Transcript Principle of Real-Time qPCR AND Applications Han-Oh Park, Ph.D.

Principle of Real-Time qPCR
AND
Applications
Han-Oh Park, Ph.D.
President & CEO
BIONEER CORPORATION
-목 차회사소개
Principle of Real-Time
qPCR AND Applications
Exicycler
Understanding
Real Time PCR Data
회사 개요
회 사 명
㈜바이오니아 (BIONEER CORPORATION)
대표이사
박한오
주
소
대전광역시 대덕구 문평동 49-3
종업원수
175명
설 립일
1992년 8월 28일
자 본금
5,675백만원
발행주식수
11,350,000주 (1주 액면가액 500원)
홈페이지
www.bioneer.co.kr
최근 매출액
116억원(’06), 100억원(’05), 92억원(’04)
인원 현황
(2007년 6월말 현재)
[직능별]
연구직
생산직
영업직
관리직
계
인원수
57
70
25
23
175
[학력별]
박사
석사
학사
기타
계
인원수
16
48
55
56
175
유전자신약
사업
BT사업
합성유전자 유전자시약
사업
사업
유전자진단시약
/시스템 사업
BIT사업
유전자장비
사업
Bio
Defense
NBT사업
나노바이오
사업
유전자
신약
기반 사업군
유전자신약 유전자
시약
Tool
합성
유전자
유전자 장비
합성
유전자
유전자 진단시약
유전자
진단시스템
Cash Cow
Bio
Defense
미생물
신약
나노
바이오
Star
1차 세계화 전략
2차 세계화 전략
3차 세계화 전략
2001~2005
2006~2010
생명공학 핵심소재인
합성 유전자를 선도사업
으로 세계 Marketing
Network 구축
세계 중요 바이오 클러스터에
유전자 사업기반 구축
글로벌 바이오텍
기업으로 성장
유전자 진단, 나노 바이오,
유전자 신약 회사로
위상 확립
Biodefense분야의 글로벌화
2011~
미국 현지공장 설립  고품질의 합성유전자 현지공급
ABADIS(Biodefense분야) 해외수출추진
미국, 유럽 현지법인 설립으로 2차 세계화 전략 진행 중
중국 판사처 설립  잠재적 거대시장 진입을 위한 사전 준비작업 진행
이 름
직 책
약
력
 서울대 화학과 졸업 / KAIST 생화학 석사,박사
 KIST 생명공학연구원 연구원/ KAIST 바이오시스템학과 겸임교수/ 서강대 바이오융합과
정
겸임교수
 BIT산업협의회 회장/ 한국산업미생물학회 부회장/ 한중생명공학 협력센타 운영위원
박한오
대표 이사
이재돈
유전자신약 사업부장
박한이
유전자시스템사업부장
박해준
유전자진단사업부장
 서울대학교 농화학과 / KAIST 생명과학과 박사
 목암생명공학연구소 진단연구실 수석연구원
김재하
나노바이오 사업부장
 연세대학교 화학공학과 / KAIST 화학공학과 박사
 대한유화㈜ 기술연구소 / KAIST 화학공학과 반응표면 연구실 연구원
최영철
유전자신약연구소장
 서울대학교 미생물학과 / Univ. of North Carolina at Chapel Hill 박사
 KAIST 유전공학센터, St. Jude Children's Research Hospital 연구원
원세연
바이오인포메틱스사업부장
문 용
중국 판사처 수석대표
유재형
영업본부장
정진평
경영지원본부장
 서울대 화학과 석사 / Univ. of Michigan 박사수료
 KAIST, Univ. of Michigan, Organic Synthesis Lab 연구원
 고려대학교 전기공학과
 삼성전기 회로사업부 연구실 / LG전자 중앙 연구소 선임연구원
 고려대학교 생물학과 / KAIST 생명과학과 박사
 과학기술정보연구원 선임연구원 / 생물정보연구소 대표
 중국 북경의과대학 / 서울대학교 보건학 박사
 중국 북경의과대 교수 / 북경의대 한국주재 대표
 영남대학교 미생물학과 / 충북대학교 미생물학과 박사
 대전보건대학 겸임교수 / ㈜씨젠 기술이사(CTO)
 충북대학교 경영학과 / 원가관리사
 오성사 기획실 / 스카이전자 경리부
도약기
2004~
04.07 합성유전자 차세대 세계일류상품
성장기
2000~2003
설립기
1992~1999
92.08 ㈜한국생공 설립
(한국생명연 연구원창업1호)
95.06
95.07
97.05
제1호 기술개발시범기업 선정
㈜바이오니아로 상호변경
97유망선진 기술기업 지정
98.04 동경 국제게놈학회
00.03
00.12
01.04
01.05
01.07
제품4종 조달청 우수제품 선정
제1회 바이오산업협회상 수상
미국 현지법인 설립
염기서열자동분석장치 출시
2001 대한민국기술대전
산업자원부 장관상 수상
02.05 월드컵 테러방지 생물학탐지기,
식별기 개발 및 상용화
02.07 Korea Technology Fast50선정
02.07 벤처기업대상 대통령 표창 수상
03.03 DTT Asia Pacific Technology
Excellent Poster Award
98.12 ISO 9001품질시스템 인증
99.01 유전자증폭시약제조기술 미국 특허
기술경쟁력 우수기업 지정(중소기업청)
FAST50 선정 (바이오텍 분야 2위)
선정 (산자부)
04.09 합성유전자 NT(신기술)인증
(산자부)
04.11 Exicycler EM마크 인증(산자부)
05.07 유럽현지법인(BEL) 설립
05.10 한국응용생물화학회 기술상 수상
05.10
05.12
06.03
06.10
06.11
100만불 수출의 탑 수상
코스닥 상장
나노바이오사업부 신설
미국 현지법인 유전자합성공장 준공
300만불 수출의 탑 수상
Principle of Real-Time qPCR
Principle of PCR
Basics of PCR
1 Cycle
Heating
Cooling
95℃
55℃
Extension
72℃
Cycling
Polymerase
Primer
Target
DNA
Cycling
Principle of PCR
Cockerill FR III. Arch Pathol Lab Med. 2003;127:1112
Disadvantages of PCR
Basics of PCR
?
Ideal graph
1 Cycle
End-Point PCR analysis is not quantitative
2 Cycle
Real graph
3 Cycle
N Cycle
Disadvantages of PCR
What is Wrong with Agarose Gels?
Poor precision
Low specificity
Size-based discrimination only
Low sensitivity/Resolution
Short dynamic range (< 2 logs)
Possibility of human errors
Cross-contamination
Results are not expressed as numbers
Ethidium bromide staining is not very quantitative
Real-Time PCR
Real-Time PCR
Real-time PCR monitors the fluorescence emitted during
the reaction as an indicator of amplicon production at
each PCR cycle (in real time) as opposed to the endpoint
detection
Principle of Real-Time PCR
Key components of Real-Time qPCR
RealTime
qPCR
1. Fluorescence
Dyes
2. Optical
Components
3. Thermal
Cycler
Fast, Accurate and Quantitative Results
Principle of Real-Time PCR
Nigel Walker, NIEHS
Advantage of Real-Time PCR
Real-time PCR vs. Conventional PCR
Real-Time PCR
PCR
Sensitivity
High
Low
Specificity
High
-use specific probes
Low
-only size discrimination
Quantitative
results
Yes
-Specific fluorescence
No
-EtBr staining
Detection
method
Probe-specific
Fluorescence
Agarose gel
Electrophoresis
Detection range
Wide range
Short range (<2 log)
Reaction time
1 hr
3-5 hr
Post-PCR steps
No
Agarose gel electrophoresis
Crosscontamination
No
-Closed system
- Single step
Yes
-Open system
- Multiple steps
Glossary of Real-Time PCR
Amplification plot: The plot of cycle number vs. fluorescence signal which
correlates with the initial amount of target nucleic acid during the
exponential phase of PCR
Baseline: The initial cycles of PCR during which there is little change in
fluorescence signal (usually cycles 3 to 15)
Fluorescence
Amplification plot
Baseline
Threshold
Ct
cycle
Glossary of Real-Time PCR
Threshold: the fluorescence measurement at which product can be
distinguished from background. Threshold should be set in the region
associated with an exponential growth of PCR product
Ct (Threshold cycle): The cycle number at which the fluorescence generated
within a reaction crosses the threshold. It is inversely correlated to the log
of he initial copy number
Fluorescence
Amplification plot
Ct
Threshold
Baseline
cycle
Detection Chemistry
Primer & Probe
Primer
Short (Often < 50 nt) oligonucleotide sequence of DNA
Complementary to the beginning and the end of the target
DNA sequence
Needed to initiate the synthesis of new DNA in a PCR
reaction
Involved in AMPLIFICATION
Probe
A single-stranded DNA with a specific base sequence
Labeled with fluorescence dyes (TaqMan probe)
Used to detect the complementary base sequence of
target DNA/RNA by hybridization
Involved in DETECTION
Reporter dye / Quencher dye
Probe
Primer
Detection Chemistry
DNA binding agents
- Intercalating method: SYBR®Green I
Fluorescent dyes
Hydrolysis Probe
- TaqMan® probe, Molecular Beacon
Hybridization Probe
- Dual oligo FRET probes
Primer based Probe
- Scorpion
SYBR Green dye
Intercalating method
1) Denaturation
F
F
2) Annealing
F
Intercalating dye fluoresces
more brightly when bound to
dsDNA.
2)
DNA binding dyes are
inexpensive compared to the
other probes.
3)
SYBR Green I, EtBr
F
F
F
1)
F
F
F
3) Extension
F
F
F
SYBR Green dye
Intercalating method
1. Advantages:
- Cheap, easy to use
- Does not inhibit the reaction of amplification
- Does not require any fluorescent probe
- Does not require any particular expertise for the design of the probes
- Is not affected by mutations in the target DNA
2. Disadvantages:
- Impossible to make sure of specificity of amplicons
- Bad pairing can lead to positive forgeries or an over-estimate of the
quantification
- Still unspecified mutagen capacity
TaqMan Probe
TaqMan® probe
1) Denaturation
1)
Fluorescent reporter dye at the 5’
end is quenched by fluorescent
quencher dye at the 3’ end.
2)
When amplification occurs the
TaqMan probe is degraded due
to the 5'-->3' exonuclease activity
of Taq DNA polymerase, thereby
separating the quencher from the
reporter during extension.
3)
The TaqMan assay accumulates
a fluorescence signal.
Q
F
2) Annealing
Taq
F
Q
3) Extension
F
F
Q
Q
TaqMan Probe
TaqMan® probe
1. Advantages:
- Increased specificity
- Better capacity of multiplexing
2. Disadvantages:
- Little expensive (dual-labeled probe)
- Less effective and less flexible compared to other techniques in the
real-time detection of specific mutation
- Require the design of probes
Molecular Beacon Probe
Molecular Beacon
1) Denaturation
1)
A molecular beacon begins as a
stem-and-loop structure. The
sequences at the ends of the
probe match and bind, creating
the stem
2)
When the probe binds to a singlestranded DNA template, the
structure unfolds, separating the
quencher from the dye and
allowing fluorescence.
F Q
2) Annealing
Taq
F
Q
3) Extension
F Q
Molecular Beacon Probe
Molecular Beacon
1. Advantages:
- Increased specificity
- High flexibility for probe design
- As the probes are not hydrolyzed, they are used at each cycle
2. Disadvantages:
- Little expensive (dual-labeled probe)
- Less effective and less flexible compared to other techniques in the
real-time detection of specific mutation
- Require the design of probes
FRET Probe
Hybridization Probe (FRET)
1) Denaturation
F
1)
FRET method designed two
specifically probe. It labeled with
different dyes, such as at the 5’
end of donor probe and at the 3’
end of acceptor probe.
2)
At close proximity, the donor dye
is excited by the light source and
the energy is transferred the
acceptor dye. Subsequently,
fluorescent light is emitted at a
different wavelength.
F
2) Annealing
Energy
transfer
Taq
F F
3) Extension
F
F
Applications of Real-Time PCR
Real-Time PCR Applications
Clinical Diagnostics
- Bacterial/ Viral pathogen detection
- Absolute pathogen quantification
Drug therapy efficacy / drug monitoring
Differential gene expression
RNAi
- siRNA validation
SNP Genotyping
Others
- Food pathogen testing
- Forensic studies
Real-Time PCR Applications
• Structural Assay
– Uses DNA, typically genomic extractions
• Single Nucleotide Polymorphism
• Functional Assay
– Uses RNA extractions
– Uses reverse transcriptase to generate cDNA templates
• Differential expression
• Diagnostics involving gene expression
• RNA interference
• Clinical Diagnostic Assay
– Uses DNA or RNA extracted from patient’s samples
• Viral/bacterial pathogens
Quantification Strategies
Absolute quantification
- Used to quantitate unknown samples by interpolating their
quantity from a standard curve.
- The standard is a known DNA sample whose concentration is
known absolutely.
- The accuracy of the absolute quantification assay is entirely
dependent on the accuracy of the standards.
Relative quantification
- Used to analyze changes in gene expression in a given sample
relative to another reference sample.
- A comparison within a sample (DNA or cDNA) is made with the
gene(s) of interest to that of an endogenous control gene.
- Quantification is done relative to the control gene.
Absolute Quantification
Pathogen Detection/Clinical Diagnostics
Detection of specific genes in pathogens (virus, bacteria, fungi, etc)
isolated from patient’s samples
Quantification: copy numbers of infected pathogens
Therapeutic Drug Monitoring/Screening
Varicella-Zoaster Virus – Real-Time PCR
106 copies
Absolute Quantification
Absolute Quantification
Ct value
Threshold
Sample 1
Ct:14
Conc: 2,500copy
The Ct value correlates strongly with the starting copy number.
It is linear with the log of starting DNA concentration.
Relative Quantification
Differential Gene Expression
Compares transcriptional levels of genes between control and
experimental samples
- Tissue distribution of target gene
- Drug screening/Drug efficacy
- Gene expression profiling after drug treatment
Endogenous controls are used to normalize the data
Endogenous controls are genes common to both control and
experimental samples that do not change their expression levels
under the experimental conditions
- GAPDH
- β-Actin
- 18s ribosomal subunit
Relative Quantification (PCR)
Experiment Design (Traditional / Relative quantification)
Cell line A
Cell line A + Drug
mRNA
mRNA
PCR using same amount of mRNA after spectrophotometer quantification
E
R
R
O
R
Relative Quantification (Real-Time PCR)
Experiment Design (Real-Time PCR /Relative quantification)
Cell line A
Cell line A + Drug
mRNA
Actin
mRNA
Target
Actin
Target
Relative Quantification
Relative Quantification (Drug-induced gene expression)
Control cell lines
Actin
Target
A > B (21 : 2 times much)
Sample A : 16 Ct
Sample B : 17 Ct
=> Delta Ct = 1 Ct
Control cell lines
+ Drug A
Actin
Target
A > B (24 : 16 times much)
Sample A : 17 Ct
Sample B : 21 Ct
=> Delta Ct = 4 Ct
Drug A treatment induced decreased target X gene expression
Relative Quantification
Relative Quantification (Tissue distribution / Delta Ct Methods)
Brain tissue
Actin
Target X
Actin : 17 Ct
Target : 21 Ct
=> Delta Ct = 4 Ct
Liver tissue
Actin
Target X
Actin : 22 Ct
Target : 26 Ct
=> Delta Ct = 4 Ct
Target gene expression level between brain and liver tissue is same
SNP Genotyping



Single Nucleotide Polymorphism (SNP)
DNA sequence variations that occur when a single nucleotide (A, T,
C, or G) in the genome sequence is altered
How many SNPs are there in humans today?
- Human mutation rate is ~ 2.5 x 108 mutation/site/generation
- ~150 mutations/diploid genome/generation
- 6.3 billion people in the world = 945,000,000,000 mutations in the
world today


The most common type of sequence difference between alleles
Provide a way to detect direct associations between
allelic forms of genes and phenotypes
SNP Genotyping (Real-Time PCR)
Allelic Discrimination Assays (Single Nucleotide Polymorphisms)
T
A
G
A
T
G
C
C
SNP Genotyping (Real-Time PCR)
G
C
T
T
A
A
G
T
A
G
C
C
RNAi: siRNA validation
RNA interference:
double stranded RNA
(siRNA) forms a complex
(RISC) that binds to target
mRNA and leads to its
degradation
Target gene expression can
be quantified by Real-Time
PCR
Multiplex Analysis
Different dyes for each target (Example: FAM, TET, VIC and JOE)
Real-time detection of four different
retroviral DNAs in a multiplex format.
Each reaction contained four sets of PCR
primers specific for unique HIV-1, HIV-2,
HTLV-I, and HTLV-II nucleotide
sequences and four molecular beacons,
each specific for one of the four
amplicons and labelled with a differently
coloured fluorophore.
HIV-1: Fluorescein
HIV-2: Tetrachlorofluorescein
HTLV-1: Tetramethylrhodamine
HTLV-II: Rhodamine
Vet JA et al. PNAS (1999)
Improving Reproducibility
• Use clean bench (hood)
•
•
•
•
Use aerosol resistant tips
Use calibrated micropipettors
Use large volumes (5µL and up)
Pipette into each reaction vessel once
Same Reagents, Different Results
Cycle
Good Technique
Cycle
Poor Technique
Special Features of ExicyclerTM
Real Time Quantitative Thermal Block
Exicycler™
The ExicyclerTM is a real time PCR system developed by Bioneer.
The ExicyclerTM is equipped with an optical system that fits above the thermal cycler.
It readily utilizes most fluorescent dyes, providing the widest choice of excitation /
emission wavelengths.
It can also be used as a standard thermal cycler for general PCR reactions.
Characteristics of ExicyclerTM
1)
Superior optic module
Light pipe technology (patented)
2)
Light source
Short Arc Lamp
Life: 3,000 hrs
High Power, high quality, low maintenance
3)
Excitation filter
Six position (blank 1, filter 5)
Wavelength range: 490-640nm
5 multiplexing
4)
Emission filter
Six position (blank 1, filter 5)
Wavelength range: 520-670 nm
5 multiplexing
5)
Powerful 2D CCD detector
High resolution and great sensitivity
Resolution : 325K pixel
6)
Thermal gradient
Superiors of ExicyclerTM
Analysis can be viewed in real time.
Melting curve can also be viewed in real time.
Thermal gradient is compatible.
Range of fluorophore excitation and emission is 490~670 nm.
It can be used for multiplexing with 5 different fluorophores.
Reliable results without using reference dye.
Not necessary to decontaminate the sample block.
96 samples can be tracked simultaneously.
All data is accessible as Excel file and jpg image.
Result reports, everyone can open on internet explorer.
Software is friendly to beginner at qPCR.
USB communication interface.
Auto loading.
Patents of ExicyclerTM
1) Real time monitoring apparatus for amplified nucleic acid product
-Appl. Con. : ROK
-Appl. Date: Jun. 18, 2002
-Appl. No. : 10-2002-0033965
Intensity Profile at Sample Reactor(Patents)
A company
2) Real time monitoring apparatus
-Appl. Con. : ROK
-Appl. Date : Apr. 3, 2003
-Appl. No. : 10-2003-0021145
BIONEER
Optic module of ExicyclerTM
Lamp
Light pipe
Camera lens
Comparison of Optics
Exicycler
Other Company
More homogeneous light intensities among wells
=> Reliable results without using reference dye
Comparison of Optics
Exicycler
Without reference dye
Other Company
Without reference dye
With reference dye
Diversity of fluorescence dyes
Excitation/Emission wavelength
Position
Excitation
Emission
Dye
1
490
520
Fluorescein, FAM, SYBR®Green
2
520
550
JOE, TET
3
550
580
TAMRA, CY3
4
580
610
Texas Red, ROX, RED610
5
640
670
CY5, RED 670
Classified by light sources
Laser
High cost and high power
Small interference
High maintenance cost
Limited number of colors
(Laser based excitation limits fluorophore range)
Can only be used for well scan type (ABI 7900)
LED
Low cost and low power
More number of colors
Inadequate for high throughput system
Normally used for well scan type (sequential data acquisition)
Chromo4, LightCycler, Opticon series, Rotor-Gene
Classified by light source
Lamp
Suitable for multi-color
Unique solution for 2 dimension sensor
(Simultaneous data acquisition)
Suitable for high throughput system
High cost optic component
ABI 7500, 7300, iCycler series, LC480, Exicycler
Classified by sensor and thermal cycling
Point sensor (PMT, photodiode) and rotating mechanism
Low price and high temperature uniformity
Difficult to handle, can’t support storage function
No gradient function to aid in optimization
Air heating not as accurate as Peltier
No system modularity
Special consumables and loading block are required
LightCycler (discontinued), Roter-Gene
Classified by sensor and thermal cycling
Point sensor (PMT, photodiode) and scanning mechanism
Cheap, simple, easy to calibrate
Time lag due to scanning
High maintenance cost
Longer running time
Chromo4, MX3005P
2D sensor (CCD)
Simultaneous detection (suitable for high throughput)
Increased material costs, hard to calibrate
ABI 7500, ABI 7300, iCycler, LC480, Exicycler
Market trend
2D CCD is the current main stream for sensor
ABI 7500, ABI 7300, iCycler, Roche LC480
High throughput
Must support plate format
Bioneer is developing 1536 well format real time PCR currently
Powerful and various analysis software
SNP typing, Absolute & Relative Quantification,
Infection Assay (using internal control)
Comparison
Name
iCycler
SmartCycler
RotorGene
Lightcycler
2
ABI7500
Opticon 2
Exicycler
Company
Bio-rad
Cepheid
Corbett
Roche
Applied
biosystem
MJ research
Bioneer
Detection
Simultaneous
Simultaneous
Sequential
Sequential
Simultaneous
Sequential
simultaneous
Type
96 well
16 tube
32, 72 tube
32 capillary
96 well
96 well
96 well
Gradient
O
×
×
×
x
O
O
Light
source
Tungsten
Halogen Lamp
LED
LED
Blue LED
Tungsten
Halogen Lamp
LED
Short Arc
lamp
Detector
10 bit CCD
Silicon
Photodetector
PMT
Photohybrid
10 bit CCD
PMT
16 bit
CCD
Ex /Em
range
400~700 ㎚
450~650 ㎚
470~660 ㎚
470~710 ㎚
488~650 ㎚
470~700 ㎚
490~670 ㎚
Multiplex
4 color
4 color
4 color
6 color
5 color
2 color
5 color
Experimental Results in ExicyclerTM
SYBR Green I data
22000
20000
18000
Without reference dye
16000
14000
12000
100000
10000
8000
6000
10000
4000
2000
1000
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
100
10
1
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Experimental Results in ExicyclerTM
TaqMan data (FAM)
20000
18000
16000
Without reference dye
14000
12000
10000
8000
100000
6000
4000
10000
2000
0
1
2
3
4
5
6
7
8
1000
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
100
10
1
1
0.1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Understanding
Real Time PCR Data
Refreshing Logarithms and
Exponentials
32
1
t=0
2
4
1
2
8
16
3
4
64
…
5
6
Refreshing Logarithms and
Exponentials
Population Size vs. time
70
Population Size (N)
60
50
40
30
20
10
0
0
1
2
3
4
Time (t)
5
6
7
Refreshing Logarithms and
Exponentials
8
f(x) = bx
6
4
f(x) = logbx
2
0
-6
-4
-2
0
-2
-4
2
4
6
8
Refreshing Logarithms and
a>1
Exponentials
• For f(x)=bxa
a>1
a=1
a<1
a<0
Regular plot
300
weight W (lbs)
250
200
150
100
50
0
50
75
100
125
150
175
200
length L (cm)
log-log plot
Semi-log plot
6
5.5
5.5
5
5
4.5
4.5
ln(W)
ln(W)
6
4
4
3.5
3.5
3
3
2.5
2.5
50
75
100
125
length L (cm)
150
175
200
4
4.2 5
4.5
4.7 5
ln(L)
5
5.2 5
DNA copy number
1.8E+09
1.6E+09
1.4E+09
1.2E+09
1.0E+09
8.0E+08
6.0E+08
4.0E+08
2.0E+08
0.0E+00
0
5
10
15
20
25
30
35
PCR cycle
10
9
8
DNA copy number (log)
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
DNA copy number
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Copies of DNA=2N
7
6
5
4
3
2
1
0
0
5
10
15
20
25
30
35
PCR cycle
PCR reagent is the limiting factor!
PCR Phases
Plateau
Linear
Ethidium-Gel
detection
Log [DNA]
Exponential
Cycle #
Variable Linear Phase
Plateau Effect
SERIES OF 10-FOLD DILUTIONS
CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA
100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY
0
1
1
1
1
1
2
2
2
2
2
4
4
3
3
3
8
7
6
5
4
16
13
10
8
5
32
25
19
14
6
64
47
34
24
7
128
89
61
41
8
256
170
110
70
9
512
323
198
119
10
1,024
613
357
202
11
2,048
1,165
643
343
12
4,096
2,213
1,157
583
13
8,192
4,205
2,082
990
14
16,384
7,990
3,748
1,684
15
32,768
15,181
6,747
2,862
16
65,536
28,844
12,144
4,866
17
131,072
54,804
21,859
8,272
18
262,144
104,127
39,346
14,063
19
524,288
197,842
70,824
23,907
20
1,048,576
375,900
127,482
40,642
21
2,097,152
714,209
229,468
69,092
22
4,194,304
1,356,998
413,043
117,456
23
8,388,608
2,578,296
743,477
199,676
24
16,777,216
4,898,763
1,338,259
339,449
25
33,554,432
9,307,650
2,408,866
577,063
26
67,108,864
17,684,534
4,335,959
981,007
27
134,217,728
33,600,615
7,804,726
1,667,711
28
268,435,456
63,841,168
14,048,506
2,835,109
29
536,870,912
121,298,220
25,287,311
4,819,686
30
1,073,741,824
230,466,618
45,517,160
8,193,466
Effects
of
Efficienc
AFTER 1 CYCLE:
100% => 2.00x
90% =>y1.90x
1,200,000,000
1,000,000,000
800,000,000
600,000,000
400,000,000
200,000,000
80% => 1.80x
0
70% => 1.70x
0
10
AFTER N CYCLES:
fold increase =
(efficiency)n
1,200,000,000
100% EFF
90% EFF
80% EFF
70% EFF
AMOUNT OF DNA
1,000,000,000
800,000,000
600,000,000
400,000,000
200,000,000
0
0
10
20
30
PCR CYCLE NUMBER
10,000,000,000
100% EFF
90% EFF
80% EFF
70% EFF
AMOUNT OF DNA
1,000,000,000
100,000,000
10,000,000
1,000,000
100,000
10,000
1,000
100
10
1
0
10
20
PCR CYCLE NUMBER
30
SERIES OF 10-FOLD DILUTIONS
Anatomy of an Amplification Plot
Sample
∆Rn
Rn
Threshold
Baseline
No Template
Control (NTC)
CT
0
10
20
cycle number
30
40
Basic Knowledge on Statistics
•
Normal distribution
x 2
1
1
(
2
 )
f ( x) 
e
 2


is the mean
is the standard deviation
Variance
is
2



79
Example: The normal distribution is the most important
distribution in Statistics. Typical normal curves with
different sigma (standard deviation) values are shown
below.
• The standardized value of x is defined
as
z
x

• It is also called a z-score.
Central Limit Theorem
• A very important result in statistics that
permits use of the normal distribution for
making inferences (hypothesis testing and
estimation) concerning the population mean.
• If a variable x (with any distribution) has a
population mean x and standard deviation ,
then: the distribution of sample means (from
samples of size n taken from the population),
has the following distribution as n tends to

infinity:
X ~ N ( ,
)
n
n
Confidence Interval for the Mean
• A way of expressing the uncertainty in x
as an estimate of .
• 95% confidence interval says that on
average 95 % of the time, if you
estimate an interval for  this way, the
true value of  will be inside the interval.
Common “Z” levels of confidence
• Commonly used confidence levels are
90%, 95%, and 99%
Confidence
Level
80%
90%
95%
98%
99%
99.8%
99.9%
Z-value
(Z-score,
Critical value)
1.28
1.645
1.96
2.33
2.58
3.08
3.27
Confidence Interval
• So, we need to count the number of
standard deviations from the mean
xz
*

n
• But we don’t know .
When  is unknown
• In most cases knowledge of the true
variability of the measurement will not be
available.
• In these cases, we proceed by substituting 
with the sample standard deviation s:
n
s
s2 
x
i
 x
2
i 1
n 1
• And basing our inference on the t distribution
with n-1 degrees of freedom (where n is the
size of the sample).
t-statistic
Sample mean – Population mean
t=
s
n
s is the
sample
standard
deviation
The t distribution
• The t distribution is symmetric, and centered around
zero.
• It has “fatter” tails compared to the standard normal
distribution.
• The t distribution is defined by n-1 degrees of
freedom (n is the sample size).
Degrees of Freedom
• Essentially the number of independent
pieces of information provided by the
sample.
• Initially, every sample has n
independent pieces of information (as
many as the number of observations).
• If we know the first n-1 observations,
we can compute the nth one, and thus
there are n-1 independent pieces of
information.
Baseline
• Background or noise signal is often
normally distributed.
• Signal is meaningful only if it is higher
enough than the background signal.
• Baseline value is a single value or a
function to represent the background
signal.
• Baseline is the cycles used to calculate
the baseline value.
Baseline Set Too Low
Baseline Set Too High
Threshold
• At least 5 to 10 z-value higher than the
mean of the distribution of background
signal.
• Many competing factors: background
noise, stabilized regions in exponential
phase for all replicates, maximizing
sensitivity
DNA copy number (log)
Log phase
Level off/ plateau
2
4
8
16
32
PCR cycle (Ct)
DRn
• Rn (normalized reporter) is the fluorescence emission
intensity of the reporter dye multiplied by the
calibration factor.
• Rn+ is the Rn value of a reaction containing all
components (the sample of interest); Rn- is the Rn
value detected in NTC.
 DRn is the difference between Rn+ and Rn-. It is an
indicator of the magnitude of the signal generated by
the PCR.
 DRn is plotted against cycle numbers to produce the
amplification curves and gives the CT value.
Types of Quantification
• End-point quantification
– SNP typing
– Pathogen detection
• Absolute quantification
• Relative quantification
• Melting Curve (for Cyber Green I)
Absolute Quantification
Generating a standard
curve
•
Absolute/Relative quantification
Used serial diluted standards of known
concentration to generate a standard
curve.
•
Standard curve is a linear relationship
between the ct and the initial amount
amounts of RNA or cDNA.
•
This allows the determination of the
concentration of unknowns based on their
ct values
Rep 1
0 hrs
12 hrs pi
24 hrs pi
Rep 2
0 hrs
12 hrs pi
24 hrs pi
Rep 3
0 hrs
12 hrs pi
24 hrs pi
Pooled sample
•
•
Assumes that all standards and samples
have equal amplification efficiencies.
The concentrations of serial dilutions
should encompass all samples and stay
within the range that can be quantified
•
Dilution 1 (10-1)
Dilution 2 (10-2)
Dilution 3 (10-3)
Carry out runs in triplicates
Comparative CT (∆∆ CT) Method
• CT (target gene, control) – CT (endog.
refer. gene, control) = ∆CT,cont (control
tissue)
• CT (target gene, exp.) – CT (endog.
refer. gene, exp.) = ∆CT,exp
(experimental tissue)
• ∆CT,exp - ∆CT,cont = ∆∆CT
Target gene exp = 2
Target gene cont
(average ∆∆CT)
Comparative CT (∆∆ CT) Method with
Efficiency Correction
• ∆∆ CT assumes similar efficiencies
Target gene exp =(Etarget )∆CT target (cont - exp)
Target gene cont
(E
)∆CT ref (cont - exp)
cont
Comparative CT (∆∆ CT) Method with
Efficiency Correction
Exist/Non-Exist Assay
• Internal Positive Control (IPC)
• Four types of reactions
1. No Amplification Control (NAC) – blocked
IPC, to calculate IPC threshold
2. No Template Control (NTC) – to calculate
target threshold
3. Target
4. Unknown sample
Exist/Non-Exist Assay
• Statistical methods for choosing
thresholds
• Three possible outcomes
1. Target below threshold, IPC above
threshold  Minus
2. Target above threshold, IPC above
threshold  Plus
3. Target below threshold, IPC below
threshold  Undetermined
SNP Typing
• Using TaqMan
– One reporter for each allele
– k-means clustering
• Using Cyber Green I
– Primers with different lengths
– Melting curve examination
Thank you !