相对定量实验设计及条件优化

Endpoint Detection for Genotyping (Dual Color)
Roche Applied Science
Melting Curve Analysis for Product Identification, Mutation Detection, and Gene Scanning

其检测特异性由引物和探针共同来决定。 通过网络设计中心www.universalprobelibrary.com选取合适探针
9
Roche Applied Science
Universal ProbeLibrary
How does it work?
Universal
Randomly picked 8-9 mer oligonucleotide in transcriptome
> 519 500
> 253 500 > 199 000 > 134 000 > 61 500
96%
99% 98% 95% 94%
Rice
Zebrafish Anopheles Yeast
Oryza sativa
Danio rerio Anopheles gambiae Saccharomyces cerevisiae
> 898 500
> 630 000 > 193 000 > 42 000
98%
98% 98% 95%
总数
> 5 000 000
13
Roche Applied Science
主要内容

检测模式及应用


对照的设置
相对定量

如何选取看家基因 相对定量实验设置

实验评判标准
Roche Applied Science
14
Controls
Enable you to understand unexpected results and verify the experiment

No template control (NTC) contains all components of the PCR reaction except the template
4
Melting Curve Analysis
Established and New Applications
SYBR Green I for product identification
Labeled Probes for Genotyping
High Resolution Melting Dye for Gene Scanning
Prevalidated Hydrolysis Probes
+
Free Online Assay Design Center
= UPL
Roche Applied Science
8
Universal ProbeLibrary通用探针库
技术要点


把传统的水解探针由25-35nt减至8-9nt，报告基团用Fluorescein标 记，并使用了低背景的BHQ（Black hole quencher）淬灭基团 熔解温度取决于所用的LNA（locked nucleic acid technology，锁 定核苷酸）技术——核苷的2、4位形成环氧亚甲基桥结构，提高短探 针Tm值，使其Tm值高于引物达到qPCR的要求。
Roche Applied Science
5
Amplification Endpoint Analysis
for SNP Genotyping with Hydrolysis Probes

Dual color approach

Typical dye combinations: FAM –
VIC/Hex

LightCycler® 480 High Resolution Melting Master LightCycler® 480 RNA Master Hydrolysis Probes

RT step done within 3 min
Roche Applied Science
7
Universal Probe Library (UPL)

Suitable Detection Formats SYBR Green I Probe-based formats — Hydrolysis Probes, e.g. Universal ProbeLibrary Probes (UPL) — Hybridization Probes
Roche Applied Science

to indicate false positive results due to DNA in the RNA sample

Internal control control sequence is amplified in the same reaction as the target and detected with a different dye (duplex approach) e.g.; for samples in bacteriology/virology to verify the PCR reaction
主要内容

检测模式及应用


对照的设置
相对定量

如何选取看家基因 相对定量实验设置
Within the human transcriptome
Each probe can hybridize to over 7000 expressed gene, each transcript could be detected by at least 16 probes.
Roche Applied Science

for qPCR with hydrolysis probes, incl. endpoint genotyping
LightCycler® 480 Genotyping Master

for melting curve-based genotyping
using HybProbe or SimpleProbe probes

Color compensation not required
6
Roche Applied Science
LightCycler® 480 Reagents
Optimized for each Application

LightCycler® 480 SYBR Green I Master LightCycler® 480 Probes Master

For initial assay validation (specificity, sensitivity, efficiency
determination)

Negative control is a sample that is negative for the target sequence of the assay

Detection of contaminating nucleic acid Detection of primer dimer formation in SYBR Green I assays

Positive control is a sample that is positive for the target sequence of the assay
T m 10°C 26°C
DNA 8-mer 5’-tgctggtg-3’ LNA 8-mer 5’-TGCTGGTG-3’
UPL probes have Tm compatible with real-time PCR
UPL probes show excellent single-mismatch discrimination
3
Amplification Analysis
for Quantification and Qualitative Detection

Typical Applications Detection and quantification of bacteria and viruses Quantification of gene expression
10
Universal Probe Library
LNA technique enable UPL Higher TM
Probe Target Perfect match 3’-acgaccac5’
Tm= 35°C Tm= 71°C
Single mismatch 3’-acggccac-5’ Tm= 25°C Tm= 45°C
Roche Applied Science
11
Primer & Probe design
Probe Finder Software - Specificity
Sepcificity is abtained by the comnination of primers and the probe.
primer
qPCR实验设计及条件优化
Roche Applied Science
1
主要内容

检测模式及应用

Hale Waihona Puke Baidu
对照的设置
相对定量

如何选取看家基因 相对定量实验设置

实验评判标准
Roche Applied Science
2
Real-Time PCR Experiments
Data Analysis depending on Application
Real-time PCR is the combination of PCR and detection within one reaction v
Dye 1 Dye 2
Amplification Analysis for Quantification and Qualitative Detection
覆盖率
99% 99% 98%
Primates
Drosophila Arabidopsis C. elegans Maize
Pan troglodytes
Drosophila melanogaster Arabidopsis thaliana Caenorhabditis elegans Zea mays
What is it?
Universal ProbeLibrary (UPL) — a unique combination of prevalidated hydrolysis probes and free online assay design that allows you to design custom gene expression assays to quantify virtually any transcript in any genome.
To verify the specificity of the assay Roche Applied Science

15
Controls,

continued
RT (reverse transcriptase) minus control for gene expression assays. Prepare a RT minus control by omitting the addition of reverse transcriptase to the cDNA synthesis reaction, or use your untranscribed sample RNA
UPL probe
Roche Applied Science
12
Universal ProbeLibrary
Only 165 probes for over 5 billion assays 种 属 实验数量
Human Mouse Rat Homo sapiens Mus musculus Rattus norvegicus > 639 500 > 509 500 > 364 000

as a control for the sample preparation if added during NA isolation, and/or as a control for the amplification reaction (PCR inhibition)
16
Roche Applied Science