM III (5108)5' MASARYK UNIVERSITY Czech Republic O ^ © Forward Primer Composition File: BRCA2 gene.seq Forward Primer AY436640 627SF19 64.2° [nearest neighbor method) 56.5# [nearest neighbor method) 70.8° [%CC method) 56' (2(A-rT)° ♦ 4(C + C)# method) Tm (RN AX 1M Na] 8T [%CC method) Tm (DNA. RNA)[ 1M Na) 74.7* [XGC method) A260/A280 1.S9 [single strand) Molecular Weight S.8K [one strand) Molecular Weight 11.7K [two strands) ug/OD 47.4 [dsDNAj Base Numbers* A 2 [10.5« C S [26.3«: c 4 [21.19Č] T 8 (42.1%) A + T 10 (52.6%) CtC 9 (47.4%) MASARYK UNIVERSITY Czech Republic ©o_e__ file: NewDatabase.cdb Oligonucleotide Database _2 Si □ # of Records: 29 # Date ID Number Sequence 3'-Dim. AC P.E / p.e. Tm /tm ,1 1 □ 21 12/02/06 AY436640:5916R19 AA 1CCC 1 CCC 1 1 IACICIC _ sc 430 430 54.1 54.5 □ 22 12/02/06 AY436640:5916R2O CAATCCCTCCCTCTACTCTC ^ 0.3 sc 366 450 SO.9 57.2 T □ 23 12/02/06 AY436640:5937R21 TCAA 1 1 1 L 1 1 1ACCTTGCCAT \___ mi™ wm. J49 449 54.7 53.1 24 12/02/06 AY436640:5937R22 TTCAA 1 1 1 L 1 1 1 ACCTTCCCAT iSm Bss 45S 55.9 53.S u 25 12/02/06 AY436640:4695U22 TCCCTTAA CAAAA CTAATCCAT 0.3 sc 432 432 54.5 53.0 u 26 12/02/06 AY436640:5325U22 AATTACCTC111CTTATCCCAA 0.3 sc 453 453 53.3 53.0 0 u 27 12/02/06 AY436640:5786L23 CTCTCCCTA CAA CATTATCACTC -0.3 sc 451 451 54.S 55.0 A u 2S 12/02/06 AY436640:5S60L19 AACAACCAAACCCAACCTC -0.9 sc 444 444 55.3 55.9 T J Oligonucleotide 5et& (64) Forward Rever&e Upper Lower # Primer Primer Ol i go Oligo 1 2 3 A 36 S 23 25 2S 42 S 24 25 28 47 14 25 27 39 9 15 25 27 33 9 16 25 27 61 9 17 25 27 4S 9 IS 25 27 This database is linked to BRCA2 gene.seq Checked Set of nested primers MASARYK UNIVERSITY Czech Republic Restriction Enzyme Sites in Protein File: BRCA2 gene.seq # EhZyme |2j4nD ^JfcDD ^ISOD ,21200 tZAhOD fiSOD !^ODD ^«DD ,2**00 flSOD ^SGDD ,2SJDD ,2SĚ.DD ^5400 |2bOD0 ^DJDO r': ^tiiDO ,2kSDD , 33 EcoRI 34 EcoRV 33 EsP3l 36 Fsel 37 Fspl 3S IgsuI 39 Hindlll 40 Hpal Kpnl 42 Mul 43 Muni 44 Nael 45 Narl 46 Ncol 47 Ndel # Enzyme Site #Cuts Positions & Fragment Sizes 4L Kpnl GT2VpzY6 S -21253 23654 68 23722 52 23774 237 24011 585 24596 L62 24758 629 25387 1219 26606 22851 J 42 Mlul TR L RVyA 7 5 -22233 22674 2824 25498 576 26074 106 26L80 244 26424 23033 43 Muni QL3Nawl5 L0 -212S7 23620 355 23975 351 24326 282 24608 242 24850 72 24922 351 25273 714 25987 187 26L74 420 26594 22863 44 Nael AC2PAxR6 7 -21S23 230B4 597 236S1 1286 24967 86 25053 573 25626 L49 25775 623 26398 23059 45 Narl CA2APZR6 L -20043 24S64 24593 f. 46 Ncol PW3HGwM5 4 -22361 22546 336 22882 887 23769 531 24300 25157 47 Ndel HM2lawY5 2 -20366 24541 1211 25752 23705 4S Nhcl A52LAK-6 L6 -22276 22631 322 22953 LS5 2313S 88 23226 27 21211 461 23714 369 240S3 312 24395 288 24683 151 24834 273 25L07 536 25643 1 402 26045 30 26075 210 26285 372 26657 22800 T ■i ■ ■ -i -i J 1- r. -.-./--I r. -í-ií- A r. i A r- ■nr. A -.-i r r.r. w-nr- -i A r.nr- Search: 22454 to 27004 End Cut Type: Blunt. Qdd, 3-overriang. S'-overhang MASARYK UNIVERSITY Czech Republic 8 O O Hybridization Time file: M13MP1S DNA Length: [ Concentration: 21 nt. 200.0 nWi L.29S Mg/mL VA = 45.4 sec T =3 rnin 47 sec MASARYK UNIVERSITY Czech Republic eo Concentrations H e: BRCA2 gene.seq © Constant Concentration Q Constant Volume © Current + Oligo: (^) Current +Oligo: C Entire Sequence (ds): (^) Forward Primer: C Reverse Primer: 0 PCR Product (ds): Upper Oligo: Q Lower Oligo: 5.OS nmol/OD, 32.5 ug/OD 4.67 nmol/ODr 30.9 ug/OD 0.001 nmol/ODh4S.L ug/OD 5.9S nmol/ODr35.0 ug/OD 5.31 nmol/ODř34.0 ug/OD 0.146 nmol/ODr 4S.L ug/OD 4.S3 nmol/ODř31.2 ug/OD 4.67 nmol/ODr 30.9 ug/OD 32.5 MS or or in L.O OD(260) 5.0S4 nmol 50S.4 uL yields LO.O uM MASARYK UNIVERSITY Czech Republic AHP2 cDNA (TAIR database)! Sequence: AT3G29350.1 Date last modified 2007-04-17 Name AT3G29350.1 Tail Accession Sequence:40 10737427 Sequence Length (lip) 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 MASARYK UNIVERSITY Czech Republic 101 CCCAATCTCC GCTTCACTCT TCTCATGGAC GCTCTCATTG CTCAGCTTCA 151 GAGACAATTT CGTGATTACA CCATTTCTCT CTACCAACAG GGGTTTTTGG 201 ATGATCAATT TACTGAGTTG AAAAAGCTAC AAGATGATGG AAGTCCTGA1 251 TTTGTGTCTG AAGTGCTTTC ACTTTTCTTT GAAGATTGTG TGAAGCTTAT 301 CAGTAACATG GCTAGAGCTT TGGACACGAC AGGAACTGTA GATTTTAGTC 351 AGGTAGGTGC TAGTGTGCAT CAATTGAAGG GTAGTAGCTC AAGTGTTGG1 401 GCCAAGAGGG TCAAAACTTT GTGTGTTAGC TTCAAGGAAT GTTGTGAAGC 451 TAAGAACTAC GAAGGGTGTG TGAGATGTTT GCAGCAAGTG GATATTGAGr 501 ACAAGGCGTT AAAGACAAAG CTTCAAGATA TGTTCAATCT TGAGAAACA" 551 ATCATTCAAG CTGGTGGTAT AGTTCCTCAA GTGGATATTA ACTAAAGAG^ EcoRI restriction site 5'......GlAATTC......3 3"......CTTAAl G......5' Design of primers AHP2ex_up y- CCG GAA TTC ATG GAC GCT CTC ATT GCT CAG - 3' AHP2ex_low 55- CCG GAA TTC TTA GTT A AT ATC CAC TTG AGG - 3' MASARYK UNIVERSITY Czech Republic 101 CCCAATCTCC GCTTCACTCT TCTCgTCGgT GCTCTCTTTC CTCAqCTTCA 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 AGTTpčHT(JAA (.ITGdATATTA ACTA^AGAGA EcoRI restriction site 5'......G| AATTC......3 3"......CTTAAl G......5 Design of primers AHP2 ex_up 5'- CCG GAA TTC ATG GAC GCT CTC ATT GCT CAG -AHP2ex_low 5'- CCG GAA TTC TTA GTT AAT ATC CAC TTG AGG - 33 MASARYK UNIVERSITY Czech Republic LITERATURE ■ 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 ■ Expert Opin Ther Pat. 2014, 24(7):801-19. Oligonucleotide delivery: a patent review (2010 - 2013). ■ AAPS Journal 2009, 11(1): 195-203. Targeted Delivery Systems for Oligonucleotide Therapeutics ■ Large-scale de novo DNA synthesis: technologies and applications Nature Methods 2014, 11 (5): 499 AutoDimer (Vallone and Butler. 2004). http://www.cstl.nist.gov/ strbase/NIJ/AutoDimer.htm CODEHOP (Kose et al.. 2003: lioycc et al.. 200*)). littps://icodehop. cphi.washington.edu/i-codehop«>« on text/'Welcome HYDEN (Linhart and Shamir. 2002; 2005). http://acgt.cs.tau.ac.il/ hyden/HYDEN.htm JCVI Primer Designer (I i et al.. 2008). http://soweeforge.net/prcyeccs primerdesigner/ MAD-DPD (Najafabadi et al.. 2008). http://bioinf.i-s.ipm.ir down load/M AD_DPD( >8 172()( )7.zip MIPS (Souvenir et al.. 2003; 2007). http://www.cs.wustl.edu/% 7 E z h a n g/ p roj ec ts / m i ps. z i p NetPrimer. http://www.preiiiitMbiosoft.com/netpriiiRT/index.html OLIGO. http://www.oligo.net PA MPS (Najafabadi et al., 2008). http://www.biomedcentral.com/ content/supplementary /1471 -2105-9-55-S1 .zip Primer3 (Rozen and Skaletsky. 2000)- http://rrodo.wi.mit.edu/primer3/ Primer3Plus (Untergasser et al.. 2oi>7). http://www.bioinforniatici.til/ cgi-bin/primer3plus/prim er3plus. cgi PrimerStation (Yamada et al.. 2o<)('>) http://ps.cb.k.u-tok\o.jic.jp Pythia (Mann at al., 2009). http://frodo.wi.mit.edu/priiner3/ ThermoBLAST™. http://dnasoftware.com/tabid/110/Defeultaspx UNAFold (MfoldM, Markham and Zuker, 2008). http://dinanielt. bioinfo.rpi.edu/download.php http://mfold.bioinfo.rpi.edu/ Vector NTIK. http://www.invitrogen.coni/site/us/en/hoine/LINNEA- Online-( !uides/LlNNEA-Coinmunities/Vector-NTI-Community/Vector- NTI.html Visual OMP™. http://dnasoftware.com/tabid/ 108/Default.avpx MASARYK UNIVERSITY Czech Republic Discovery is not in seeking new landscapes, but in having new eyes... Marcel Proust