TAKARA 1. PCR 2. qpcr 2014. 02. 13. 최유미
DNA?
DNA 생물체 > 기관 > 세포 >DNA DNA (Deoxyribonucleic acid) 세포내에서생물의유젂정보를보관하는물질 구성 : Adenine, Thymine, Cytosine, Guanine 종특이적, 개체특이적
PCR (Polymerase Chain Reaction) PCR 용도 현재생물학에서쓰이지않는곳이없다고해도과얶이아닐정도로광범위하게사용 개체구별 : 친자확인, 동일종내우수품종확인 유젂자증폭 : 다른실험을위해원하는유젂자증폭, 질병진단 PCR 재료 Template DNA: 증폭대상이되는 DNA 핚쌍의 primer: 증폭핛부분을잡는짧은 oligonucleotide Taq polymerase : Thermus aquaticus 약자로높은온도에서도 DNA 합성가능효소 dntp : 유젂자합성재료 nucleotide, datp, dctp, dgtp, dttp
PCR 효소의선택 TaKaRa Taq HS Perfect Mix
왜 high fidelity 효소를써야하나? 일반 Taq polymerase DNA 합성시에러가존재 유젂자존재여부확인시에적합 High fidelity 효소 정확핚 sequence 를요하는연구 DNA sequence 분석, 단백질발현 개체간특이 sequence 분석에오류발생가능성 하나의염기변화로발현시키고자하는유젂자가바뀔가능성
일반적인 High Fidelity 효소의단점 증폭효율이 Taq polymerase만큼좋지않다. 반응시간이길다. DNA, cdna template 양에영향을받는다. 긴단편인경우증폭이잘안된다.
PrimeSTAR GXL DNA Polymerase Components PrimeSTAR GXL DNA Polymerase(1.25 U/μl) 200 μl 5X PrimeSTAR GXL Buffer(Mg2+ plus) 1 ml x 2 dntp Mixture(2.5mM each) 800 μl 1. 25pg 2. 250pg 3. 2.5ng 4. 25ng 5. 250ng 6. 500ng 7. 750ng 10. 2ug 8. 1ug 9. 1.5ug Human genomic DNA (100ng/50ul reaction) 1. APOE 746bp(GC 74%) 2. TGF β1 2005bp(GC 69%) T. Thermophilus HB8 genomic DNA 3. 2029bp(GC 74%) 4. 4988bp(GC 74%) GC-rich template, excess template, and amplifying Long products 뛰어난신장성 (~30 kb) GC rich template에높은증폭율높은농도의 template에서도증폭기본 Extension은 1min/kb 효소를 2 배첨가시신장시간 10sec/kb의고속 PCR 가능 GC rich template은 standard protocol로진행
PrimeSTAR Max DNA Polymerase Components PrimeSTAR Max Premix(2X) 625 μl x 4 Mismatch 9/230,129 반응에필요한구성물 (buffer, dntp) 을미리섞어놓은 master mix PrimeSTAR Max Maximum Efficiency: Highest Fidelity Maximum Speed: Very Fast (5sec/kb) Premix: Saves Time 다량의 template 적용성 다량의 template DNA 을포함핚반응도 1min/kb 로설정하면증폭이가능 1 : 25 ng total RNA 2 : 50 ng total RNA 3 : 125 ng total RNA 4 : 250 ng total RNA 5 : 500 ng total RNA 6 : 750 ng total RNA 7 : 1 μg total RNA ~6kb 증폭가능 (Human genomic DNA)
PrimeSTAR HS DNA Polymerase Components PrimeSTAR HS DNA Polymerase (2.5 units/μl) 100 μl 5X PrimeSTAR Buffer (Mg2+ plus) 1 ml X 2 dntp Mixture (2.5 mm each) 800 μl Mismatch 12/249,94 높은정확성과증폭성 매우강력핚 3' 5' exonuclease 활성 ~8.5kb 증폭가능 (Human genomic DNA) PCR 조건 Tm value ( )=2 (NA+NT)+4 (NC+NG)-5 Tm 55 5 sec, Tm < 55 15 sec 다량의 template 적용성 3step 추천 98 10sec 55 5sec 1 or 55 30sec 2 72 1min/kb]30cycles High priming efficiency 1 : 100 pg 2 : 1 ng 3 : 10 ng 4 : 100 ng Excellent amplification efficiency with higher specificity
PrimeSTAR 요약! PrimeSTAR HS DNA Polymerase (R010) PrimeSTAR Max DNA Polymerase (R045) PrimeSTAR GXL DNA Polymerase (R050) 정확도 GC or AT rich 스피드 넓은범위의양 타겟길이 (human genome) 말단모양 (Blunt 또는 3'A end) 8.5 kb Blunt ( )* 6 kb Blunt 30 kb Blunt * extension time 을길게설정할경우 (- 1 min/kb)
PrimeSTAR GXL and Max 경쟁사 브랜드제품명 Code 횟수소비자가 Reaction 당가격 Takara R050A 200 290,000 1,450 PrimeSTAR GXL DNA Polymerase R050B 800 928,000 1,160 R045A 100 250,000 2,500 PrimeSTAR Max DNA Polymerase R045B 400 800,000 2,000 KAPA HiFi HotStart DNA Polymerase KK2501 100 76,000 760 KK2502 250 164,000 656 HiFi HotStart Ready Mix KK2601 50 57,000 1,140 KK2602 250 281,000 1,124 TOYOBO KOD Plus-Neo KOD-401 200 202,400 1,012 KOD FX-Neo KFX-201 200 270,000 1,350 Phusion Hot Start II High Fidelity F-549S 100 171,000 1,710 Thermo DNA Polymerase F-549L 500 678,000 1,356 Fisher Phusion Flash High Fidelity PCR F-548S 40 143,000 3,575 Master Mix F-548L 200 565,000 2,825 12355- Life 100 214,000 2,140 012 Pfx50 DNA Polymerase Technologies 12355-500 901,000 1,802 036 Fidelity Compared to Competitor Enzymes
Convenient Premixes: EmeraldAmp
EmeraldAmp 시리즈 종류 EmeraldAmp GT PCR Master Mix (Cat. # RR310) EmeraldAmp PCR Master Mix (Cat. # RR300) 특징 편리함 : 효소, buffer, dntp, loading dye 포함하는 2 Premix type 시간젃약 : Primer와 template DNA및 dh 2 O를첨가후 PCR GC rich 영역증폭에적합 저렴핚가격 : EmeraldAmp GT PCR Master Mix
EmeraldAmp GT 경쟁사 브랜드제품명 Code 횟수소비자가 Reaction 당가격 Takara EmeraldAmp GT PCR Master Mix RR310 400 80,000 200 PromegaGo Taq Green Master Mix Bioneer AccuPower PCR PreMix, 0.2 ml thin-wall 8 -strip tubes with attached cap M7122 100 67,000 670 M7123 1000 560,000 560 K-2012 96 47,000 490 K-2016 480 186,000 388
PCR 효소의선택
SYBR green qpcr 시약시리즈
Real time PCR Plateau Stage (End-point) End-Point PCR 은정확한정량분석이불가능하다
검출방법 특이성다중검정사용상용이성비용 TaqMan Probe 검출특이성이높다가능 Probe 제작이까다롭다고가 TaqMan Probe
검출방법 SYBR Green I 특이성 검출특이성이높지않다 다중검정 - Melting Curve 가능 사용상용이성 초기 setting이쉽다 비용 저렴 SYBR Green I SYBR 의단점을보완하기위핚 Melting Curve ( 형광신호변화 / 온도변화 ) Primer dimer 나 non-specific product Specific product
Takara 시약선택가이드 Long product Fast qpcr SYBR Premix Ex Taq (Tli RNaseH Plus) <Code RR420> SYBR Premix Ex Taq II (Tli RNaseH Plus) <Code RR820> SYBR Premix DimerEraser (Perfect Real Time) <Code RR091> Premix Ex Taq (Probe qpcr) <Code RR390> Specificity gdna template 제품추천가이드 qpcr 실험을하는고객에게첫번째추천시약 RR820 Fast/ 긴단편의 qpcr 실험에 RR420 을추천 SYBR Premix Ex Taq GC (Perfect Real Time) <Code RR071> RR420, RR820 보다높은반응특이성을원하는고객에게 RR091
Tli RNaseH? - Tli RnaseH qpcr reagents TakaRa qpcr reagents + Tli RnaseH 타사제품에는없는오직다카라제품에만있는강점 2x Premix 시약에내열성 RNase H (Tli RnaseH) 첨가 역젂사반응후 Rnase H처리과정 (37, 20분 ) 불필요 *Tli RNaseH: 호열성세균유래의내열성 RNaseH
Tli RNaseH 효과 : VGLL4(GC 65%) RR081 RR820 Ct 값 : RR820<RR081 RR081: Tli RNaseH 불포함 RR820: Tli RNaseH 포함 mrna 제거를통해증폭효율상승!! GC 함량이높을수록 mrna inhibition 이높음. RR081+RNaseH Ct 값 : RR820=RR081
반응특이성비교 : RR820 vs RR420 RR820 YWHAZ (38%) RR420 X X 폭넓은타겟, 특이성이높다! 길이가길고고속모드기기!! 특이성 : RR820>RR420
반응효율비교 : RR820 vs RR420 Extension: 30s (TP900) RR420 RR420 RR420 RR820 RR820 RR820 ACTB : 186bp ACTB : 381bp ACTB : 533bp RR420 standard curve 증폭효율 :RR420 >RR820 RR820 standard curve
경쟁사성능비교 _Fast PCR(Abi) Takara VS Power SYBR FAST(Abi) RR420 ACTB : 186bp FastSYBR X ACTB : 381bp X Although RR420 is as fast as FastSYBR, RR420 is more specific than FastSYBR. ACTB : 533bp X
Rox dye 가포함된 SYBR green premix Abi qpcr reagent를 Takara로 Switch!! Abi 기기점유율을이용하자! Rox dye 가포함되어 Abi 기기에편리하게적용 (RR39LR, RR82LR, RR42LR) Abi 뿐만아니라다양한기기에최적화된 protocol PRTSS 서비스를통한최적화된 primer 제작 Cat. # 구성기기적용 RR820, RR420, RR390 SYBR Premix Ex Taq II (Tli RNaseH Plus) 50X ROX Reference Dye 50X ROX Reference Dye II Abi 7000/7700, 7300 StepOnePlus Abi 7500, 7500 Fast Takara Thermal Cycler Dice Real Time System II Roche Smart/LightCycler Bio-rad CFX96 RR82LR, RR42LR, RR39LR SYBR Premix Ex Taq II (2X) (Tli RNaseH Plus), ROX plus Abi 7000/7700, 7300 StepOnePlus
SYBR 을이용핚 qpcr: 최적의 primer 필요 실험결과향상을위핚 tool TAKARA Perfect Real Time Support System Human, rat, mouse, cow, dog, chicken의 Refseq등록유젂자에대핚프라이머서열보유 온라인으로간편하게검색 Takara 제품과는반응조건의최적화과정불필요 UCSC Genome Browser http://takara.co.kr/web01/product/customservice02.asp
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