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© 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
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© 2009 Applied Biosystems
TaqMan® Sample-to-SNP™
One hour. One kit. Any sample
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© 2009 Applied Biosystems
野生型 (wt)
突变型 (mut)
等位基因鉴定机理
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© 2009 Applied Biosystems
After each cycle . . .
● Theoretically, amount of target in reaction tube doubles ● Reporter fluorescence increases by a proportional amount
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© 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
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© 2009 Applied Biosystems
SNP 研究策略
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© 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)
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)
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© 2009 Applied Biosystems
TaqMan® Sample-to-SNP™——One hour. One kit. Any Sample
任何样本
Contact Information
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© 2009 Applied Biosystems
高通量TaqMan SNP 分型平台 ——TaqMan OpenArray
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.
FAM™
VIC®
Homozygote for FAM™-specific allele
(G/G)
FAM™ VIC® Heterozygote (G/A)
VIC®
FAM™
Homozygote for VIC®-specific allele
(A/A)
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© 2009 Applied Biosystems
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© 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.
– 1:2 in VIC + FAM cluster
– 2:2 in FAM cluster
NTC
Heterozygotes
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© 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
Taqman® Sample-to-SNP™ SNPShot OpenArray™
Illumina Affymetrix
SOLiD
384
768
3000
300000
GB
Increasing Throughput
Data points
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© 2009 Applied Biosystems
技术流程/应用
Markers
• 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
100,000 10,000
SNPShot™
以实时PCR为基础的平台
1,000 100
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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
500,000
100,000
发现
10,000 1,000 100
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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
R 报告荧光 NFQ 无荧光淬灭基团 MGB 小沟结合物
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© 2009 Applied Biosystems
∆Tm决定探针杂交特异性
TAMRA MGB
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
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.
● 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
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© 2009 Applied Biosystems
Genotyping Throughput Landscape: AB Has a Broad Range of Options
● 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
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)