TaKaRa Real Time PCR System · 2011. 5. 12. · Basics of Real-time PCR z To accuratelyyq y g...

TaKaRa Real Time PCR System

TaKaRa Korea Biomedical., IncSales & Marketing Department.

pg. 1 © 2010 TaKaRa

Contents

1.1. Principle of RealPrinciple of Real--Time PCRTime PCR1.1. Principle of RealPrinciple of Real Time PCRTime PCR

2.2. Process of RealProcess of Real-- Time PCRTime PCR2.2. Process of RealProcess of Real Time PCRTime PCR

3.3. Introduction ofIntroduction of TaKaRaTaKaRa Real time SystemReal time System3.3. Introduction of Introduction of TaKaRaTaKaRa Real time SystemReal time System


Principle of RealPrinciple of Real--Time PCRTime PCRPrinciple of RealPrinciple of Real--Time PCRTime PCR


Basics of Real-time PCRTo accurately quantify starting amounts of nucleic acid during the y q y g gPCR reaction without the need for post-PCR analyses.

A fluorescent reporter is used to monitor the PCR reaction as itA fluorescent reporter is used to monitor the PCR reaction as it occurs; the reporter can be of a nonspecific or specific nature.

The fluorescence of the reporter molecule increases as productsThe fluorescence of the reporter molecule increases as products accumulate with each successive round of amplification.

+

Traditional PCR + fluorescence detectorpg. 4 © 2010 TaKaRa

Traditional PCR + fluorescence detector

Traditional PCRdetection; EtBr Gel Detection

PCR Phase

Plateau

; EtBr Gel Detection

[DNA] Exponential Linear[ ]

[Cycle #]Area of detection f R l Ti PCR


for Real Time PCR

Plateau Stage (End-point) ☞g ( p )

10ng 1ng 10-1ng 10-2ng 10-3ng 10-4ng 10-5ngInitial Stage ☞


SYBR® Green I


Melt Curves (Only SYBR)

Following amplification temperature is slowly increasedFollowing amplification temperature is slowly increased (ie. 60°C to 95°C, 0.5°C/sec)

Strands denatureStrands denature

Dye released, fluorescence decreases

Melting curve; Temperature (x) vs. Fluorescence (y)

Sharp downward turn denotes TmSharp downward turn denotes Tm

Tm function of %GC and length


Dual-Labeled Probe


Absolute vs. Relative quantification

Absolute – produces data with quantity amountsRequires a standard whose concentration(or copy number) is knownRequires a standard whose concentration(or copy number) is known absolutelyIdentical amount of sample must be assayed for all unknowns (complex)(complex)

Relative – makes comparisons of quantity (no units)Requires endogenous control target (standard curve not used)Normalizes for amount of sample addedStandard Curve methodStandard Curve methodComparative method(or ∆∆Ct)=> 2∆∆Ct


Process of RealProcess of Real--Time PCRTime PCRProcess of RealProcess of Real--Time PCRTime PCR


Overview of Real Time Assay Process

Cell / Tissue…

extract RNA

copy into cDNA(reverse transciptase)(reverse transciptase)

Real time PCRReal-time PCR

A l ltpg. 12 © 2010 TaKaRa

Analyze results

Perfect Real Time Series

2-step reaction 1-step reaction


TaKaRa Dice Real Time System OverviewOctober 19, 2010


,

1-4). Real Time System Line-up 사양

Excitation Emission Channel Range

(nm)Range (nm)

Simplex Dyes Notice

1 482 536 FAM/SYBR TP850

2 562 624 ROX/TxR TP800

3 525 585 HEX/VIC TP800(option)


(option)

4 628 692 Cy5 TP800(option)

1-5). TaKaRa Real Time System 특징

■ 고성능 하드웨어

◀ 96 well에서 높은 균일성 실현

- 안정적 온도 제어 시스템

◀ 2배 농도차까지 검출 가능

- 정확한 광학 검출 시스템



■ 쉽게 사용할 수 있는 편리한 소프트웨어

▼ 초보자도 쉽게 사용 가능

▶ 2개 화면으로 분리, 결과 분석이 용이

- 독립조작을 통해 다양한 시점에서분석 가능



■ 다양한 분석방법 제공

▼ SNP Genotyping Assay

◀▼ 측정치의 분포 Scatter Plot 제공◀▼ 측정치의 분포 Scatter Plot 제공종합적으로 판단 Assay 결과 판정

▶ Plus/Minus Assay


1-8). 대규모 해석이 가능한 「Multiplate RQ」 소프트웨어

■ 복수의 Reference 유전자를 이용한 보정 가능

■ 복수 l t 를 한번의 분석으로 해석 정량이 가능■ 복수 plate를 한번의 분석으로 해석, 정량이 가능

pg. 20 © 2010 TaKaRa * MRQ는 TP860/870에 기본 탑재, TP800 시리즈 전용 S/W

TaKaRa Real Time PCR System · 2011. 5. 12. · Basics of Real-time PCR z To accuratelyyq y g...

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