UNI Design of PCR Primers fGdCTTCTG CTCAATCTTT ctACAACCAA AGCTCTGTCT TGAA> GTCAI UU I I U I ÚUACUAI U ATCATGTTTT CCTTGATATC ATGT "GCTTCAACA CTCCAAATAC AGAGGTAATT AAATATTATT ATCA kATCAACC/Vi TTAQOATOAA TQAQAAmAA TTGTAAACAC TTCA 0"GGTGATTT TAAAGAATAT GTTTTACTTA TGTTATGAAC TATC" TGTGAAATA TTTCATAACT AATGTGGAAA ACTATATAAC CCCT ^AAACGTAAG TAAAATTTAT GAAATCCTAT CA1 1 I I I AAA GGTT/ -0-« T Step 3. Capping DMT- O - O- p _o -, - o OR N Stop 4. Oxidation O II cmj- c - o - e Step 3. Capping Liquid Chromatography UNI LC option: Perfusion Chromatography classical sorbent POROS 1341ong.bio - 20.0ul 0.15H 2 6 0 n m 0.10- 0.05^ 0.00- 0510 15 20 25 30 35 Min <--W Min 134.bio - 20.0ul 0.1W 2 6 0 ■ m O.IOJ 0.05H forot &C( * i i —e/- 0.0 2.5 Min M U l\l I Other tools for Quality Control LC- M S * additional literature for interested An automated compliance-ready LC-MS workflow for mass confirmation of both modified and unmodified oligonucleotides, Sep 2020 https://www.waters.com/webassets/cms/library/docs/720006820en.pdf PAGE Mass/charge Capillary ckctiophoresiB trace MUNI YIELD Yield 85 85 75 75 65 65 crude purified crude purified crude purified MUNI PURIFICATION • Sephadex • RP cartridge • HPLC 190.bio - 20-Ovl MUNI • manual • computer assisted * Additional literature for interested https://www.sciencedirect.com/science/article/pii/S2001037019300844 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 UNI OLIGO 6 PCR primers hybridisation probes sequencing primers OLIGO 7 (from 2008) TaqMan probes primers for nested PCR molecular beacons siRNA * Additional resources for interested http://oligo.net/tutorials.html MUNI Terminology of PCR primers forward primer... part of the + string reverse primer... part of the - string pos: 350 tm: 57.1 113 ,3€0 .370 CCTGGCTCTGACTACTCAjr h h h h......h......h......► ► ► ► i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 hhhhhhthhth TTAATG C CTG G CTGTGACTACTCACWtt EAAGGATGGTATTGAG C CTATGTG G G AAG A GAGAAAAACAAAC GGGGAGGAC GATGG CTAATTACATTGAACAAACAH ACACACG AAGTGAtCTC AATTAC G GAC C GACACTGATGAGTGAAAAATTC CTAC CATAACTC G GATACAC C CTTCTACTC11111GTTTG C C C CTC CTG CTAC C GATTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG LMPGtDYSLFKLJ-GIEPMiELJEKNK 1 T~ Uli 1 \ 1 NKQQRR5DL UPPER - FORWARD - LEFT 5'-► + 5' 3' - 3'-5' <-5' LOWER - REVERSE - RIGHT MUNI Terminology forward primer., reverse primer.. part of the + string part of the - string pos: 350 tm: 57.1 , 2E€ _ , 270 _ , 2G0 _ ,,290,, ^33 1.3 120 110 !'-■) 110 100 170 TTAATG C CTG G CTGTGACTACTCAC1 111IAAGGATGGTATTGAGCCTATCTGGGAAGATGACAAAAACAAACGGGGmGGACGA GGCTAATTACATTGAACAAACAGCACACACGAAGTGACC 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 + 5] ^^^^=1 3' polymerase * 5' LOWER - REVERSE - RIGHT MUNI Annealing of PCR primers pos: 350 tm: 57.1 , 2G0 _ , 29D _ 300 Ö.3 3?0 33Ü 3^0 373 TTAATG C CTG G CTGTGACTACTCAC t 111IAAGGATGGTATTGAGCCTATCTGGGAAGATGACAAAAACAAACGGGGAGGACGA GGCTAATTACATTGAACAAAtAGCAGAGACGAAGTGACC C AATTAC GGACCGACACTGATGAGTGAAAAATTCCTAC CATAACTCGGATACACCCTTCTACTCTTTTTGTTTG CCCCTCCTGCTACCGATTAATGTAACTTGTTTGTCGTCTCTG CTTCACTG GAG 4 4 4 1 4 H < ACTC G GATACAC CCTTCTACTC LMPGCDY5 L F K D G I E P M W E DE KNKRGGRW I T L NKQQRR5DL UPPER - FORWARD - LEFT + 5'-- 3'- 3' 5' 5' LOWER - REVERSE - RIGHT MUNI 5' CTT CTG CTC AAT CTT TCT AC 3' FORWARD read from left to right + C ' 1 ATGdCTTCTG CTCAATCTTT CTACf\ACCAA AGCTCTGTCT TGAAAATCAA 51 TGTCAII I U IUUAUUAIU AI ÜATGTTTT 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 T A AC CA AT AT GATATATAAA 451 GTTTATTTTC TTTTTGTCAA ACCATACTTT ATACTATGTA ACTTTTTTAA 501 GAGATTATTG AAAATAGTTT ATTTATAAAA TAGTAACCTA TTGTTGAATT 551 AAAAAAAAAA A a a a a ATraT a a ArarraTT rar, a a a r.n Ar. ATGTGATTTA 601 TCTTAGTTTA /|A ACT AG CTG ATATTCTTCAlAATCGACTGT TCTTATAAGT 651 AATCAACCAA t IAUUAIUAA I UAUAAI AAA 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'TGAAGAATATCAGCTAGTTT 3' REVERSE read from right to left complementary MUNI «oe Sequence DNA Sequence selected Oligo Position Lcngtn^Ü Sequence Length: LS6S nt m Forward Primer 259 IS Reading Frame: + 1 ID ifia£rse Primer 32S ^^Jä^r Current Oligo Length: 21 nt B UpperUngo^™ Position: 356 ID _B_ Lower Oligo 294 22_ ID W 593°C ID PGR Product S7nt # Feature Location 1 source -IE..1S5Ö- CD5 1..651 =1 m pos: I 350 tm: | 57,1 ......... ,270 ,230 I I.........I I I I ,290 ,300 I.........I...... ,310 ,320 ,330 ,340 I I I I.........I....... 350 ,3E0 ,370 I.........I...... CC~CGC~C~CAC~AC~C^ TTAATG C CTG G CTCTC AtTAtTt AtTTTTTAAG GATG GTATT G AG C CTATGTG G G AAG ATGAG AAAAACAAAC G G G GAG GAC GA~G G C^PTTAC ATTG AAC AATTAC GGACC G AC ACTG ATG AGTG AAAAATTC CTAC CATAACTC GGATACACC CTTCTACTCTTTTTGTTTG C C C CTC CTG CT^yfrTAATGTAACTTGTTTGTC GTCTCTG CTTCACTG GAG .........ACTC G GATACACCCTTCTACTC ^ ....................... CCTCCTGCTACC GATTAA I LMPGCDYSLFKDGIEPMWEDEKHKRGGRW ITL NKQQRR5DL _ I Search for Primers & Probes 7 — Search Options Subsearches Search in: iVf + Strand 5? - Strand Search Mode: @ Select Verify 5? Complex Substrate ^ PCR Primers. Compatible with the _ Forward Primer O Reverse Primer 0 TaqMan Probes & PCR Pairs Compatible with the Upper Probe w Lower Probe O Molecular Beacons & PCR Pairs O Nested Primers Sequencing Primers C Hybridization Probes siRNA Probes After successfull search show: All Results Search ( Cancel ) ( Apply ) \ Parameters ( Ranges ( Defaults ) I Search for Primers & Probes Search Options ~ Subsearches 1 Search method: Compatible Pairs 0 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 q Oligonucleotides within Selected Tm Limits Hairpin-free Oligonucleotides 5! Eliminate Mono- and Di-Nucleotide Repeats 0 Detect Sequence Repeats B Eliminate Frequent Oligonucleotides Omit High Secondary Structure Regions in the Template Check Primers/Probe Sequence Constraints 0 Restrict the Number of C Bases 0 Eliminate False Priming Oligonucleotides and_ Continue Above Search in Other File(s) _ Consensus Primers I Search ) ( Cancel ) ( Apply ) ( Parameters ) ( Ranges ) ( Defaults ) MUNI File: oo PGR File: Human 4E.seq Optimal Annealing Temperature: 50.S T (Max: 66.3 °Q Position and Length Uppe Lower Oligo CC P.E# Score 7B.9 29.6 n/a 697 56.9 45.5 47L/47L S40 55.3 29.6 4S9 / 4S9 S34 55.5 33.3 479 / 479 9L7 55.4 39.1 45? f 451 S4L Product tm - Reverse Primer Tm: Z3.6 Primers Tm difference: L.6 °C Comments: Concentration Forward Primer 200.0 nM Reverse Primer 2ÜO jO 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: mM MUNI ©60 Selected Primers File: BRCA2 gene.seq AY436640:1543SF22 5' CA ATA TATA C C CTA CTC C C CTA 3' Length: 22-mer 5core: 802 points 5' Position: L5438 WW 53.4 52.6 °C AC/Ag (25 °C)\ -30.5 -29.2 kcal/rnol A5/As: -472.1 -449.5 cal/°K*mol AH/Ah: -L7L.3 -L63.2 kcal/mol 3'AC: -6.5 kcal/mol Degeneracy: ^^^^^ P.E.#: ^43/4431 L/E: ^.Iffnni ol /A2 G0 31.1 pg/A260 AY436640:15917R20 5' CACCTACATATTACCCCACA 3' Length: 20-mer Score: 914 points 3' Position: 15917 Tm/tm: 53.1 53.S °C AC/Ag (25 ÜC): -28.6 -28.5 kcal/rnol AS/As: -430.5 -419.6 cal/°K * mol AH/Ah: -L57.0 -153.6 kcal/rnol 3'AC: -6.9 kcal/mol Degeneracy: ^^^^^1 P.E.#: ^77/47^ 1/E: ^^*9!ff?nni ol /A^ q n 31.0 Mg/A2C0 Priming Efficiency PE Score UNI Secondary structures eoe Current Oligo Duplexes file: BRCA2 gene.acq Current Oligo 2L-mer [5042] [Currentt Oligo! - The most stable 3-dinier: # of hydrogen bonds = 10: AC = -0t7 kcal/mol 5 cAArTACArAAA^cAM^ra a- DIMER intermolecular 3' ArTAAACrTAAATAC-ATTAAC:- [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 = 2.2irC 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 5'?ttTTfiGA HAIRPIN intramolecular 3 ' ATTAAACTTAAAT-1 MUNI 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 GAATTÄG-, III A 3 'ATTAAACTTAAAT-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 Ma LCT 5055 1 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 A!?T AAA- III 668 Reverse Primer False Priming Sites I 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 MUM 3'(626) agcaaatggtc—tgctgc (610)5' Priming efficiency 193 (above the threshold) 5'(6328) GGTTTTCCCAGTCACGACG (6310)3' III I I I I III III 3'(5125) Ictaagtggtcagtg-tgc (5108)5' © ^ O Forward Primer Composition File: BRCA2 gene.seq Forward Primer AY436640 6275F19 64.2° (nearest neighbor method] Tm 56.5# (nearest neighbor method] Tm 70.8° (*GC method) Tm 56# (2(A+T)° + 4(C + C)0 method) Tm(RNA)(lM Na] 81* |*CC method) Tm (DNA. RNA)[ 1M Na) 74.7° [*GC method) A260/A280 1.59 (single strand] Molecular Weight S.8K (one strand) Molecular Weight 11.7K (two strands] ug/OD 47.4 IdsDNA] Base Number** A 2 (10.5*1 C 5 (26.3*] C | 4 (21.1%) T 8 (42.1*) A + T 10 (S2.6*| G + C 9 (47.4*1 MUNI file: NewDatabase.cdb O'iqor'Lcleotide Database 5? Si □ # of Records: 29 # Date ID Number Sequence 3-Dirn. AC P.E / p.e. Tm /tm ,1 1 □ 21 12/02/06 AY436640:5916R19 AA 1CCC 1 CCC 1 1IACICIC _ 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 \— laň J49 449 54.7 53.1 24 12/02/06 AY436640:5937R22 TTCAA 1 1 1 L 1 1 1 ACCTTCCCAT UM »ss 45S SS.9 S3.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 AATTACCTC1 1 1CTTATCCCAA 0.3 sc 453 453 53.3 53.0 0 u 27 12/02/06 AY436640:5786L23 CTCTCCCTA CAA CATTATCACTC -0.3 sc 4SI 4SI 54.S 55.0 A u 2S 12/02/06 AY436640:5S60L19 AACAACCAAACCCAACCTC -0.9 sc 444 444 55.3 55.9 T Oligonucleotide 5et& (64) □ □ □ U U □ 36 42 47 39 33 61 4S Forward Primer S S 9 9 9 9 Rever&e Primer —T~ 23 24 14 15 16 17 IS Upper Ol i go 3 25 25 25 25 25 25 2S Lower Ol i go 4 2S 23 27 27 27 27 27 Checked Set of nested primers This database is linked to BRCA2 gene.seq MUNI Restriction Enzyme Sites in Protein File: BRCA2 gene.seq # EhZyme |2j4nD lZlh0D ^ISOD ^aODD tZAhOD fiSOD !^ODD ^«DD ,24i.DD ^-1 tLÚ j^SGDD ,2SJDD ,2SĚ.DD ^SiDO |2bOD0 ^DJDO r': ,2ti,D0 ,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 162 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 26174 420 26594 22863 44 Nael AC2PAxR6 7 -21S23 230B4 597 236S1 1286 24967 86 25053 573 25626 149 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. -.-./--lr. -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 O O O Hybridization Time file: M13MP1S DNA Length: Concentration: 21 nt. 200.0 nM L.298 MQ/mL TO = 45.4 sec T =3 rnin 47 sec I eo Concentrations i fi e: BRCA2 gene.seq © Constant Concentration Q Constant Volume © Current + Oligo: l3 Current +Oligo: C Entire Sequence (ds): (3) 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/00,34.0 ug/OD 0.146 nmol/ODr 4S.L ug/OD 4.S3 nmol/ODr31.2 ug/OD 4.67 nmol/ODr 30.9 ug/OD 32.5 MS or or in L.O OD(260J 5.0S4 nmol 50S.4 uL yields LO.O uM MUNI 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 MUNI 101 CCCAATCTCC GCTTCACTCT TCTCATGGAC GCTCTCATTG CTCAGCTTCA 151 GAGACAATTT CGTGATTACA CCATTTCTCT CTACCAACAG GGGTTTTTGG 201 ATGATCAATT TACTGAGTTG AAAAAGCTAC AAGATGATGG AAGTCCTGAT 251 TTTGTGTCTG AAGTGCTTTC ACTTTTCTTT GAAGATTGTG TGAAGC TT AT 301 CAGTAACATG GCTAGAGCTT TGGAC AC GAC AGGAACTGTA GATTTTAGTC 351 AGGTAGGTGC TAGTGTGCAT CAATTGAAGG GTAGTAGCTC AAGTGTTGGT 401 GCCAAGAGGG TCAAAACTTT GTGTGTTAGC TTCAAGGAAT GTTGTGAAGC 451 TAAGAACTAC GAAGGGTGTG TGAGATGTTT GCAGCAAGTG GATATTGAGT 501 ACAAGGCGTT AAA GAG A AAG CTTCAAGATA TGTTCAATCT TGAGAAACAC 551 ATCATTCAAG CTGGTGGTAT AGTTCCTCAA GTGGATATTA ACT AAA GAGA EcoRI restriction site 5'......GlAATTC......3 3"......CTTAAI G......5' Design of primers AHP2ex_up y- 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' I 10: 1 CCCAATCTCC GCTTCACTCT TCTCgTCGgT GCTCTCATTG 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 AGTTpiHTc_AA (.ITGGATATTA ACTA^AGAGA EcoRI restriction site 5'......G| AATTC......3 3"......CTTAAl G......5' Design of primers AHP2ex_up 53- CCG GAA TTC ATG GAC GCT CTC ATT GCT CAG - 33 AHP2ex_low 5'- CCG GAA TTC TTA GTT AAT ATC CAC TTG AGG - V U l\l I Additional resources for interested Computational and Structural Biotechnology Journal 17, 2019,1056-1065. In-silico Design of DNA Oligonucleotides: Challenges and Approaches, includes a list ofoligo design tools https://www.sciencedirect.com/science/article/pii/S2001037019300844 www.protocol-online.org/prot/Research Tools/Online Tools/Oligo Design/index.html OLIGO 7 Power Point Presentation http://oligo.net/tutorials.html OLIGO Primer analysis software, Version 7 http://oligo.net/tutorials.html https://langdalelab.files.wordpress.com/2015/07/degenerate-primer-design.pdf http://www.genomecompiler.com/tips-for-efficient-primer-design/ (2015) Nature Methods 2014, 11 (5): 499. Large-scale de novo DNA synthesis: technologies and applications Expert Opin Ther Pat. 2014, 24(7):801-19. Oligonucleotide delivery: a patent review (2010 - 2013). Laboratory Methods in Enzymology 529: DNA Explanatory Chapter: PCR Primer Design (2013) PCR Primer Design; IMBB Workshop, N. Ndegwa (2013) PCR Primer Design; A. Yuryev (2010) AAPS Journal 2009, 11(1): 195 - 203. Targeted Delivery Systems for Oligonucleotide Therapeutics iCODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer)PCR primer design (2009) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2703993/ Bioinformatic tools and guideline for PCR primer design. Kamel A. Abd-Elsalam (2003) Artificial DNA: Methods and Applications; Khudyakov, Y.E., Fields, W.A., Ed. (2003) PCR Primer: A Laboratory Manual (2003) III Discovery is not in seeking new landscapes, but in having new eyes... Marcel Proust