Oligonucleotide - AccuOligo Pre-made Primers QC & Order system DNA Oligonucleotide FAQs User Protocol DNA/RNA Oligonucleotide Synthesis Phone:

Oligonucleotide - AccuOligo Pre-made Primers QC & Order system DNA Oligonucleotide FAQs User Protocol DNA/RNA Oligonucleotide Synthesis Phone: 1588-9788(ext.4->1) Email: Oligo-support@bioneer.co.kr

DNA/RNA Oligonucleotide Synthesis Oligonucleotide - AccuOligo 3 Standard Oligonucleotide 4 HT-Oligo (High Throughput Oligonucleotide) 6 Modification Oligonucleotide 7 Dual-Labeled Probes 13 EBQ-Next Generation Dark Quencher Modifications 16 Extendamers 18 Large Scale Oligonucleotide 19 Custom RNA 20 Premade Primers 22 General Research Primer 22 Sequencing Primer 24 Random Primer 25 QC & Order System 26 DNA Oligonucleotide FAQs 27 User Protocol 32

Oligonucleotide - AccuOligo Overview 바이오니아는국내최대의 DNA 및 RNA 합성회사로서, 최고품질의올리고를제공합니다. 대전 DNA 합성센터는세계 3 위의합성능력을보유하였습니다. 바이오니아의올리고합성서비스는주문부터합성, QC, 포장까지전공정을 Computer Aided Manufacturing (CAM) 시스템 에의해자동화함으로써휴먼에러를최소로하여고품질의올리고공급에최선을기하고있습니다. 특히, 독자개발한 Bio-RP 초고순도정제시스템 은고순도올리고생산을가능하게함으로써, PCR, sequencing 등의일반적인실험을수행하는연구기관및회사뿐아니라, SNP typing, gene synthesis, DNA chip 등다양한응용분야의세계적인연구기관및회사에도당사올리고를공급하고있습니다. 바이오니아는올리고를자동정량, 분주시스템과 AccuOligo 기술에의해정확한농도로제공하고, 또고객들이안심하고사용할수있도록총 7 대의 MALDI-TOF 분자량측정장치와 96 CE 시스템을도입하여합성된올리고를전수검사하여통과된고품질의올리고를공급합니다. AccuOligo Technology 일반적으로공급되는올리고는합성, 정제, QC, 분주과정을거쳐건조된후고객에게배송됩니다. 그런데건조된올리고는운송과정중에흔들림과충격에의해튜브의바닥에서떨어져나와벽이나뚜껑에묻어있게되며특히건조된상태의올리고는정전기를띄고있는경우가많아이러한상태로그대로개봉을하게되면용기내에서이탈되어손실이발생할수있습니다. Plate 로공급할경우더심각하여상호교차오염이발생할수도있습니다. 이런경우에는올리고의농도가맞지않아실험결과를신뢰할수없게됩니다. 이러한문제점들은그동안인식되어왔지만, 불안정한용액상태로보내는것이외에는해결책이없었습니다. 바이오니아연구진은이러한올리고이탈손실문제를해결하기위해심한진동에서도튜브바닥에서건조된올리고가떨어지지않는 AccuOligo 기술을개발하였습니다. ( 신규건조올리고뉴클레오티드조성물 특허출원 2006 등록번호제 10-0777249 호 ). AccuOligo 의특징은아래와같습니다. 올리고이탈방지제는 PCR, sequencing, enzyme cutting 등의실험에전혀영향을주지않음 제조또는운송중올리고가이탈손실되는현상을방지 미량올리고도뚜껑에부착되어이탈손실되는현상을방지 올리고를육안으로확인할수있음 Plate 내각 well 간의교차오염방지바이오니아에서제공하는모든올리고는상기 AccuOligo 시스템이적용되며 clean room 에서생산함으로써 DNase, RNase, DNA free 한상태로고객여러분께공급되고있습니다. Bio-RP 정제 : 합성기에서올리고를합성해나가는과정중 coupling monomer 로쓰여지는 phosphoramidite 는 5 -OH 그룹이 DMT 기로보호되어있는데이는합성과정중연속 coupling 반응이일어나는것을방지하기위해서입니다. 합성기상에서올리고합성의마지막 cycle 이끝난뒤 DMT 기를떼어내지않고합성과정을완료하면 ( trityl-on mode) 원하는길이의 N-mer 올리고에만 5 -DMT 기가붙어있고나머지부산물들인 N-1, N-2, N-3 mer 에는 DMT 기가붙어있지않은상태로합성이완료됩니다. DMT 기는 hydrophobic 한성질을가지고있습니다. 바이오니아의연구진들은이러한 DMT 기에강하게부착되는 RP resin 을개발하고컬럼공정을자동화한 Bio-RP 정제방법 을개발하였습니다. 바이오니아는 Bio-RP 정제방법으로원하는 N-mer 올리고만효율적으로분리하고 N-1, N-2, N-3 mer 들을제거함으로써고순도의 N-mer 올리고를정제할수있습니다. HPLC 정제 : Cloning, site directed mutagenesis 또는 quantitative gene detection 등의실험을수행할때에는고순도의합성올리고가필요합니다. 이경우, desalting 이나 Bio-RP 정제만으로는충분하지않은경우가많습니다. 고순도의올리고로정제하기위해서는 HPLC 방법이자주사용됩니다. 정제 resin 으로는 anion-exchange resin 또는 reversed-phase resin 이사용됩니다. anion-exchange chromatography 는 30 mer 가량의올리고정제에적합하며 90% 이상의순도를나타냅니다. RP-HPLC 를사용한정제도 anionexchange HPLC 를사용했을때와유사한정제효율을나타냅니다. 두방법모두합성올리고의길이에따라정제효율이영향을받기때문에보통 35 mer 이상의합성올리고는고순도로정제하기가어렵습니다. PAGE 정제 : 긴올리고를고순도로정제할때사용되는정제방법입니다. Polyacrylamide gel 에전기영동하여보통 130 mer 정도까지분리할수있으며정제후순도는 95% 이상입니다. 전기영동을이용한정제방법은매우높은분리기능을가지지만, 정제된올리고를얻기위해서 gel 상에서원하는밴드를잘라추출해야하고, 또한 desalting 과정을거쳐야하므로최종수율이낮아집니다. Purifications Bio-RP, HPLC, PAGE 등의다양한정제방법을선택할수있습니다. 바이오니아는 Bio-RP 정제를무료로서비스해드립니다. 언제든지고객의실험에맞는올리고의정제방법을상담해드립니다. www.bioneer.co.kr 3

Standard Oligonucleotide Description Standard oligonucleotide 합성서비스는 130 mer 이하의올리고합성서비스입니다. 다양한합성스케일을선택할수있으며정제방법또한용도에따라 Bio-RP, HPLC 또는 PAGE 등다양하게선택가능합니다. Features and Benefits 철저한품질관리 - 총 7 대의 MALDI-TOF mass spectrometer 를사용하여 QC 전수검사시행. 50 mer 이상의올리고는 CE (Capillary Electrophoresis) 장비를사용하여정량 QC 함으로써완벽한품질관리 고품질의올리고 - Bio-RP 초고순도정제시스템으로 n-1 mer 를포함한 truncated failures 제거, clean room 에서생산함으로써 DNase, RNase, DNA free 한상태로제공 정확한농도자동정량분주기와 AccuOligo 기술을적용한정확한농도의올리고제공 자동화생산 - 세계최초로자동화생산라인공정구축 대량생산능력 - 1 일 30,500 개의합성유전자생산 경제적인가격 - 원재료자체생산으로안정적인납기와품질의균일성을보장 Purifications 용도에따른정제방법 Application Standard PCR / RT -PCR Purification Bio-RP PAGE HPLC Multiplex PCR Real-Time PCR Sequencing Genotyping / SNP Cloning (e.g. chemical linkers) Mutagenesis Gene Synthesis DNA Microarray Probes Antisense Primer Extension cdna Library Generation End Labelling & FISH Northern & Southern Blotting Gel Shift Assay Specifications 용도에따른정제방법 합성 Scale ( 합성시작기준 ) 최대합성길이 선택가능한정제방법 QC & QA Guaranteed Yield 25 nmol, 50 nmol, 0.2 μmol, 1.0 μmol, 10 μmol, 15 μmol 5~130 mer Bio-RP, HPLC and PAGE MALDI-TOF Mass Spectrometer & CE (Capillary Electrophoresis) 올리고의합성수율은염기의구성, 정제방법, 올리고길이, 합성 scale 또는 modification 여부에따라달라질수있습니다. 바이오니아는최저올리고공급수율을 기준수율 로정하여기준수율이상을공급하고있습니다. Synthesis Scales 최소 Guaranteed O.D. 25 mer 기준 Bio-RP PAGE HPLC 0.025 μmole 2 1 1.5 0.05 μmole 4 2 2.5 0.2 μmole 8 6 7 1 μmole 30 18 25 10 μmole 300 150 200 15 μmole 문의 Guaranteed Quality DMT-monitoring QC, PAGE QC, HPLC QC 방법이일반적이지만모두간접적인방법이기때문에, fail 올리고및 modification 성공률등을정확히확인할수없습니다. MALDI-TOF QC (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometer 를이용하면올리고의정확한분자량을측정할수있으므로기존방법들과달리합성올리고의정확한품질을평가할수있습니다. 7 대의 MALDI- TOF mass spectrometer 를도입하여모든올리고를 MALDI-TOF QC 하여고품질의올리고를공급합니다. MALDI-TOF QC 를수행한 QC Data ( 합성올리고 sheet) 를제공함으로써올리고의분자량을직접확인할수있습니다. 50 mer 이상의긴올리고는 HPCE (High Performance Capillary Electrophoresis) Oligo-pro TM 로순도를측정합니다. Delivery PAGE 정제올리고및 modification 올리고를제외하고는주문후이틀내발송을원칙으로합니다. ( 재합성또는택배회사의운송스케줄에따라배송이지연될수도있습니다.) BIONEER 4

Standard Oligonucleotide Next-Day Delivery Service 바이오니아는고객이오전 8 시부터오후 5 시까지주문한올리고를익일수령할수있는, 빠르고편리한 Next-day delivery service 를운영하고있습니다. 서비스대상 35 mer 이하의 Bio-RP 정제올리고 주문접수시간월요일에서목요일까지오전 8 시부터오후 5 시주문건만가능합니다. 배송시간주문익일오후 3 시 ~6 시사이수령 ( 운송업체의사정으로납품이지연될수있습니다.) Set-up Charge 또는운송료 10,000 ~ 30,000 원 (VAT 별도 ) 청구 ( 일부지역에는운송료추가비용이발생할수있습니다.) Standard Oligonucleotide Price Synthesis Scale (μmole) Price / Base ( 원 ) 합성가능 Base 수 (mer) Purification ( 원 ) Guaranteed Yield (O.D.) 20 base 기준 Delivery Time ( 일 ) Bio-RP PAGE HPLC Bio-RP PAGE HPLC Bio-RP PAGE HPLC 0.025 550 15-34 무료 25,000 25,000 2 1 1.5 2 3 3 0.05 620 10-75 무료 25,000 25,000 4 2 2.5 2 3 3 0.2 1,000 5-110 무료 30,000 30,000 8 6 7 2 3 3 1 2,500 5-130 무료 40,000 40,000 30 18 25 2 3 3 10 20,000 5-50 무료문의문의 300 150 200 2 3 3 15 30,000 5-50 무료문의문의문의문의문의 3 4 4 * 35 mer 이상은 0.05 μmole 이상으로주문하시기바라며, 75 mer 이상은 0.2 μmole 이상으로주문하시기바랍니다. 또한 50 mer 이상의올리고는반드시 PAGE 정제를하시기바랍니다 Additional Service Price Post-Handling Tube ( 원 ) Plate ( 원 ) Concentration normalized - Standard 500 250 (per well) Concentration normalized - Custom 750 350 (per well) Bar-code label No Charge No Charge Dried No Charge No Charge Aliquoting to daughter plates 750 350 (per well) Mixing primers 750 350 (per well) Individual tube plate-labeled - 250 (per well) Custom plate - 10,000 Double strand (Annealing service) 20,000 / Pair - MALDI-TOF (QC) No Charge No Charge MALDI-TOF (SNP or Genotyping) 2,500 2,500 (per well) Shipping charge Shipping charge may apply Shipping charge may apply Hand mix service (MIX base Code) 100,000 / (1~250 base) 50,000 / (1~250 base) Remainder in plate service - 75,000 (96 well) / 150,000 (384 well) Circular ssdna service 250,000 / sample 150,000 / sample www.bioneer.co.kr 5

HT- Oligo (High Throughput Oligonucleotide) Description HT-Oligo TM 합성서비스는여러개의올리고를한번에합성하실때유용한서비스입니다. 바이오니아는대량올리고를합성할수있는시스템을자체개발하였습니다. 한번에합성하는개수가 96 개이상일경우이서비스를이용하시면빠르고합리적인가격으로올리고를구매하실수있습니다. Specifications 합성 Scale ( 합성시작기준 ) 최대합성길이선택가능한정제방법 QC & QA 25 nmol, 50 nmol, 0.2 μmol, 1.0 μmol, 10 μmol, 15 μmol 5~130 mer Bio-RP, HPLC and PAGE MALDI-TOF Mass Spectrometer & CE (Capillary Electrophoresis) HT-Oligo TM Price Synthesis Scale (μmole) Price / Base ( 원 ) 합성가능 Base 수 (mer) Purification ( 원 ) Guaranteed Yield (O.D.) 20 base 기준 Delivery Time ( 일 ) Bio-RP PAGE HPLC Bio-RP PAGE HPLC Bio-RP PAGE HPLC 0.025 350 15-34 무료 25,000 25,000 2 1 1.5 2 3 3 0.05 450 10-75 무료 25,000 25,000 4 2 2.5 2 3 3 0.2 700 5-110 무료 30,000 30,000 8 6 7 2 3 3 1 2,000 5-130 무료 40,000 40,000 30 18 25 2 3 3 10 15,000 5-50 무료문의문의 300 150 200 문의문의문의 15 20,000 5-50 무료문의문의문의문의문의문의문의문의 BIONEER 6

Modification Oligonucleotide Description 바이오니아는회사설립이래축척된연구개발과생산노하우를바탕으로우수한올리고합성기술을보유하고있습니다. 바이오니아의숙련된기술진과연구진은다양한종류의 modification oligonucleotide 합성기술을개발하였고, 더욱유용한 modification oligonucleotide 를만들기위해최선을다하고있습니다. 현재 biotin-labeled primer 부터 Real-Time PCR 에사용되는다양한 fluorescence-labeled primer 까지수십종의 modification oligonucleotide 합성서비스를제공하고있습니다. Guaranteed Yield Modification oligonucleotide 합성용 phosphoramidites 는 modification 종류에따라합성 coupling yield 차이가있어, 공급량이상이할수있습니다. Delivery 일부품목을제외한 modification oligonucleotide 는주문일로부터 4 일 (Working day 기준 ) 내발송합니다. Modification Oligos Price 3 Modification Modification 50 nmole 200 nmole Price / Synthesis Scale ( 원 ) 1 μmole 10 μmole 15 μmole DIG 90,000 110,000 165,000 450,000 750,000 4 C6 Amine 30,000 40,000 60,000 180,000 300,000 4 Phosphate 30,000 40,000 60,000 180,000 300,000 4 Biotin 65,000 80,000 140,000 420,000 720,000 4 C3 spacer 50,000 60,000 80,000 240,000 400,000 4 C6 spacer 50,000 60,000 80,000 240,000 400,000 4 C12 spacer 50,000 60,000 80,000 240,000 400,000 4 C18 atom spacer 50,000 60,000 80,000 240,000 400,000 4 ds spacer 65,000 80,000 140,000 700,000 980,000 4 FAM 70,000 90,000 160,000 480,000 800,000 4 TAMRA 120,000 150,000 220,000 660,000 1,100,000 4 Thiol 150,000 180,000 250,000 750,000 1,250,000 4 Texas Red 180,000 220,000 330,000 900,000 1,500,000 4 JOE 180,000 220,000 330,000 900,000 1,500,000 4 ROX 180,000 220,000 330,000 900,000 1,500,000 4 Cy5 150,000 175,000 250,000 1,250,000 2,450,000 4 Cy3 150,000 175,000 250,000 1,250,000 2,450,000 4 Dabcyl 120,000 150,000 220,000 660,000 1,100,000 4 BHQ1 125,000 150,000 220,000 660,000 1,100,000 4 BHQ2 125,000 150,000 220,000 660,000 1,100,000 4 Cholesteryl 90,000 110,000 350,000 1,500,000 2,100,000 4 Inverted dt 45,000 60,000 95,000 350,000 700,000 4 3'-Inverted da 45,000 60,000 95,000 350,000 700,000 4 3'-Inverted dc 45,000 60,000 95,000 350,000 700,000 4 3'-Inverted dg 45,000 60,000 95,000 350,000 700,000 4 Ara-dC 90,000 110,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 5-F-dU 90,000 110,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 2',3'-ddC 90,000 110,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 AlexaFluor 488 225,000 250,000 350,000 1,750,000 2,450,000 4 AlexaFluor 532 315,000 350,000 550,000 2,680,000 3,850,000 4 AlexaFluor 546 315,000 350,000 550,000 2,680,000 3,850,000 4 AlexaFluor 594 360,000 400,000 650,000 3,250,000 4,550,000 4 Delivery Time( 일 ) www.bioneer.co.kr 7

BIONEER 8 Modification Oligonucleotide AlexaFluor 647 225,000 250,000 350,000 1,750,000 2,450,000 4 AlexaFluor 660 360,000 400,000 650,000 3,250,000 4,550,000 4 AlexaFluor 750 360,000 400,000 650,000 3,250,000 4,550,000 4 N6-methyl- 2'-deoxyadenosine 120,000 150,000 220,000 3 개미만 (Set-up Charge: 175,000) 4 DNP (2,4-dinitrophenyl)-TEG 125,000 175,000 275,000 3 개미만 (Set-up Charge: 175,000) 4 Cy3.5 225,000 250,000 350,000 1,750,000 2,450,000 4 Cy5.5 225,000 250,000 350,000 1,750,000 2,450,000 4 Puromycine 90,000 110,000 350,000 1,500,000 2,100,000 4 Yakima Yellow 120,000 150,000 275,000 Inquire 4 3'-dA 250,000 275,000 350,000 1,750,000 2,450,000 4 3'-dC 250,000 275,000 350,000 1,750,000 2,450,000 4 3'-dG 250,000 275,000 350,000 1,750,000 2,450,000 4 3'-dT 250,000 275,000 350,000 1,750,000 2,450,000 4 5-Nitroindole 125,000 175,000 250,000 562,500 1,125,000 4 2'-F-rA 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rC 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rG 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rU 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 Maleimide 50,000 65,000 95,000 350,000 700,000 4 2-Aminopurine 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2,6-Diaminopurine 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 Dithiol 150,000 175,000 275,000 1,500,000 2,100,000 4 EDTA-C2-dT 120,000 150,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 Thymidine Glycol 150,000 175,000 275,000 3 개미만 (Set-up Charge: 175,000) 4 Zebularine 75,000 90,000 125,000 3 개미만 (Set-up Charge: 250,000) 4 3' Methylene Blue 150,000 175,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 3' AMCA (amino-methyl-coumarinacetate) 125,000 175,000 275,000 3 개미만 (Set-up Charge: 175,000) 4 3' Deoxypurine (2'-DeoxyNebularine) 75,000 90,000 125,000 3 개미만 (Set-up Charge: 250,000) 4 3' PEG-2000 Inquire 4 3' Azide 150,000 175,000 275,000 1,500,000 2,100,000 4 3' Acrylamide (acrydite) 90,000 110,000 350,000 1,500,000 2,100,000 4 O6-Methyl 2'-dG 75,000 90,000 125,000 375,000 625,000 O4-Methyl-dT 75,000 90,000 125,000 375,000 625,000 dt-alkyne 225,000 250,000 350,000 1,750,000 2,450,000 Rhodamine 6G 225,000 225,000 350,000 1,750,000 2,450,000 Epoch Eclips Quencher 225,000 225,000 350,000 1,750,000 2,450,000 EBQ 112,500 135,000 198,000 594,000 990,000 4 Pyrene-dU 150,000 175,000 275,000 650,000 750,000 Perylene-dU 150,000 175,000 275,000 650,000 750,000 C8-Alkyne-dT 150,000 175,000 275,000 650,000 750,000 C8-Alkyne-dC 150,000 175,000 275,000 650,000 750,000 Desthiobiotin TEG 150,000 175,000 275,000 750,000 875,000 N3-Methyl dc 150,000 175,000 275,000 650,000 750,000 N4-Ethyl dc 150,000 175,000 275,000 650,000 750,000 Marina blue New 125,000 175,000 250,000 1,250,000 NA Azobenzene New 125,000 175,000 250,000 1,250,000 NA

Modification Oligonucleotide 5 Modification Modification Price / Synthesis Scale ( 원 ) 50 nmole 200 nmole 1 μmole 10 μmole 15 μmole DIG 90,000 110,000 165,000 450,000 750,000 4 C6 Amine 20,000 30,000 50,000 150,000 250,000 4 Phosphate 20,000 30,000 50,000 150,000 250,000 4 Biotin 40,000 50,000 120,000 360,000 600,000 4 Thiol 125,000 150,000 220,000 660,000 1,100,000 4 C3 spacer 50,000 60,000 80,000 240,000 400,000 4 C6 spacer 50,000 60,000 80,000 240,000 400,000 4 C12 spacer 50,000 60,000 80,000 240,000 400,000 4 18 atom spacer 50,000 60,000 80,000 240,000 400,000 4 ds spacer 65,000 80,000 140,000 700,000 980,000 4 FAM 70,000 90,000 160,000 480,000 800,000 4 TAMRA 120,000 150,000 220,000 660,000 1,100,000 4 HEX 125,000 150,000 300,000 1,500,000 2,100,000 4 TET 125,000 150,000 300,000 1,500,000 2,100,000 4 Texas Red 180,000 220,000 330,000 900,000 1,500,000 4 JOE 180,000 220,000 330,000 900,000 1,500,000 4 ROX 180,000 220,000 330,000 900,000 1,500,000 4 Cy5 150,000 175,000 250,000 1,250,000 2,450,000 4 Cy3 150,000 175,000 250,000 1,250,000 2,450,000 4 IRD700 70,000 90,000 160,000 800,000 1,120,000 4 IRD800 70,000 90,000 160,000 800,000 1,120,000 4 C2 Aldehyde 125,000 150,000 220,000 660,000 1,100,000 4 Cy5.5 225,000 250,000 350,000 1,750,000 2,450,000 4 Cholesteryl 90,000 110,000 350,000 1,500,000 2,100,000 4 Dabcyl 220,000 250,000 350,000 1,750,000 2,450,000 4 Ara-dC 90,000 110,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 5-F-dU 90,000 110,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 2',3'-ddA 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2',3'-ddC 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2',3'-ddG 125,000 150,000 375,000 3 개미만 (Set-up Charge: 300,000) 4 2',3'-ddT 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 AlexaFluor 488 225,000 250,000 350,000 1,750,000 2,450,000 4 AlexaFluor 532 315,000 350,000 550,000 2,680,000 3,850,000 4 AlexaFluor 546 315,000 350,000 550,000 2,680,000 3,850,000 4 AlexaFluor 594 360,000 400,000 650,000 3,250,000 4,550,000 4 AlexaFluor 647 225,000 250,000 350,000 1,750,000 2,450,000 4 AlexaFluor 660 360,000 400,000 650,000 3,250,000 4,550,000 4 AlexaFluor 750 360,000 400,000 650,000 3,250,000 4,550,000 4 N6-methyl- 2'-deoxyadenosine 120,000 150,000 220,000 3 개미만 (Set-up Charge: 175,000) 4 PC(photo-cleavable) Amine Linker 120,000 150,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 PC(photo-cleavable) Biotin Linker 120,000 150,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 DNP (2,4-dinitrophenyl)-TEG 125,000 175,000 275,000 3 개미만 (Set-up Charge: 175,000) 4 Cy3.5 225,000 250,000 350,000 1,750,000 2,450,000 4 3'-dA 250,000 275,000 350,000 1,750,000 2,450,000 4 Delivery Time( 일 ) www.bioneer.co.kr 9

BIONEER 10 Modification Oligonucleotide 3'-dC 250,000 275,000 350,000 1,750,000 2,450,000 4 3'-dG 250,000 275,000 350,000 1,750,000 2,450,000 4 3'-dT 250,000 275,000 350,000 1,750,000 2,450,000 4 5-Nitroindole 125,000 175,000 250,000 562,500 1,125,000 4 2'-F-rA 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rC 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rG 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rU 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 Maleimide 50,000 65,000 95,000 350,000 700,000 4 2-Aminopurine 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2,6-Diaminopurine 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 Dithiol 150,000 175,000 275,000 1,500,000 2,100,000 4 BHQ2 180,000 220,000 330,000 900,000 1,500,000 4 EDTA-C2-dT 120,000 150,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 Thymidine Glycol 150,000 175,000 275,000 3 개미만 (Set-up Charge: 175,000) 4 Zebularine 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 5' Methylene Blue 150,000 175,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 5' AMCA (amino-methyl-coumarinacetate) 125,000 175,000 275,000 3 개미만 (Set-up Charge: 175,000) 4 5' Deoxypurine (2'-DeoxyNebularine) 75,000 90,000 125,000 3 개미만 (Set-up Charge: 250,000) 4 5' PEG-2000 Inquire 4 5'-BromoHexyl (Br) 150,000 175,000 275,000 1,500,000 2,100,000 4 5' Acridine 125,000 175,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 5' Acrylamide (acrydite) 90,000 110,000 350,000 1,500,000 2,100,000 4 5'-Yakima Yellow 250,000 275,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 O 6-Methyl 2'-dG 75,000 90,000 125,000 375,000 625,000 - O 4-Methyl-dT 75,000 90,000 125,000 375,000 625,000-5 C3-Amine 50,000 60,000 80,000 240,000 400,000-5 C12-Amine 75,000 90,000 125,000 375,.000 625,000 - dt-alkyne 225,000 250,000 350,000 1,750,000 2,450,000 - Rhodamine 6G 225,000 250,000 350,000 1,750,000 2,450,000 - Pyrene-Cap 225,000 250,.000 350,000 1,750,000 2,450,000 - Epoch Eclips Quencher 225,000 250,000 350,000 1,750,000 2,450,000-5 -Hexynyl 50,000 60,000 80,000 240,000 400,000 - Pyrene-dU 150,000 175,000 275,000 650,000 750,000 - Perylene-dU 150,000 175,000 275,000 650,000 750,000 - C8-Alkyne-dT 150,000 175,000 275,000 650,000 750,000 - C8-Alkyne-dC 150,000 175,000 275,000 650,000 750,000 - AminoOxy 125,000 150,000 220,000 550,000 650,000 - Desthiobiotin TEG 150,000 175,000 275,000 750,000 875,000 - N3-Methyl dc 150,000 175,000 275,000 650,000 750,000 - N4-Ethyl dc 150,000 175,000 275,000 650,000 750,000-5 -OMe-dT 75,000 95,000 150,000 375,000 475,000 - C6-Psoralen New 225,000 250,000 350,000 1,750,000 NA - Marina blue New 125,000 175,000 250,000 1,250,000 NA - DBCO TEG New 180,000 220,000 330,000 900,000 NA - Azobenzene New 125,000 175,000 250,000 1,250,000 NA -

Modification Oligonucleotide Internal Modification Modification Price / Synthesis Scale ( 원 ) 50 nmole 200 nmole 1 μmole 10 μmole 15 μmole Internal Amino Modifier C6 dt 150,000 180,000 250,000 1,250,000 1,750,000 4 Internal Biotin-dT 180,000 200,000 300,000 1,500,000 2,100,000 4 Fluorescein dt 220,000 250,000 350,000 1,750,000 2,450,000 4 C3 spacer 50,000 60,000 80,000 320,000 480,000 4 C6 spacer 50,000 60,000 80,000 320,000 480,000 4 C12 spacer 50,000 60,000 80,000 320,000 480,000 4 18 atom spacer 50,000 60,000 80,000 320,000 480,000 4 ds spacer 65,000 80,000 140,000 700,000 980,000 4 Phosphorothioate (per insertion) - 4,500 7,500 37,500 52,500 4 5-Methyl dc 60,000 75,000 110,000 330,000 770,000 4 Inosine 8,000 10,000 20,000 60,000 100,000 4 Deoxy Uridine 9,000 12,000 18,000 71,000 90,000 4 2'-O-Methyl 9,000 12,000 18,000 71,000 90,000 4 5-Bromo du 70,000 90,000 160,000 800,000 1,120,000 4 8-Oxo-dA 150,000 180,000 250,000 1,250,000 1,750,000 4 8-Oxo-dG 150,000 180,000 250,000 1,250,000 1,750,000 4 Ferrocene-dT 180,000 200,000 300,000 1,500,000 2,100,000 4 5-hydroxymethyl-dC 180,000 200,000 300,000 1,500,000 5-Hydroxy-dU 145,000 175,000 240,000 680,000 5-hydroxymethyl-dU 145,000 175,000 240,000 680,000 3 개미만 (Set-up Charge: 250,000) 3 개미만 (Set-up Charge: 250,000) 3 개미만 (Set-up Charge: 250,000) Ara-dC 90,000 110,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 5-F-dU 90,000 110,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 N6-methyl-2'-deoxyadenosine 90,000 110,000 350,000 3 개미만 (Set-up Charge: 250,000) 4 PC(photo-cleavable) Linker 120,000 150,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 DNP(2,4-dinitrophenyl)-TEG 125,000 175,000 275,000 3 개미만 (Set-up Charge: 175,000) 4 3'-dA 250,000 275,000 350,000 1,750,000 2,450,000 4 3'-dC 250,000 275,000 350,000 1,750,000 2,450,000 4 3'-dG 250,000 275,000 350,000 1,750,000 2,450,000 4 3'-dT 250,000 275,000 350,000 1,750,000 2,450,000 4 5-Nitroindole 125,000 175,000 250,000 562,500 1,125,000 4 2'-F-rA 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rC 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rG 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2'-F-rU 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2-Aminopurine 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 2,6-Diaminopurine 75,000 90,000 125,000 3 개미만 (Set-up Charge: 175,000) 4 Dithiol 150,000 175,000 275,000 1,500,000 2,100,000 4 EDTA-C2-dT 120,000 150,000 275,000 3 개미만 (Set-up Charge: 250,000) 4 Thymidine Glycol 150,000 175,000 275,000 3 개미만 (Set-up Charge: 175,000) 4 Delivery Time( 일 ) 4 4 4 www.bioneer.co.kr 11

Modification Oligonucleotide Zebularine 75,000 90,000 125,000 3개미만 (Set-up Charge: 250,000) 4 Biotin-TEG 67,500 85,000 115,000 510,000 850,000 4 HEX-dT 125,000 150,000 300,000 1,500,000 2,100,000 4 Methylene Blue 150,000 175,000 275,000 3개미만 (Set-up Charge: 250,000) 4 Tamra-dT 220,000 250,000 350,000 1,750,000 2,450,000 4 BHQ1-dT 250,000 275,000 350,000 1,750,000 2,450,000 4 BHQ2-dT 250,000 275,000 350,000 1,750,000 2,450,000 4 Dabcyl-dT 250,000 275,000 350,000 1,750,000 2,450,000 4 Deoxypurine (2'-DeoxyNebularine) 75,000 90,000 125,000 3개미만 (Set-up Charge: 250,000) 4 Azide 150,000 175,000 275,000 1,500,000 2,100,000 4 Trebler Branching 220,000 250,000 350,000 1,750,000 2,450,000 4 O 6-Methyl 2'-dG 75,000 90,000 125,000 375,000 625,000 - O 4-Methyl-dT 75,000 90,000 125,000 375,000 625,000 - dt-alkyne 225,000 250,000 350,000 1,750,000 2,450,000 - Epoch Eclips Quencher 225,000 225,000 350,000 1,750,000 2,450,000 - Thiol-dT 225,000 225,000 350,000 1,750,000 2,450,000 - Cy3 da 250,000 275,000 420,000 1,950,000 2,750,000 - Cy3 dc 250,000 275,000 420,000 1,950,000 2,750,000 - Cy3 dg 250,000 275,000 420,000 1,950,000 2,750,000 - Cy3 dt 225,000 250,000 350,000 1,750,000 2,450,000 - Cy5 da 250,000 275,000 420,000 1,950,000 2,750,000 - Cy5 dc 250,000 275,000 420,000 1,950,000 2,750,000 - Cy5 dg 250,000 275,000 420,000 1,950,000 2,750,000 - Cy5 dt 225,000 250,000 350,000 1,750,000 2,450,000 - Pyrene-dU 150,000 175,000 275,000 650,000 750,000 - Perylene-dU 150,000 175,000 275,000 650,000 750,000 - C8-Alkyne-dT 150,000 175,000 275,000 650,000 750,000 - C8-Alkyne-dC 150,000 175,000 275,000 650,000 750,000 - Desthiobiotin TEG 150,000 175,000 275,000 750,000 875,000 - N3-Methyl dc 150,000 175,000 275,000 650,000 750,000 - N4-Ethyl dc 150,000 175,000 275,000 650,000 750,000 - Azobenzene New 125,000 175,000 250,000 1,250,000 NA - 8-Br-dA New 150,000 180,000 250,000 1,250,000 NA - 8-Br-dG New 150,000 180,000 250,000 1,250,000 NA - 8-Br-dC New 150,000 180,000 250,000 1,250,000 NA - 6-thio-dG New 225,000 250,000 350,000 1,750,000 NA - 4-thio-dT New 225,000 250,000 350,000 1,750,000 NA - BIONEER 12

Dual-Labeled Probes Description 저희바이오니아의 Dual-Labeled Probe 는일반적으로 5 말단에 fluorescent dye 가위치하고, 3 말단에 Quencher 가위치하여 Real-Time PCR 에사용됩니다. Dual-labeled Probe 는유전자의서열에특이적으로반응하여매우민감한유전자정성및정량분석을가능하게하고여러가지의형광물질을사용함으로써한번에여러개의유전자를검색하는 Multiplex Analysis 도가능합니다. Features and Benefits 바이오니아의 Dual-Probe 는 Fluoroskan 을이용한형광테스트를수행함으로써고품질을보장합니다. MALDI-TOF Mass Spectrometer QC 방법으로품질을보장합니다. Dual-Probe 디자인서비스를무상으로제공합니다. 해외주문시보다빠른배송및저렴한가격을보장합니다. Delivery 모든 Dual-Labeled Probe 는주문일로부터 4~5 일 (Working day 기준 ) 내발송됩니다. Spectra of Fluorescent Dyes Dye Excitation Max (nm) Extinction Coefficient (L/mole Cm) Emission Max (nm) 6-FAM 494 83,000 520 Fluorescein-dT 494 83,000 522 TET 521 73,000 541 HEX 535 73,000 553 TAMRA 556 91,000 580 Cy3 546 150,000 563 Cy3.5 581 150,000 596 Cy5 646 250,000 662 Cy5.5 675 250,000 694 Cy7 743 250,000 767 JOE 529 71,000 555 ROX 588 82,000 608 Texas Red 598 116,000 617 NED 546-575 VIC 538-554 IR700 685 170,000 705 IR800 787 200,000 807 Rhodamine 6G 524 116,000 550 DABCYL 478 32,000 - BHQ-1 534 34,000 - EBQ 570 41,360 - BHQ-2 579 38,000 - (Reference) UV/ Visible spectra of the variety of dyes (emission) and quenchers (absorption) currently available. www.bioneer.co.kr 13

Dual-Labeled Probes Combination of Dual-Probe Dye Excitation Max (nm) Emission Max (nm) 6-FAM 494 520 JOE 529 555 TET 521 541 HEX 535 553 VIC 538 554 Cy3 546 563 NED 546 575 TAMRA 556 580 Cy3.5 581 596 ROX 588 608 Texas Red 598 617 Cy5 646 662 Cy5.5 675 694 Compatible Quencher Dabcyl tamra BHQ1 BHQ2 EBQ BIONEER 14

Dual-Labeled Probes Dual-Labeled Probe Price Modification Price / Synthesis Scale ( 원 ) 50 nmole 200 nmole 1 μmole 10 μmole 15 μmole Delivery Time( 일 ) 5'-FAM-3'-TAMRA 200,000 250,000 350,000 1,400,000 1,750,000 4 ~ 5 5'-HEX-3'-TAMRA 250,000 300,000 500,000 2,000,000 2,500,000 4 ~ 5 5'-TET-3'-TAMRA 250,000 300,000 500,000 2,000,000 2,500,000 4 ~ 5 5'-JOE-3'-TAMRA 300,000 350,000 550,000 2,200,000 2,750,000 4 ~ 5 5'-FAM-3'-DABCYL 200,000 250,000 350,000 1,400,000 1,750,000 4 ~ 5 5'-HEX-3'-DABCYL 250,000 300,000 500,000 2,000,000 2,500,000 4 ~ 5 5'-TET-3'-DABCYL 250,000 300,000 500,000 2,000,000 2,500,000 4 ~ 5 5'-TAMRA-3'-DABCYL 250,000 300,000 500,000 2,000,000 2,500,000 4 ~ 5 5'-JOE-3'-DABCYL 300,000 350,000 550,000 2,200,000 2,750,000 4 ~ 5 5'-FAM-3'-BHQ1 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-HEX-3'-BHQ1 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-TET-3'-BHQ1 300,000 350,000 550,000 2,200,000 2,750,000 4 ~ 5 5'-JOE-3'-BHQ1 300,000 350,000 550,000 2,200,000 2,750,000 4 ~ 5 5'-TAMRA-3'-BHQ1 300,000 350,000 550,000 2,200,000 2,750,000 4 ~ 5 5'-ROX-3'-BHQ1 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-Texas Red-3'-BHQ1 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-Cy3-3'-BHQ1 400,000 450,000 650,000 2,600,000 3,250,000 4 ~ 5 5'-FAM-3'-BHQ2 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-HEX-3'-BHQ2 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-TET-3'-BHQ2 300,000 350,000 550,000 2,200,000 2,750,000 4 ~ 5 5'-JOE-3'-BHQ2 300,000 350,000 550,000 2,200,000 2,750,000 4 ~ 5 5'-TAMRA-3'-BHQ2 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-ROX-3'-BHQ2 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-Texas Red-3'-BHQ2 275,000 320,000 520,000 2,080,000 2,600,000 4 ~ 5 5'-Cy5-3'-BHQ2 400,000 450,000 650,000 2,600,000 3,250,000 4 ~ 5 5'-Cy3-3'-BHQ2 400,000 450,000 650,000 2,600,000 3,250,000 4 ~ 5 5'-FAM-BHQ1-dT-Amine-3' 450,000 565,000 750,000 3,000,000 5,250,000 4 ~ 5 5'-FAM-Tamra-dT-PO4-3' 450,000 565,000 750,000 3,000,000 5,250,000 4 ~ 5 5'-FAM-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-HEX-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-TET-3'- EBQ 270,000 315,000 495,000 1,980,000 2,475,000 4 ~ 5 5'-JOE-3'- EBQ 270,000 315,000 495,000 1,980,000 2,475,000 4 ~ 5 5'-TAMRA-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-ROX-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-Texas Red-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-Cy5-3'-EBQ 360,000 405,000 585,000 2,340,000 2,925,000 4 ~ 5 5'-Cy3-3'- EBQ 360,000 405,000 585,000 2,340,000 2,925,000 4 ~ 5 www.bioneer.co.kr 15

EBQ Next Generation Dark Quencher Modifications Description EBQ(Excellent Bioneer Quencher) 는바이오니아에서직접개발한새로운개념의 Dark Quencher 입니다. EBQ 는기존의 Quencher 보다강하고넓은흡수영역을가지고있습니다. 이를바탕으로 Dual-Probe 영역에서기존의 Quencher 보다더다양한 Reporter- Dye(fluorophore) 를효율적으로 Quenching 할수있으며, 열및 ph 변화에도안정한구조를가지고있습니다. 바이오니아에서새롭게선보이는차세대 Quencher 인 EBQ 를이용하여다양한연구에이용하시길바랍니다. Features and Benefits 넓은흡수파장영역 : FAM 부터 Cy5.5 까지커버가능한 400~700 nm 의넓은흡수파장영역 ( 최대 570 nm 의흡수파장영역 ) 안정성 : 열및 ph 변화에안정한구조 다양한 dye 적용가능 : Marina Blue, Pacific Blue, Oregon Greens, Cy dyes, AMCA, Bodipy 계열 Dye, Fluorescein 계열, FAM, JOE, TET, HEX, VIC, Cy3, NED, TAMRA, Cy3.5, ROX, Texas Red, Cy5, Cy5.5 등 400~700 nm 파장 dye Application Real-Time PCR, RT-qPCR, Molecular diagnostics, in situ hybridization, etc. Figure 1. EBQ 로형광제어 (Quenching) 가능한 Reporter-dye 종류 BIONEER 16

EBQ Next Generation Dark Quencher Modifications Technical / Specifications EBQ 는기존의 Quencher 보다강하고넓은흡수영역을가지고있습니다. 아래비교자료는기존두 Quencher 의형광제어 (quenching) 영역을단지 EBQ 하나로형광제어가능함을의미합니다. Table 2. S1 nuclease 처리를통한형광제어 (quenching efficiency) 효과비교. Dual-Labeled Probe Fluorescence Signal Intensity Before After Difference Efficiency (%) EBQ-FAM 4.41 93.17 88.76 113.0 BHQ1-FAM 4.20 82.74 78.54 100.0 EBQ-ROX 1.89 65.25 63.37 124.7 BHQ2-ROX 1.14 51.94 50.80 100.0 FAM-Excitation: 494 nm, Emission: 520 nm ROX-Excitation: 588 nm, Emission: 608 nm *EBQ *BHQ1 *EBQ *BHQ1 Figure 2. Real-Time PCR 에서 ΔRn 값측정을통한형광제어 (quenching efficiency) 효과비교. ΔRn value= a fluorescence quenching capacity of Quencher at Real-Time PCR Ordering Information Modification Price / Synthesis Scale ( 원 ) 50 nmole 200 nmole 1 μmole 10 μmole 15 μmole Delivery Time( 일 ) 5'-FAM-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-HEX-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-TET-3'- EBQ 270,000 315,000 495,000 1,980,000 2,475,000 4 ~ 5 5'-JOE-3'- EBQ 270,000 315,000 495,000 1,980,000 2,475,000 4 ~ 5 5'-TAMRA-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-ROX-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-Texas Red-3'- EBQ 247,500 288,000 468,000 1,872,000 2,340,000 4 ~ 5 5'-Cy5-3'-EBQ 360,000 405,000 585,000 2,340,000 2,925,000 4 ~ 5 5'-Cy3-3'- EBQ 360,000 405,000 585,000 2,340,000 2,925,000 4 ~ 5 상담안내 : oligo-support@bioneer.com 메일또는 042-930-8574 전화로문의하여주시기바랍니다. www.bioneer.co.kr 17

Extendamers Description Extendamers TM (Long oligonucleotides) 합성서비스는 130 ~ 200 bases 에이르는긴길이의올리고를합성해드리는서비스입니다. 합성된 long oligonucleotide 는바이오니아가자체개발한 Bio-RP resin 을이용하여정제하여공급하며, 고순도정제를원할경우에는 PAGE 정제도가능합니다. Long oligomers 는 cloning, shrna, gene construction 등의용도로적합하며, 전체 target fragment 를합성함으로써 Application 중발생하는에러를줄일수있고많은비용과시간을절약할수있습니다. Features and Benefits 130 ~ 200 bases 까지의 Long DNA Oligomers 다양한 Modification Extendamers TM (Long oligonucleotides) 서비스가능 Cloning, shrna 또는 gene construction 에사용하기에적합 Bio-RP Extendamers TM 는 5 ~ 6 일, PAGE Extendamers TM 는 7 ~ 8 일이내배송 Specifications Coupling Efficiency 최대합성길이 선택가능한정제방법 QC & QA 99.0% 이상 130~200 mer Bio-RP, PAGE PAGE Gel QC & Capillary Electrophoresis Extendamers TM s Modification Price Modification Price ( 원 ) 5'-Phosphate 30,000 5'-Biotin 50,000 5'-Amine 30,000 5'-Fluorescein 90,000 5'-Tamra 150,000 Inosine 10,000 * 130 mers 이상의올리고는 Extendamers TM (Long oligonucleotides) 의합성가격이적용됩니다. Extendamers TM Price Price / Base ( 원 ) 합성가능 Base 수 (mer) Purification ( 원 ) Guaranteed Yield Delivery Time ( 일 ) Bio-RP PAGE Bio-RP PAGE Bio-RP PAGE 1,250 130-200 무료 40,000 3 ~ 4 0.25 ~ 0.3 5 ~ 6 7 ~ 8 BIONEER 18

Large Scale Oligonucleotide Description Large Scale Oligonucleotide (Bulk Oligo) 는 15 μmol scale 이상의올리고합성서비스입니다. 자체개발한 Bulk 올리고용합성기로 g 단위스케일의올리고합성이가능하며 Antisense 연구및 Decoy, RNAi 올리고등대용량 scale 의연구에필요한올리고합성기술과특수정제기술을확보하고있습니다. Specifications 합성 Scale (Final yield) 최대합성길이 선택가능한정제방법 QC & QA 50 mg ~ 1 g 5 ~ 35 mer Bio-RP, HPLC MALDI-TOF Mass Spectrometer & CE (Capillary Electrophoresis) Large Scale Oligonucleotide Synthesis DNA Price Final Yield (mg) DNA Price ( 원 ) DNA Price ( 원 ) Bio-RP HPLC 50 1,500,000 1,800,000 100 1,750,000 2,100,000 250 2,400,000 2,600,000 500 4,200,000 4,400,000 1,000 6,600,000 6,800,000 S-DNA Price (Phosphorothioate) Final Yield (mg) DNA Price ( 원 ) DNA Price ( 원 ) Bio-RP HPLC 50 2,100,000 2,350,000 100 2,500,000 2,700,000 250 3,350,000 3,600,000 500 5,750,000 6,150,000 1,000 8,500,000 9,500,000 www.bioneer.co.kr 19

Custom RNA Description 바이오니아의올리고는자동정량, 분주시스템과기술에의해정확한농도로제공되고있으며, 또고객들이안심하고사용할수있도록총 7 대의 MALDI-TOF 분자량측정장치와 96 CE 시스템으로전수검사하여통과된고품질의올리고입니다. 다양한 modification, 추적가능한바코드, chimeric RNA/DNA 합성등실험에가장적합한상태로제공합니다. 보장수율, 이용가능한 modification, 정제와서비스옵션은제품주문페이지에서더자세히확인하실수있습니다. Features and Benefits 철저한품질관리 - 총 7 대의 MALDI-TOF mass spectrometer 를사용하여 QC 전수검사시행. 50 mer 이상의올리고는 CE (Capillary Electrophoresis) 장비를사용하여정량 QC 함으로써완벽한품질관리 고품질의올리고 - Bio-RP 초고순도정제시스템으로 n-1 mer 를포함한 truncated failures 제거, clean room 에서생산함으로써 DNase, RNase, DNA free 한상태로제공 정확한농도자동정량분주기와 AccuOligo 기술을적용한정확한농도의올리고제공 자동화생산 - 세계최초로자동화생산라인공정구축 대량생산능력 - 1 일 30,500 개의합성유전자생산 경제적인가격 - 원재료자체생산으로안정적인납기와품질의균일성을보장 Custom RNA Price Custom RNA Synthesis Cat.no. Guaranteed Yield Base Limitation Base Price ( 원 ) Bio-RP HPLC ( 원 ) SR-10 10 nmole 30 mer 3,800 Free 30,000 SR-1002 20 nmole 30 mer 6,000 Free 40,000 SR-1003 50 nmole 30 mer 8,300 Free 50,000 SR-1004 100 nmole 30 mer 13,800 Free 60,000 SR-1005 10 nmole 31-35 mer 5,700 Free 30,000 SR-1006 20 nmole 31-35 mer 9,000 Free 40,000 SR-1007 50 nmole 31-35 mer 12,500 Free 50,000 SR-1008 100 nmole 31-35 mer 20,700 Free 60,000 BIONEER 20

Custom RNA Modification for Custom RNA Modification 10 nmole ( 원 ) 20 nmole ( 원 ) 50 nmole ( 원 ) 100 nmole ( 원 ) 5'-Amine 45,000 81,000 108,000 153,000 5 -Biotin 41,000 50,000 73,000 100,000 5 -Cy3 180,000 220,000 260,000 340,000 5 -Cy5 180,000 220,000 260,000 340,000 5 -Fluorescein 70,000 90,000 120,000 180,000 5 -PEG 2000 Inquire Inquire Inquire Inquire 5 -Phosphorylation 22,000 30,000 41,000 53,000 5 -TAMRA 135,000 170,000 200,000 290,000 5 -Thiol 125,000 200,000 320,000 450,000 3 -Amine 60,000 100,000 140,000 180,000 3 -Biotin 130,000 230,000 300,000 410,000 3 -Cholesterol 130,000 150,000 180,000 250,000 3 -DABCYL 250,000 450,000 600,000 850,000 3 -Fluorescein 80,000 120,000 145,000 210,000 3 -PEG 2000 Inquire Inquire Inquire Inquire 3 -Phosphorylation 60,000 100,000 140,000 180,000 3 -TAMRA 135,000 170,000 200,000 290,000 3'-Thiol 125,000 230,000 350,000 500,000 Internal 2 -F(A) 28,000 32,000 38,000 50,000 Internal 2 -F(C) 28,000 32,000 38,000 50,000 Internal 2'-F(G) 28,000 32,000 38,000 50,000 Internal 2'-F(U) 28,000 32,000 38,000 50,000 Internal 2'-OMe 8,500 11,000 16,000 22,000 Internal Chimeric DNA 3,500 5,000 7,500 12,000 Internal Deoxy-abase 49,000 58,000 70,000 94,000 Internal Inosine 17,000 19,000 25,000 31,000 Internal Phosphorothioate - 5,500 9,000 12,000 www.bioneer.co.kr 21

Premade Primers Overview Description 바이오니아만의합성유전자생산기술을바탕으로고객이원하는진단및연구용 premade primer 를 130 여종이상합성하여이를상용화하였습니다. 일반초보실험자및모든연구영역에서바이오니 아의 Premade Primer 로연구할수있습니다. General Research Primer Purification: PAGE Purification Formats: 2 nmole of lyophilized oligo in 1.5 ml tubes Human HLA Polymorphism Study Cat. no. Product Description Price ( 원 ) N-1043 HUMAN LEUCOCYTE ANTIGEN (HLA), DP-ALPHA / 290 (bp) 35,000 N-1045 HUMAN LEUCOCYTE ANTIGEN (HLA), DQ-ALPHA / 139 (bp) 35,000 N-1046 HUMAN LEUCOCYTE ANTIGEN (HLA), DQ-BETA / 212 (bp) 35,000 Human Cytokines & Growth Factors BIONEER 22 Cat. no. Product Description Price ( 원 ) N-1047 HUMAN T-CELL SURFACE MARKERS, CD4 (T4) / 169 (bp) 35,000 N-1048 HUMAN T-CELL SURFACE MARKERS, CD8 (LEU-2/T8) / 287 (bp) 35,000 N-1049 HUMAN ENDOTHELIAL CELL GROWTH FACTOR (ECGF) / 153 (bp) 35,000 N-1050 HUMAN EPIDERMAL GROWTH FACTOR (EGF) / 107 (bp) 35,000 N-1051 HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR / 216 (bp) 35,000 N-1052 HUMAN GRANULOCYTE COLONY STIMULATING FACTOR (GM-CSF) / 424 (bp) 35,000 N-1053 HUMAN INTERFERON-ALPHA / 283 (bp) 35,000 N-1054 HUMAN INTERFERON-BETA / 282 (bp) 35,000 N-1055 HUMAN INTERFERON-GAMMA / 397 (bp) 35,000 N-1056 HUMAN INSULIN-LIKE GROWTH FACTOR (IGF) / 471 (bp) 35,000 N-1057 HUMAN INTERLEUKIN 1-ALPHA (IL-1-alpha) / 442 (bp) 35,000 N-1058 HUMAN INTERLEUKIN 1-BETA (IL-1-beta) / 426 (bp) 35,000 N-1059 HUMAN INTERLEUKIN 2 (IL-2) / 398 (bp) 35,000 N-1060 HUMAN INTERLEUKIN 3 (IL-3) / 358 (bp) 35,000 N-1061 HUMAN INTERLEUKIN 4 (IL-4) / 375 (bp) 35,000 N-1062 HUMAN INTERLEUKIN 5 (IL-5) / 202 (bp) 35,000 N-1063 HUMAN INTERLEUKIN 6 (IL-6) / 486 (bp) 35,000 N-1064 HUMAN INTERLEUKIN 7 (IL-7) / 454 (bp) 35,000 N-1065 HUMAN INTERLEUKIN 8 (IL-8) / 300 (bp) 35,000 N-1066 HUMAN INTERLEUKIN 2 RECEPTOR / 282 (bp) 35,000 N-1067 HUMAN INTERLEUKIN 6 RECEPTOR / 203 (bp) 35,000 N-1068 TRANSFORMING GROWTH FACTOR (TGF)-ALPHA / 263 (bp) 35,000 N-1069 TRANSFORMING GROWTH FACTOR (TGF)-ALPHA / 256 (bp) 35,000 N-1070 TRANSFORMING GROWTH FACTOR (TGF)-BETA / 161 (bp) 35,000 N-1071 TRANSFORMING GROWTH FACTOR (TGF)-BETA / 228 (bp) 35,000 N-1072 HUMAN TUMOR NECROSIS FACTOR (TNF) - ALPHA / 518 (bp) 35,000 N-1073 HUMAN TUMOR NECROSIS FACTOR (TNF) - BETA / 179 (bp) 35,000

Premade Primers Oncogene & Drug Resistant Gene Cat. no. Product Description Price ( 원 ) N-1074 HUMAN P53 GENE, RNA / 430 (bp) 35,000 N-1075 HUMAN K-RAS ONCOGENE / 112 (bp) 35,000 N-1076 HUMAN DRUG RESISTANT, H-RAS 1 GENE / 542 (bp) 35,000 N-1077 HUMAN DRUG RESISTANT, DNA POLYMERASE BETA GENE / 128 (bp) 35,000 N-1078 HUMAN DRUG RESISTANT, FOS GENE / 122 (bp) 35,000 N-1079 HUMAN DRUG RESISTANT, THYMIDYLATE SYNTHASE GENE / 171 (bp) 35,000 Miscellaneous Oligo Cat. no. Product Description Price ( 원 ) N-1080 HUMAN BETA-ACTIN, RNA / 300 (bp) 35,000 N-1082 HUMAN Y CHROMOSOMAL TESTIS-EXPRESSED PROTEIN / 799 (bp) 35,000 N-1083 HUMAN Y CHROMOSOME, SPECIFIC (SRY) REGION / 419 (bp) 35,000 Mouse Cytokines & Growth Factors Cat. no. Product Description Price ( 원 ) N-4003 MOUSE T CELL SURFACE MARKER, CD4 (L3T4) / 239 (bp) 35,000 N-4004 MOUSE T CELL SURFACE MARKER, CD8 (LYT-2) / 251 (bp) 35,000 N-4005 MOUSE GRANULOCYTE COLONY STIMULATING FACTOR (GM-CSF) / 258 (bp) 35,000 N-4006 MOUSE INTERFERON-GAMMA / 244 (bp) 35,000 N-4007 MOUSE INTERFERON-GAMMA / 397 (bp) 35,000 N-4008 MOUSE INTERLEUKIN 1-ALPHA (IL-1-ALPHA) / 309 (bp) 35,000 N-4009 MOUSE INTERLEUKIN 1-BETA (IL-1-BETA) / 291 (bp) 35,000 N-40 MOUSE INTERLEUKIN 2 (IL-2) / 222 (bp) 35,000 N-41 MOUSE INTERLEUKIN 3 (IL-3) / 293 (bp) 35,000 N-42 MOUSE INTERLEUKIN 5 (IL-5) / 243 (bp) 35,000 N-43 MOUSE INTERLEUKIN 6 (IL-6) / 155 (bp) 35,000 N-44 MOUSE INTERLEUKIN 10 (IL-10) / 417 (bp) 35,000 N-45 MOUSE TUMOR NECROSIS FACTOR (TNF) - ALPHA / 300 (bp) 35,000 N-46 MOUSE TUMOR NECROSIS FACTOR (TNF) - ALPHA / 366 (bp) 35,000 N-47 MOUSE TUMOR NECROSIS FACTOR (TNF) - ALPHA / 368 (bp) 35,000 N-48 MOUSE TUMOR NECROSIS FACTOR (TNF) - BETA / 355 (bp) 35,000 N-49 MOUSE TUMOR NECROSIS FACTOR (TNF) - BETA / 316 (bp) 35,000 N-4020 MOUSE TUMOR NECROSIS FACTOR (TNF) - BETA / 387 (bp) 35,000 Miscellaneous Mouse Oligo Cat. no. Product Description Price ( 원 ) N-4021 MOUSE BETA-ACTIN, RNA / 395 (bp) 35,000 www.bioneer.co.kr 23

Premade Primers Sequencing Primer Purification : PAGE Purification Formats : 2 nmole of lyophilized oligo in 1.5 ml tubes pbr322 Sequencing Primers Cat. no. Product Description Price ( 원 ) N-81 pbr322, EcoRⅠ Primer (cw) / 16 mer 30,000 N-8102 pbr322, Hind Ⅲ Primer (cw) / 16 mer 30,000 N-8103 pbr322, PstⅠ Primer (cw) / 16 mer 30,000 N-8104 pbr322, PstⅠ Primer (ccw) / 15 mer 30,000 N-8105 pbr322, SalⅠ Primer (cw) / 15 mer 30,000 N-8106 pbr322, SalⅠ Primer (ccw) / 15 mer 30,000 N-8107 pbr322, BamHⅠ Primer (cw) / 20 mer 30,000 N-8108 pbr322, BamHⅠ Primer (ccw) / 16 mer 30,000 RNA Polymerase Promoter Sequencing Primers Cat. no. Product Description Price ( 원 ) N-8109 SP6 Promoter Primer / 19 mer 30,000 N-8110 T7 Promoter Primer / 20 mer 30,000 PUC/M13 Sequencing Primers Cat. no. Product Description Price ( 원 ) N-8112 PUC/M13 (-40) Forward Primer / 17 mer 30,000 N-8113 PUC/M13 Reverse Primer / 17 mer 30,000 N-8114 PUC/M13 (-47) Forward Primer / 24 mer 30,000 N-8115 PUC/M13 Reverse Primer / 22 mer 30,000 Lambda gt10 Sequencing Primers Cat. no. Product Description Price ( 원 ) N-8116 Lambda gt10 Forward Primer / 27 mer 30,000 N-8117 Lambda gt10 Reverse Primer / 31 mer 30,000 Lambda gt11 Sequencing Primers Cat. no. Product Description Price ( 원 ) N-8118 Lambda gt11 Forward Primer / 24 mer 30,000 N-8119 Lambda gt11 Reverse Primer / 24 mer 30,000 BIONEER 24

Premade Primers Universal Sequencing Primers Cat. no. Product Description Price ( 원 ) N-8200 T7 terminator 30,000 N-82 T3 30,000 N-8202 SP6 30,000 N-8203 EBV-R 30,000 N-8204 BGH-rev 30,000 N-8205 pgex5 30,000 N-8206 pgex3 30,000 N-8207 pqe-forward 30,000 N-8208 pqe-reverse 30,000 N-8209 EGFP-C 30,000 N-8210 EGFP-N 30,000 N-8211 RVprimer3 30,000 N-8212 RVprimer4 30,000 N-8213 GLprimer1 30,000 N-8214 GLprimer2 30,000 N-8215 CMV-F 30,000 N-8216 CMV30 30,000 N-8217 CMV24 30,000 N-8218 Gal4AD 30,000 N-8219 Gal4BD-F 30,000 N-8220 Gal4BD-R 30,000 N-8221 MATCHMAKER3 30,000 N-8222 pbad-for 30,000 N-8223 pbad-rev 30,000 N-8224 SV40-pArev 30,000 N-8225 SV40-pAF 30,000 N-8226 malef 30,000 N-8227 27F 30,000 N-8228 1492R 30,000 Random Primer Purification : PAGE Purification Formats : 2 nmole of lyophilized oligo in 1.5 ml tubes Cat. no. Product Description Price ( 원 ) N-7051 Random Hexamer,pd(N)6 35,000 N-7052 Random Nonamer,pd(N)9 35,000 N-7053 Oligo dt 20 mer 35,000 N-80-8083 RAPD 용 (Random Amplification Polymorphic DNAs) 10 mer, 83 종각 1 OD (7500 원 / 개당 ) 7,500 www.bioneer.co.kr 25

QC and Order System QC System Oligo 자동정량분주시스템 (UV spectrophotometer): 자체개발한정량 / 분주시스템을사용합니다. PAGE(PolyAcrylamide Gel Electrophoresis): 합성유, 무및간접적인순도측정방법입니다. HPLC: 정제와동시에 n-1 mer (failed oligo) 를확인하는방법입니다. MALDI-TOF & CE Oligo mass 를확인함으로써예상분자량과측정분자량을분석하여 depurination, N-1 failed oligo, modification success 를확인할수있는장비입니다. 일반적인 genomics 연구뿐만아니라 Large scale genomics 연구 (DNA chip, gene-therapy, SNP detection 등 ) 에는반드시 MALDI-TOF 통과한올리고를사용하셔야합니다. 50 mer 미만의올리고는모두 MALDI-TOF QC 검사가진행됩니다. 50 mer 이상의올리고는 HPCE 를이용한 QC 로순도측정데이터를제공합니다. MALDI-TOF QC Data Figure 2. Examples of a typical 30 mer and 23 mer oligo spectrum, in this case employing a Kratos MALDI-TOF system. Oligo Order System 바이오니아홈페이지에서주문접수, 합성, QC 및발송여부등일련의진행과정을확인할수있습니다. 고객의주문올리고정보는비밀관리됩니다. 배송업체의화물추적시스템을연결하여, 수령일정을미리확인할수있습니다. SMS 문자서비스를통하여제품의합 / 부판정여부를알려드립니다. Figure 1. Typical Oligo Datasheet with MALDI-TOF Information. BIONEER 26

DNA Oligonucleotide FAQs 취급및보관 1. 올리고는어떻게보관하며얼마나오래가나요? 일반적으로올리고는 -20 에서 1 년이상안정합니다. 특히건조한상태에서는매우안정적이며오래보관하실수있습니다. 용액상태에서도안정하지만 nuclease 등의 contamination 에의해 degradation 될수도있습니다. 또한올리고는 얼렸다녹였다 를반복하게되면분해를촉진시켜품질의이상이발생될수있으므로합성올리고는받으신후에여러개로분주하여각각을보관하신뒤필요량만큼꺼내어사용하시는것이바람직하며, 용액상태로보관할때에는멸균증류수에녹이는것보다 TE (10 mm Tri-HCl (ph8.0), 0.1 mm EDTA) Buffer 에녹여두시면더오래보관할수있습니다. DNA 의경우는산성조건에서는 depurination 에의해 decompose 될수있으니 ph 를낮추지말아야하며 RNA 의경우는높은 ph 에서는 hydrolysis 에의해 phosphodiester bond 가깨질수있으니주의를요합니다. Oligo 형상및보관방법에따른저장수명 Storage Condition Shelf Life (Month) at RT in water 2 at 4 in water 9 at -20 in water 18 at -20 (Dry) 24 2. 올리고는어떻게용해시키나요? 올리고는일반적으로 dry 된형태로공급됩니다. 깨끗하게 dry 된올리고는 aqueous solution 에대개잘녹지만간혹약간의시간이더걸리실수있습니다. 그럴때는약간의 NaOH 용액을넣어주면용해시킬수가있습니다. 그리고용액상태로보관할때에는멸균증류수에녹이는것보다 TE (10 mm Tri-HCl (ph 8.0), 0.1 mm EDTA) Buffer 에녹여두시면더오래보관할수있습니다. 3. Fluorescent dye modified 올리고는어떻게보관해야하나요? 빛에노출되면형광이약해질수있으므로빛이차단된용기에보관하시고, 저장하는장소도빛이들어오지않는장소에서보관하시는것이좋습니다. Cy3, Cy5 는 ph 9 이상에서는 degrade 될수있으므로 Cy3, Cy5 modified 올리고는보관시 basic 한환경을피하도록하셔야합니다. 4. 올리고용액을상온에서일주일이상방치했는데괜찮을까요? Contamination 만되지않았다면큰문제는없습니다. 정량및농도 1. 올리고의 O.D. 값은무엇을의미하나요? 합성올리고는매우미량이라어느정도의양이있는지무게를재서알아내기는매우힘듭니다. 따라서올리고의빛의흡광도를측정하여간접적으로합성올리고의정량을하게되는데이때빛의흡수량을 O.D. (Optical density) 값으로표시합니다. 2. 합성올리고의양을 O.D. 값으로부터어떻게얻어내나요? 합성올리고를파장 260 nm 의 UV Light 에서 O.D. 값을측정한뒤다음과같은수식을이용하여합성된올리고의양을얻어냅니다. O.D. = εc ε 은 Extinction Coefficient 로서물질이빛을흡수하는정도를나타내는상수로물질에따라고유한값을가지며 C 는올리고의농도를의미합니다. 올리고의 Extinction Coefficient 값을안다면 ε 값과 O.D. 값을상기의식에적용하여용액의 concentration, 즉올리고의양을알수있게됩니다. 올리고의 ε 값은각 base 의 Extinction Coefficient 값의 (Table 1) 합으로계산하는방법과 base 간의 sequence 간섭에의한차이를고려한 Extinction Coefficient 값의 (Table 2) 합으로계산하는방법이있는데각각의 260 nm UV Light 에서의각 base 의 Extinction Coefficient 값과간섭에의한 Extinction Coefficient 값은다음과같습니다. Table 1. Table 2. da 15,400 dc 7,400 dg 11,500 dt 8,700 5' -> 3' da dc dg dt da 27,400 21,200 25,000 22,800 dc 21,200 14,600 18,000 15,200 dg 25,200 17,600 21,600 20,000 dt 23,400 16,200 19,000 16,800 따라서전체올리고의 Extinction Coefficient 값은계산방식에따라편차가발생하게됩니다. www.bioneer.co.kr 27

DNA Oligonucleotide FAQs BIONEER 3. 다음과같이올리고 (3G, 4C, 5A, 6T / GGGCCCCAAAAATTTTTT) 18 mer 의전체 O.D. 값이 0.7 이었다면올리고의양은얼마나되나요? 올리고의양은흡광계수 (Extinction Coefficient) 를구하는방법에따라다음과같이계산됩니다. Table 1 에의한흡광계수 (Extinction Coefficient) ε = G 의염기개수 * 11,500 + C 의염기개수 * 7,400 + A 의염기개수 * 15,400 + T 의염기개수 * 8,700 = 11,500 x 3 + 7,400 x 4 + 15,400 x 5 + 8,700 x 6 = 193.3 (L/ mole) 따라서 O.D. = εc 에대입하여계산하면 C = 0.7 /193.3 = 0.003621 (mole/l) = 3.6 (nmole/μl) 이됩니다. Table 2 에의한흡광계수 (Extinction Coefficient) ε=(gg + GG + GC + CC + CC + CC + CA + AA + AA + AA + AA + AT + TT + TT + TT + TT + TT)-(G + G + G + C + C + C + C + A + A + A + A + A + T + T + T + T + T + T) = (21,600 + 21,600 + 17,600 + 14,600 + 14,600 + 14,600 + 21,200 + 27,400 + 27,400 + 27,400 + 27,400 + 22,800 + 16,800 + 16,800 + 16,800 + 16,800 + 16,800) - (11,500 + 11,500 + 11,500 + 7,400 + 7,400 + 7,400 + 7,400 + 15,400 + 15,400 + 15,400 + 15,400 + 15,400 + 8,700 + 8,700 + 8,700 + 8,700 + 8,700 + 8,700) = (342,200) - (193,300) = 148,900 (L/mole) 따라서 O.D. = εc 에대입하여계산하면 C = 0.7 /148.9 = 0.0047142(mole/L) = 4.70 (nmole/μl) 가됩니다자사에서는 Table 2 에의한방법을도입하여적용하고있으며, 이결과는 Oligo Synthesis Report 상의 Total nmole 수치로나타납니다. 4. 합성올리고의분자량 (Molecular Weight) 측정은어떻게하나요? 다음의수식에각 base 의수를대입하여계산합니다. M.W. =(NA x 249.2) + (NC x 225.2) + (NG x 265.2) + (NT x 240.2) + (oligolength-1) x 63.98) + 2.02 NA = Total number of A NC = Total number of C NG = Total number of G NT = Total number of T 5. 합성 Scale 의 μmole 단위를 ng 단위로변환하는방법은어떻게되나요? 일반적으로올리고의합성 scale 은 μmole 단위로신청되며합성된결과도보통 nmole 단위로 report 됩니다. 하지만, 사용하고자하실때 ng 또는 μg 단위로하신다면 mole 을 g 으로환산하시면됩니다. Report 에올리고의분자량이표시되기때문에쉽게바꿀수있습니다. 분자량 M.W. (g) X mole 수 (nmole) = 올리고의양 (ng) 6. 긴올리고는 Base Composition 을몰라정량하기가어려운데대략적으로알수는없을까요? 대략적으로 single strand 올리고의경우 1 O.D. 값이약 33 μg 이며 double strand DNA 경우는약 50 μg 정도가됩니다. 하지만짧은길이의올리고의경우는이값과차이가많이나기때문에정확한정량을위해서는계산을해서사용하시는것이좋습니다. 7. 합성올리고의 Tm 값은어떻게계산하나요? Tm (melting temperature) 는 oligonucleotide 중 50% 가 duplex 형태를이루고나머지 50% 는 single-strand 형태를지닐때의온도를말하며, 이에영향을미치는 factor 로는 oligonucleotide 의 sequence 와반응에사용되는 oligonucleotide 의농도, 그리고반응시 salt 농도등이있습니다. Tm 계산방법은여러가지가있으나이중바이오니아에서는 nearestneighbor 방법 (PNAS 83, 3746-50) 을이용합니다. 이방법이나오기전에는몇가지예측방법이있었는데, 15 base 미만에서잘예측되는방법으로 A 와 T 에는 2 를곱하고 G 와 C 에는 4 를곱해서이들값을더하는방법 (Wallace rule) 이있었습니다. Tm = 2 C (A + T) + 4 C (G + C) 또다른방법으로 GC content 를기초로한방법으로긴 sequence 에서잘예측되는방법이있습니다. Tm = 81.5 + 0.41 (%GC) - 500 /[L] + 16.6 log [M] ([L]; oligonucleotide 길이, [M]; monovalent cation 의농도 ) 하지만이와같은방법들은 base stacking effect 를고려하지못하고있고예측의정확도가떨어져서현재로는 nearest neighbor 방법이가장많이사용되고있습니다. ( 물론 nearest neighbor 방법도 60~70 bp 이상에서와 15 bp 이하에서는예측의정확성이떨어집니다.) 바이오니아에서사용하는 nearest neighbor 방법의가장큰특징은 base sequence 에따라 hybridization 에미치는영향이다르다는것을감안하여예측하는방법으로 2 개의 base 간의 hybridization 효과를모두열역학적으로측정하여이를통해 Tm 값을예측하는것입니다. 일례로 5 -GC-3 인 sequence 와 5 -CG-3 의열역학적측정치는서로다릅니다. 이와같이 2 base 간에이룰수있는가능한조합을모두측정하여변수로사용하고있습니다. nearest neighbor 방법의계산식은아래와같습니다. 여기서계산되는 Enthalpy 값과 Entropy 값은위에서미리 2 base 간에측정된열역학값을이용하여해당 oligonucleotide 의 sequence 에따라결정됩니다. 이때 [salt] 는반응에참여하는 monovalent cation 의농도를말하며 [Oligo] 는반응에들어가는올리고의농도를말합니다. R 은 Gas Constant (1.987 cal/k mole) 를의미합니다. 당사의 Tm 값은이같은식에의거하여계산되며, Salt 농도는 50 mm 로그리고올리고농도는 1 nm 을기준으로계산되었습니다. 28

DNA Oligonucleotide FAQs 이상과같은방법으로바이오니아는합성유전자의 Tm 값을예측하여고객에게제공하고있습니다. 하지만 Tm 값은예측치일뿐이지절대적인값은아닙니다. 특히실험자가 template 를준비하는과정에서생기는여러가지불순물과 salt 들은위의식에의해예측할수없기때문에실험자는 Tm 값을참고자료로실험을수행하는것이좋으며, 실험결과가나오지않을경우해당 Tm 값보다 4-5 낮은조건으로실험에이용하는것이좋습니다. 물론기준 Tm 값에서 non-specific product 가많은경우에는 annealing temperature 를높여가면서원하는정확한 product 를얻을수있는실험조건을찾아야합니다. 8. 본인이제작한올리고의 Tm 값과바이오니아에서제공한 Tm 값이차이가나는이유는무엇인가요? 바이오니아에서사용하는 TM calculator 는일반적으로 A, G, C, T 의개수에일정값을곱하기하는단순한계산방식과는다른것으로서 A, G, C, T 의배열순서에따라 Tm 값은차이가나게됩니다. 즉, 한염기의뒤에오는 sequence 가무엇이냐에따라전체염기수는같아도 Tm 은다르게되며더욱정밀하다고할수있습니다. 9. 합성올리고의농도맞추는방법에대한설명 우선올리고합성시트에쓰여있는 volume for 100 pmole/μl 라는말은 100 pmole/μl 의농도로만들기위해첨가해야할 TE buffer 나 D.W 의양을이야기합니다. 받으신리포트용지에각각 189.0 과 209.2 라고적혔다면, 각 oligomer tube 에적힌대로각각 D.W. 등을 189.0 μl 와 209.2 μl 를각각첨가했을때농도가 100 pmole/μl 가된다는것입니다. 즉, 각 tube 에는 oligomer 가 189.0 μl x 100 pmole/μl = 18,900 pmole = 18.9 nmole, 209.2 μl x 100 pmole/μl = 20,920 pmole = 20.92 nmole 씩들어있습니다. 사용하시는목적에따라 primer 의농도를알맞게설정하여야하지만, 일반적으로 PCR 이나 sequencing 의경우 100 pmole/μl 정도의농도로사용하실때편리하기에본사에서제공하는 sheet 에는농도를 100 pmole/μl 로만들기위한 volume 을제공하는것입니다. 0.5 μm 로 primer 를사용하시려고하신다면, 본사의 sheet 에서제시하는것처럼 primer 의농도를결정하였을때, pmole/μl 농도를 μm 농도로바꾸어주면 100 pmole/μl = 100 x 10 6 pmole/10 6 μl = 100 x 10 6 x 10-12 mole/l = 10-4 mole/l = 10-4 x 10 6 μmole/l = 10 2 μmole/l = 100 μm 즉, 100 μm 가됩니다. 이농도는사용하시려는 0.5 μm 의 200 배농도가되므로, 최종반응 volume 의 1/200 만큼 primer 를사용하시면원하시는 0.5 μm 가됩니다. 50 μl 로 PCR 을한다면, 50/200 = 0.25 μl 만큼을첨가하시면원하시는농도로반응을할수있습니다. 만일 0.25 μl 를 pipetting 하기힘들다면, 100 μm 의 primer 를 1/10 혹은 1/20 으로 dilution 한후사용하신다면, 각각 2.5 μl (1/10 의경우 ), 혹은 5 μl (1/20 의경우 ) 를최종반응에사용하면됩니다. 10. Morlarity 와 mole 의단위변환 Oligomer 의기본농도는 M (mole/l) 이고, 경우에따라서그앞에붙는 μ (micro), n (nano), p (pico) 등의첨자들은 M 을보조하는단위들입니다. 이단위들은다른길이, 질량등의 unit 들과도함께사용되므로이에대해서는다음을참조하시기바랍니다. 10-1 = deci[d] 10 1 = deca[da] 10-2 = centi[c] 10 2 = hecto[h] 10-3 = milli[m] 10 3 = kilo[k] 10-6 = micro[μ] 10 6 = mega[m] 10-9 = nano[n] 10 9 = giga[g] 10-12 = pico[p] 10 12 = tera[t] 10-15 = femto[f] 10 15 = peta[p] 1 pmole/μl = 1x10-12 mole / 1x10-6 L = 1x10-6 mole / L = 1 μmole/ L = 1 μm 이므로, μm 과 pmole/μl 는같은단위가됩니다. 합성및주문 1. 올리고는어떻게합성되는지알고싶습니다. 현재 oligonucleotide 합성기에서가장보편적으로사용되는합성방법은 Koster 에의해개발된 cyanoethyl phosphoramidite 를이용하여 DNA 구조의골격을이루는 phosphodiester 결합을연결해가는 phosphite triester 방법입니다. (Nucl. Acids Res. 1984, 12, 4539; Tetrahedron Lett. 1983, 24,5843) 이를통해원하는 oligonucleotide 를높은효율로합성할수있으며 ( 합성효율 : >98%) 합성시사용되는 phosphoramidite monomer 는 coupling 을위해활성화되기전에는상당히안정되어있어서장기보관도가능하고또한이를이용한 oligonucleotide 의합성시간도다른방법들에비해짧다는장점을지니고있습니다. 합성과정은 nucleoside 가부착된고형지지체상으로부터시작하여 deblocking, coupling, oxidation, capping 으로이루어지는 cycle 을반복함으로써원하는길이의 oligonucleotide 를얻게됩니다. <Figure 1> www.bioneer.co.kr 29

DNA Oligonucleotide FAQs Figure 1 A. Deblocking 합성 cycle 의첫단계인 deblocking 과정은고형지지체로부터 DMT 기를떼어냄으로써시작되며이때산성조건이요구되어보통 3% trichloroacetic acid 를사용합니다. 한편산성조건하에서는 purine 계열염기와 sugar ring 사이의결합이끊어지는 depurination 반응이일어날수있으며특히염기가 adenosine 인경우에는그경향이더심할수있다고보고된바있습니다. Trichloroacetic acid 는매우강한산으로 (pka : ~1.5) deblocking 에사용할때 depurination 시킬가능성이있어너무오랫동안 deblocking 반응을하지않도록주의가요구됩니다. 상기의문제점을최소화하기위해 trichloroacetic acid 대신약한산인 dichloroacetic acid 가쓰여지기도합니다. Deblocking 시고형지지체로부터떨어져나가는 DMT 양이온은진한주황색을띄는데이의흡광도를통해올리고합성시의결합효율을측정하는데에이용할수있습니다. B. Coupling Deblocking 과정을통해생성된고형지지체내의 5 -hydroxyl 기는 nucleoside phosphoramidite monomer 와 coupling 반응을통해원하는 sequence 의 oligonucleotide 를합성하게됩니다. 이때사용되는 nucleoside phosphoramidite monomer 는 Figure 2 에서보듯이염기부분의 amine 기는보통 benzoyl 기 (adenosine, cytidine 경우 ) 또는 isobutyryl 기로 (guanosine 경우 ) 보호되어있으며모든 monomer 는 5 -hydroxyl 기가 DMT 로보호되어있어계속되는결합반응의연결부분역할을하게됩니다. <Figure 2> 사용되는 phosphoramidite 구조는그자체로는상당히안정되어있으므로고형지지체의 5 -hydroxyl 기와결합하기위해서는활성화과정이필요합니다. 보편적으로사용되는 activator 로는 tetrazole 로서, phosphoramidite 와반응하여 nitrogen 부분을 protonation 시킨뒤 diisopropyl amino 기를치환시켜매우반응성이강한 tetrazolide 구조로변환시킵니다. 결과적으로반응성이강한 tetrazolide 와고형지지체의 5 -hydroxyl 기간의결합반응을통해 oligonucleotide 의사슬이이어지게되는데이때약간의수분이라도존재하면반응성이강한 tetrazolide 와반응하여원하지않는부산물을생성하여합성수율을저하시키기때문에 coupling 과정에서는무수조건이필수적입니다. Figure 2 C. Oxidation Phosphoramidite 와 5 -hydroxyl 기사이의 coupling 과정을거쳐생성된결합구조는본래의 phosphodiester 구조가아닌 phosphite triester 구조로, 더안정된 phosphate 로변환시키는과정이필요합니다. 이를위해서는 iodine 을사용한 oxidation 반응이사용되며결과적으로 phosphite 는 phosphate 로바뀌게됩니다. D. Capping Coupling 반응은반응물농도에선형적으로비례하지않기때문에 ( 보통 >98%) coupling 후고형지지체에는반응하지않은 5 -hydroxyl 기가남아있게되며이는다음 cycle 의 coupling 과정에참여하여원하지않는염기배열을갖는 (N-1)mer 를생성하는주원인이됩니다. 이렇게생성된부반응물들은합성완료후원하는 oligonucleotide 의정제를어렵게만들기때문에 coupling 과정후반응하지않고남아있는고형지지체내의 5 -hydroxyl 기를 blocking, 다시말해 capping 할필요가있습니다. 이를위해 acetic anhydride 와 N-methylimidazole 을사용하여 acetylation 시킴으로써반응하지않고남아있는고형지지체내의 5 -hydroxyl 기를 capping 시키게됩니다. 원하는길이의 oligonucleotide 의합성은상기의과정들을반복하여실시함으로써이루어지며합성이완료되면최종적으로 ammonia 처리를하여합성된 oligonucleotide 를고형지지체로부터떼어낸뒤정제과정을거쳐순수한 oligonucleotide 로얻게됩니다. BIONEER 30