Lec 9 基因芯片技术
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第九章 基因芯片 /DNA 微阵列
Gene Chip /DNA Microarray
1
一、基因芯片简介
Introduction to gene chip
二、基因芯片工作原理
How to work for gene chip
三、基因芯片的应用
Application to gene chip
2
一、基因芯片简介
Personalized medicine
Developmental Biology
Toxicology
Prediction of Drug Efficacy/Toxicity
12
二、基因芯片工作原理
How to work for gene chip
• 基因芯片的工作流程
(workflow) • 基因芯片的设计和制备 (design and preparation) • 杂交信号的检测 (hybridized signal detection)
23
例一： 例一：基于芯片的基因测序
24
基因芯片流程（ 基因芯片流程（二）
4. 芯片杂交 芯片杂交（将用Cy3和Cy5荧光标记的对照组 和实验组的cDNA 等量混合，与芯片进行杂交） 5. 芯片扫描 芯片扫描（采用激光扫描仪，分别用532nm 和635nm波长激光扫描芯片，对于每张芯片， 得到Cy3和Cy5通道两幅图象）
10
Uses and Applications
• Gene Chip used in wide range of genomic analyses, such as: - gene expression detection - drug discovery - mutation detection • Diverse fields from health care to environmental sciences
22
Probe design
Target seq：---TCACGCCATAAATCAAATAGGTTTGGTCCTAGCCT--Compl seq：---AGTGCGGTATTTAGTTTATCCAAACCAGGATCGGA--： Probe Probe Probe Probe Probe Probe 1：---AGTGCGGTATT 2：-------CGGTATTTAGTT 3：----------- TTTAGTTTATCC 4：-----------------TTTATCCAAACC 5：------------------------AAACCAGGATC 6：----------------------------CAGGATCGGA
emission
cy3 cy5
510 nm
Differential dye incorporation 562 nm cy5 less well than cy3 Light sensitivity: cy5 more easily degraded
emission
cy3
26
Cy3 and Cy5-labeled DNA
Target
AAA AAA AAA Control AAA AAA AAA
RNA
Cy5
RT
Cy3
cDNA
27
Fluorophore Cy3 and Cy5 （荧光素） 荧光素）
cy3 and cy5: Commonly used dyes
cy5
664 nm
4
DNA Chip
• genes or gene fragments attached to a chip (glass) • Two dyes to label sample • Hybridized to chip • Image analyzed Tens of thousands of spots
5
基因芯片
基因芯片是将大量DNA或寡核苷酸密集排列 所形成的探针阵列固定于支持物上，再与标记的 数以千记的DNA片段同时进行杂交处理，通过检 测每个探针分子的杂交信号强度进而获取样品分 子的数量和序列信息。
6
The Beginnings of Microarray Technology
Lockhart et al., 1996 Nature Biotechnology “Expression monitoring by hybridisation to high-density oligonucleotide arrays” Schena et al., 1995 Science “Quantitative monitoring of gene expression patterns with a complementary DNA microarray”
18
Expressed Genes = mRNA
DNA
transcription
High throughput protein assays complicated
mRNA
translation
We measure transcript level!!!
Is a gene expressed? Is protein produced
Introduction to gene chip Synonymous（同义名） （同义名）
Gene chip DNA microchip DNA array DNA microarray Oligonucleotide array
3
What is DNA chip
1.DNA chip consists of 100 - 1 million DNA probes attached to a surface of 1x1cm2 chip. 2.By hybridisation, they can detect DNA or RNA 3. If the hybridised DNA or RNA is labelled fluorescently it can be quantified by scanning of the chip.
28
cDNA spotted microarrays
பைடு நூலகம்29
DNA microarrays can be manufactured by: • Photolitography (Affymetrix, Febit, Nimblegen) • Inkjet (Agilent, Canon) • Robot spotting (many providers)
17
Spotted array experiment
1. Prepare sample.
Control
4. Print microarray.
Test
2. Label with fluorescent dyes.
5. Hybridize to microarray.
3. Combine cDNAs. 6. Scan.
7
核酸体外杂交技术
8
Why DNA Chip?
• Functional Genomics : get information about genes that is unavailable from sequence • Understand how cells/organisms react to external stimuli • Understand gene regulation networks • Determine what makes the difference between healthy and diseased tissue • Simply do 15,000 Northern blots at a time
13
The blueprint of gene chip development
Target determination Probe sequence determination Test sample prep. Probe array prep. Detection device
Hybridized signal detection & data analysis
14
基因芯片研制的总体蓝图
研制方向的确定 基因组序列分析与待检基 因探针序列的确定 检测样品 的制备 检测设备的 研制
探针阵列的 准备
杂交检测与数据分析
15
基因芯片的相关技术示意图
提出问题 芯片设计 芯片制作 点样方法 ● 在片合成
●
表达差异分析 ●多态性分析 ●再测序
●
生物信息学 ●数学优化 ●数据库
11
Uses and Applications
Pathway mapping Target identification
Gene screening
Disease characterization
Mechanism of Action Studies
Molecular Diagnosis of Disease
Protein
Ideally measure protein levels
19
Expressed Genes = Currently Transcribed
Extract total RNA Isolate mRNA’s mRNA mRNA
20
Sample preparation
• Testing objects:
25
Spotted array experiment
1. Prepare sample.
Target
4. Print microarray.
Reference
2. Label with fluorescent dyes.
5. Hybridize to microarray.
3. Combine cDNAs. 6. Scan.
●
试样处理
PCR扩增 扩增 ●靶基因标记
●
芯片杂交
实际应用
数据分析
杂交检测
16
基因芯片流程（ 基因芯片流程（一）
1. 实验设计 2. 样品制备 样品制备（指mRNA或总RNA样品，包括对 照组和实验组。将mRNA或总RNA分别进行 逆转录生成cDNA，然后将对照组和实验组 cDNA分别标记Cy3和Cy5荧光信号） 3. 芯片制备 芯片制备（寡核苷酸探针或 cDNA探针，包 括PCR，纯化，点样等步骤）
transcription complementation
DNA
(gene) CCTT
mRNA
GGAA
cDNA
experimental
CCTT
controlled
Referential cDNA
21
Creating Targets
mRNA
Reverse Transcriptase
cDNA
PCR Amplification of DNA In Vitro transcription to create cRNA
9
Functional Genomics
• There may be 100,000 different transcripts in human cells • We only have sound information on 12,000 genes • All cells have the same genome, but there are more than 200 cell types in a single organism • Gene expression determined the cell type (neuron, lymphocyte, fibroblast etc) and directs development of an organism (by spatial/temporal patterns) • DNA chip technology promises to solve such unanswered questions
30
DNA Array Manufacturing Process
Treat substrate with chemically protected linker molecules Selectively expose array sites to light Flush chip’s surface with solution of protected A, C, G, T
Repeat last two steps until desired probes are synthesized
Gene Chip /DNA Microarray
1
一、基因芯片简介
Introduction to gene chip
二、基因芯片工作原理
How to work for gene chip
三、基因芯片的应用
Application to gene chip
2
一、基因芯片简介
Personalized medicine
Developmental Biology
Toxicology
Prediction of Drug Efficacy/Toxicity
12
二、基因芯片工作原理
How to work for gene chip
• 基因芯片的工作流程
(workflow) • 基因芯片的设计和制备 (design and preparation) • 杂交信号的检测 (hybridized signal detection)
23
例一： 例一：基于芯片的基因测序
24
基因芯片流程（ 基因芯片流程（二）
4. 芯片杂交 芯片杂交（将用Cy3和Cy5荧光标记的对照组 和实验组的cDNA 等量混合，与芯片进行杂交） 5. 芯片扫描 芯片扫描（采用激光扫描仪，分别用532nm 和635nm波长激光扫描芯片，对于每张芯片， 得到Cy3和Cy5通道两幅图象）
10
Uses and Applications
• Gene Chip used in wide range of genomic analyses, such as: - gene expression detection - drug discovery - mutation detection • Diverse fields from health care to environmental sciences
22
Probe design
Target seq：---TCACGCCATAAATCAAATAGGTTTGGTCCTAGCCT--Compl seq：---AGTGCGGTATTTAGTTTATCCAAACCAGGATCGGA--： Probe Probe Probe Probe Probe Probe 1：---AGTGCGGTATT 2：-------CGGTATTTAGTT 3：----------- TTTAGTTTATCC 4：-----------------TTTATCCAAACC 5：------------------------AAACCAGGATC 6：----------------------------CAGGATCGGA
emission
cy3 cy5
510 nm
Differential dye incorporation 562 nm cy5 less well than cy3 Light sensitivity: cy5 more easily degraded
emission
cy3
26
Cy3 and Cy5-labeled DNA
Target
AAA AAA AAA Control AAA AAA AAA
RNA
Cy5
RT
Cy3
cDNA
27
Fluorophore Cy3 and Cy5 （荧光素） 荧光素）
cy3 and cy5: Commonly used dyes
cy5
664 nm
4
DNA Chip
• genes or gene fragments attached to a chip (glass) • Two dyes to label sample • Hybridized to chip • Image analyzed Tens of thousands of spots
5
基因芯片
基因芯片是将大量DNA或寡核苷酸密集排列 所形成的探针阵列固定于支持物上，再与标记的 数以千记的DNA片段同时进行杂交处理，通过检 测每个探针分子的杂交信号强度进而获取样品分 子的数量和序列信息。
6
The Beginnings of Microarray Technology
Lockhart et al., 1996 Nature Biotechnology “Expression monitoring by hybridisation to high-density oligonucleotide arrays” Schena et al., 1995 Science “Quantitative monitoring of gene expression patterns with a complementary DNA microarray”
18
Expressed Genes = mRNA
DNA
transcription
High throughput protein assays complicated
mRNA
translation
We measure transcript level!!!
Is a gene expressed? Is protein produced
Introduction to gene chip Synonymous（同义名） （同义名）
Gene chip DNA microchip DNA array DNA microarray Oligonucleotide array
3
What is DNA chip
1.DNA chip consists of 100 - 1 million DNA probes attached to a surface of 1x1cm2 chip. 2.By hybridisation, they can detect DNA or RNA 3. If the hybridised DNA or RNA is labelled fluorescently it can be quantified by scanning of the chip.
28
cDNA spotted microarrays
பைடு நூலகம்29
DNA microarrays can be manufactured by: • Photolitography (Affymetrix, Febit, Nimblegen) • Inkjet (Agilent, Canon) • Robot spotting (many providers)
17
Spotted array experiment
1. Prepare sample.
Control
4. Print microarray.
Test
2. Label with fluorescent dyes.
5. Hybridize to microarray.
3. Combine cDNAs. 6. Scan.
7
核酸体外杂交技术
8
Why DNA Chip?
• Functional Genomics : get information about genes that is unavailable from sequence • Understand how cells/organisms react to external stimuli • Understand gene regulation networks • Determine what makes the difference between healthy and diseased tissue • Simply do 15,000 Northern blots at a time
13
The blueprint of gene chip development
Target determination Probe sequence determination Test sample prep. Probe array prep. Detection device
Hybridized signal detection & data analysis
14
基因芯片研制的总体蓝图
研制方向的确定 基因组序列分析与待检基 因探针序列的确定 检测样品 的制备 检测设备的 研制
探针阵列的 准备
杂交检测与数据分析
15
基因芯片的相关技术示意图
提出问题 芯片设计 芯片制作 点样方法 ● 在片合成
●
表达差异分析 ●多态性分析 ●再测序
●
生物信息学 ●数学优化 ●数据库
11
Uses and Applications
Pathway mapping Target identification
Gene screening
Disease characterization
Mechanism of Action Studies
Molecular Diagnosis of Disease
Protein
Ideally measure protein levels
19
Expressed Genes = Currently Transcribed
Extract total RNA Isolate mRNA’s mRNA mRNA
20
Sample preparation
• Testing objects:
25
Spotted array experiment
1. Prepare sample.
Target
4. Print microarray.
Reference
2. Label with fluorescent dyes.
5. Hybridize to microarray.
3. Combine cDNAs. 6. Scan.
●
试样处理
PCR扩增 扩增 ●靶基因标记
●
芯片杂交
实际应用
数据分析
杂交检测
16
基因芯片流程（ 基因芯片流程（一）
1. 实验设计 2. 样品制备 样品制备（指mRNA或总RNA样品，包括对 照组和实验组。将mRNA或总RNA分别进行 逆转录生成cDNA，然后将对照组和实验组 cDNA分别标记Cy3和Cy5荧光信号） 3. 芯片制备 芯片制备（寡核苷酸探针或 cDNA探针，包 括PCR，纯化，点样等步骤）
transcription complementation
DNA
(gene) CCTT
mRNA
GGAA
cDNA
experimental
CCTT
controlled
Referential cDNA
21
Creating Targets
mRNA
Reverse Transcriptase
cDNA
PCR Amplification of DNA In Vitro transcription to create cRNA
9
Functional Genomics
• There may be 100,000 different transcripts in human cells • We only have sound information on 12,000 genes • All cells have the same genome, but there are more than 200 cell types in a single organism • Gene expression determined the cell type (neuron, lymphocyte, fibroblast etc) and directs development of an organism (by spatial/temporal patterns) • DNA chip technology promises to solve such unanswered questions
30
DNA Array Manufacturing Process
Treat substrate with chemically protected linker molecules Selectively expose array sites to light Flush chip’s surface with solution of protected A, C, G, T
Repeat last two steps until desired probes are synthesized