ABI SNP分型研究

TaqMan OpenArray基因分型系统
22
© 2009 Applied Biosystems
• 5uL 1500 rxns • 10uL 750 rxns
– 以基因组DNA 或PCR产物为模板 – 2条引物, 2条MGB探针 – FAM和VIC标记 – 终点读板实验
¾4,500,000 Pre-designed
¾160,000 Validated
¾70,000 cSNP
¾2,600 DME
3
© 2009 Applied Biosystems
SNP 研究意义
● 作为遗传图谱用于致病基因的定位 ● 应用于进化和种群多样性的研究，农业品种鉴定 ● 在药物基因组学、诊断学和生物医学研究中起重要作用 ● 个体化医疗和基因身份证
4
© 2009 Applied Biosystems
SNP 研究策略
or
or
or
or
+
Sample Prep: 5 minutes;
Multiple sample types
PCR About 60 minutes
Analysis
(Actual genotyping data from crude lysate from
whole blood)
Genotyping made simple!
17
© 2009 Applied Biosystems
No Compromise in Call Rate with DNA Lysate
DNA Lysate
Purified DNA
18
© 2009 Applied Biosystems
TaqMan® Sample-to-SNP™ Kit Contents
Assay Chemistry: 通过散点图显示结果
● 平台期终点的荧光信号的散点图： – VIC (allele 1) on X-axis – FAM (allele 2) on Y-axis
Homozygotes
● 每个数据集代表一种基因型：每种 纯合子、杂合子和无模板对照（ VTC）
– 1:1 in VIC cluster
– 1:2 in VIC + FAM cluster
– 2:2 in FAM cluster
NTC
Heterozygotes
13
© 2009 Applied Biosystems
450万个SNP分型试剂盒
– 187.5ul of 20 x mix – CD containing:
» Protocol » Product Insert » Assay information » Data sheet
(non-fluorescent quencher) NFQ
R
Q
Q
AGGCCTTGAGAGATAT MGB
(minor groove binder)
提高探针Tm值
R AGGCCTTGAQGAGATAT
| | | || | | | | | | | | | | |
GCTACACAGTCCGGAACTCTCTATAGCATCACAC
16
© 2009 Applied Biosystems
Universal Protocol for Any Sample
Buccal Swab
Mouse tail
Corn Leaf
FFPE
Sample types tested during development:
Blood, mouse liver, mouse ear clip, mouse tail, hair, nail, seeds, leaf tissue, FTA paper, blood cards, fin clips, fish scales, FFPE, buccal swabs.
14
© 2009 Applied Biosystems
实验分两步
PCR试剂 + 引物探针 + 2 ng样品 实时循环
终点读板
ABI 7300 7500 7900 StepOne定量 PCR仪
15
© 2009 Applied Biosystems
TaqMan® Sample-to-SNP™
One hour. One kit. Any sample
● Taqman MGB 探针 – TaqMan® Assays——低通量 – TaqMan® OpenArray™——超高通量
● 片断分析 – SNaPshot——中通量
● 测序 – 测序仪 – 下一代测序仪SOLID
5
© 2009 Applied Biosystems
Genotyping Throughput Landscape: AB Has a Broad Range of Options
500,000
100,000
发现
10,000 1,000 100
7
验证
候选基因 / 临床研究
筛选
100
1,000
10,000
© 2009 Applied Biosystems
Samples
100,000
AB公司基因分型产品技术平台
Markers
SOLiDTM 系统
500,000
以测序仪为基础的平台
AmpliTaq® Fast DNA Polymerase, UP
Ultra Pure DNA polymerase for improved detection of bacterial targets. This instant hot-start enzyme is inactive at room temperature so reactions can be set up on the benchtop.
● 包括单碱基的转换、颠换，以及单碱基的插入／缺失等
ATGCATGCATGCATGCATGC ATGCATGCATACATGCATGC
This G/A is a SNP
● SNP在人基因组中的发生频率比较高（不低于1%，低于1%算点突变）， 大约平均每100个碱基中就有一个多态位点，是继微卫星之后的第三代遗 传标记。有些SNP位点影响基因的功能，导致生物性状改变甚至致病
100,000 10,000
SNPShot™
以实时PCR为基础的平台
1,000 100
8
TaqMan® SNP Assays
TaqMan® OpenArray™
100
1,000
10,000
© 2009 Applied Biosystems
Samples
100,000
TaqMan MGB探针法——SNP分型的金标准
• Goal of project: To develop a genome-wide haplotype map for identifying haplotype blocks, common haplotypes and haplotype tag SNPs in these populations.
TaqMan® Sample-to-SNPTM Kit includes:
DNA Extract All Lysis Reagents
TaqMan® GTXpressTM Master Mix
Contains the Lysis Solution, an enhanced alkaline solution with the ability lyse virtually any type of sample , and the DNA Stabilizing Solution, which has been specifically formulated to preserve the DNA lysate and neutralize a wide range of inhibitors.
19
© 2009 Applied Biosystems
TaqMan® Sample-to-SNP™——One hour. One kit. Any Sample
任何样本
Contact Information
20
© 2009 Applied Biosystems
高通量TaqMan SNP 分型平台 ——TaqMan OpenArray
Tracking Dye
The GTXpress Master Mix contains a nonfluorescent tracking dye for easier loading on multiwell plates..
ROX™ dye
Passive internal reference based on proprietary ROX™ dye for increased precision on Applied Biosystems real-time instruments.
10
© 2009 Applied Biosystems
野生型 (wt)
突变型 (mut)
等位基因鉴定机理
11
© 2009 Applied Biosystems
After each cycle . . .
● Theoretically, amount of target in reaction tube doubles ● Reporter fluorescence increases by a proportional amount
R 报告荧光 NFQ 无荧光淬灭基团 MGB 小沟结合物
9
© 2009 Applied Biosystems
∆Tm决定探针杂交特异性
TAMRA MGB源自文库
Tm=65 Tm=65
Tm=59 Tm=49
(∆Tm = 6°C) (∆Tm = 16°C)
70°C
65°C
60°C
55°C
50°C
∆Tm = Tm 配对 - Tm 不配对 要有效地区分等位基因，退火/延伸温度必须落在∆Tm 区间内
Note: TaqMan® Sample-to-SNP™ and the GTXpress™ Master Mix are in product line SDR and plan code FJD (same as Gene expression and Genotyping master mixes)
FAM™
VIC®
Homozygote for FAM™-specific allele
(G/G)
FAM™ VIC® Heterozygote (G/A)
VIC®
FAM™
Homozygote for VIC®-specific allele
(A/A)
12
© 2009 Applied Biosystems
Taqman® Sample-to-SNP™ SNPShot OpenArray™
Illumina Affymetrix
SOLiD
384
768
3000
300000
GB
Increasing Throughput
Data points
6
© 2009 Applied Biosystems
技术流程/应用
Markers
• Provides a tool to enable researchers to find genes and genetic variations affecting health and disease.
HapMap SNPs will be the Golden Standard for association studies
ABI SNP分型研究平台
张远涛 Ph.D.
Application Specialist 4006209660 cnmcbsupport@lifetech.com
SNP (Single Nucleotide Polymorphisms)
单一碱基序列多态性
● 是由于单个核苷酸改变而导致基因组核苷酸水平上的变异引起的DNA序 列多态性
2
© 2009 Applied Biosystems
HapMap Project SNPs
• Validated SNP genotype data for > 3.2 million genome-wide SNPs in 44-60 individuals from:
• Yorubans - Ibadan, Nigeria, Africa (YRI) • Caucasian - North & West European ancestry; Utah, USA (CEU) • Japanese - Tokyo, Japan (JPT) • Chinese - Han Chinese; Beijing, China (CHB)