Transcript （from基因公司黄文晋）.ppsx

疾病相关基因鉴定的技术
路线与方法学进展
基因有限公司市场部
黄文晋
Genomics
Sequencing
DNA
Polymorphism, eg SNP…
Epigenetics
…
Transcriptomics
DNA Microarrays
(genechips)
…
Proteomics
RNA
2D-MS
…
Protein
疾病相关基因鉴定的技术路线
1，1)通过全基因组扫描分析，首先鉴定与特定疾病相关的
特定染色体区域
2)然后对特定染色体区域进行精细扫描,鉴定特定疾病相
关的基因
2，通过全基因组扫描分析，直接鉴定特定疾病相关的基因
关联分析（association）
疾病相关基因鉴定的方法
1，1)通过全基因组扫描分析，首先鉴定与特定疾病相关的
特定染色体区域
a, 全基因组STR扫描：连锁分析（linkage）
b, Affymetrix全基因组10/50k SNP芯片扫描：连锁分析
e, Affymetrix全基因组10/50/100/250/500k SNP芯片扫描：
分析基因组片段的缺失，扩增，LOH
c, 核型分析：分析易位,缺失，插入,倒位,复制/扩增
d, 比较基因组杂交：分析基因组片段的缺失，扩增
2)然后对特定染色体区域进行精细扫描,鉴定特定疾病相
关的基因
a,特定染色体区域加密STR分析
b,特定染色体区域中通量SNP分析: Pyrosequencing技术
定量PCR HRM技术
c,特定染色体区域高通量SNP分析:
Affymetrix MegAllele技术(1500-20000 SNP)
2，通过全基因组扫描分析，直接鉴定特定疾病相关的基因
关联分析（association）：Affymetrix 100/250/500K SNP芯片
Identifying disease genes by linkage:
STR（ ~400 str markers）
2
-2
0
NPL score (N~(0,1)
4
1. Whole genome STR scan
0
50
100
150
200
2
1
Distance (cM)
2. Fine-mapping
With more markers:
SNP或加密STR
5-10 Mb linkage region
3. Candidate gene analysis
Identifying disease genes by linkage:
SNP (~10k or 50k snp markers)
2
-2
0
NPL score (N~(0,1)
4
1. Whole genome SNP scan
0
50
100
150
200
2
1
Distance (cM)
2. Fine-mapping
With more markers:
SNP或加密STR
5-10 Mb linkage region
3. Candidate gene analysis
Science 26 May 2006
Vol. 312. no. 5777, pp. 1228 - 1230
Pituitary Adenoma Predisposition Caused by Germline Mutations
in the AIP Gene
50k SNP芯片
连锁分析鉴定
PAP相关染色
体区域
Identifying disease genes by Association: SNP
(~100k or 250k or 500k snp markers)
1. Whole genome SNP scan
Candidate gene
2. Candidate gene analysis
500k SNP芯片关联分析鉴定记忆相关基因的SNP
Pyrosequencing技术分析该基因SNP在两组人不同个体中的基因型
科学家利用100K SNP芯片技术,通过对96个病人和50个正常人的全基因组SNP扫
描,发现complement factor H (CFH)基因的一个内含子内的一个SNP与AMD有很强
的关联性.通过生物信息学分析,确认一个完全位于CFH基因内的haplotype block是
AMD的risk haplotype.作者对96个人的CFH基因进行再测序,发现CFH基因的一个
导致CFH 402位氨基酸改变(tyrosine-histidine)的一个SNP可能是疾病原因.该论文
发表在Science. 2005 Apr 15;308(5720):385-9.03-04年有6篇国际论文在寻找AMD
的相关基因,由于这6篇论文都用基于家系的全基因组STR连锁扫描技术,因此没
有找到AMD相关基因,只证明1号染色体的1q31区域与AMD有关。SNP芯片找到
的AMD相关基因CFH就在1q31区域.
科学家利用100K SNP芯片技术进行694个人的全基因组关联分析,发现INSIG2基
因附近的一个SNP与儿童和成人的肥胖病关联.该SNP位于INSIG2基因转录起始
位点上游10kb处.该论文发表在Science 14 April 2006: Vol. 312. no. 5771, pp. 279
-283.以往对小鼠和人的连锁分析已经暗示INSIG2基因区域是肥胖病的一个因
-素,而只有SNP芯片技术可以直接找到与肥胖相关的SNP.
• 100/250/500K SNP基因芯片技术可以进行基于
Case-Control或家系的全基因组关联分析
(association),可以从全基因组DNA水平上直接
找到与疾病相关的基因.在该技术出现之前,没
有任何其他技术可以进行全基因组的关联分析.
• 传统的全基因组STR技术可以做家系的连锁分
析(linkage),但只能找到与疾病相关的某段染色
体区域;由于某段染色体区域可以包含数以百计
个基因,因此还必须通过对该区域的精细扫描
(fine mapping)技术才能找到与疾病相关的基
因.
• 10/50K SNP芯片可以做家系的连锁分析,与STR
技术比较,其优势是不会漏选某些与疾病相关的
染色体区域
Affymetrix全基因组10/50/100/250/500k SNP芯片扫描
分析基因组片段的缺失，扩增，LOH
研究者利用Affymetrix SNP微阵列进行了肿瘤LOH等研究,包括乳
腺癌2-6, 膀胱癌7,8, 前列腺癌9,10, 骨癌11, 口腔癌12 和肺癌1,3,13。
1. Lindblad-Toh, K. et al. Loss-of-heterozygosity
analysis of small-cell lung carcinomas using
single-nucleotide polymorphism arrays.
Nat Biotechnol 18, 1001-5 (2000).
2. Huang, J. et al. Whole genome DNA copy number
changes identified by high density oligonucleotide
arrays. Hum Genomics 1, 287-99 (2004).
3. Zhao, X. et al. An integrated view of copy number and
allelic alterations in the cancer genome using single
nucleotide polymorphism arrays. Cancer Res 64, 3060-71
(2004).
4. Schubert, E. L. et al. Single nucleotide polymorphism
array analysis of flow-sorted epithelial cells from
frozen versus fixed tissues for whole genome analysis
of allelic loss in breast cancer.
Am J Pathol 160, 73-9 (2002).
5. Wang, Z. C. et al. Loss of heterozygosity and its
correlation with expression profiles in subclasses of
invasive breast cancers. Cancer Res 64, 64-71 (2004).
6. Paez, J. G. et al. Genome coverage and sequence
fidelity of phi29 polymerase-based multiple strand
displacement whole genome amplification.
Nucleic Acids Res 32, e71 (2004).
7. Primdahl, H. et al. Allelic imbalances in human bladder
cancer: genome-wide detection with high-density singlenucleotide polymorphism arrays.
J Natl Cancer Inst 94, 216-23 (2002).
8. Hoque, M. O., Lee, C. C., Cairns, P., Schoenberg, M. &
Sidransky, D. Genome-wide genetic characterization of
bladder cancer: a comparison of high-density singlenucleotide polymorphism arrays and PCR-based
microsatellite analysis. Cancer Res 63, 2216-22 (2003).
9. Lieberfarb, M. E. et al. Genome-wide loss of
heterozygosity analysis from laser capture
microdissected prostate cancer using single nucleotide
polymorphic allele (SNP) arrays and a novel
bioinformatics platform dChipSNP.
Cancer Res 63, 4781-5 (2003).
10. Dumur, C. I. et al. Genome-wide detection of LOH in
prostate cancer using human SNP microarray technology.
Genomics 81, 260-9 (2003).
11. Wong, K. K. et al. Allelic imbalance analysis by highdensity single-nucleotide polymorphic allele (SNP)
array with whole genome amplified DNA.
Nucleic Acids Res 32, e69 (2004).
12. Zhou, X., Mok, S. C., Chen, Z., Li, Y. & Wong, D. T.
Concurrent analysis of loss of heterozygosity (LOH)
and copy number abnormality (CNA) for oral
premalignancy progression using the Affymetrix 10K
SNP mapping array. Hum Genet 115, 327-30 (2004).
13. Janne, P. A. et al. High-resolution single-nucleotide
polymorphism array and clustering analysis of loss of
heterozygosity in human lung cancer cell lines.
Oncogene 23, 2716-26 (2004).
14. Rauch, A. et al. Molecular karyotyping using an SNP array for
genomewide genotyping. J Med Genet 41, 916-22 (2004).
以上仅是05年前的部分文献。
500k SNP芯片鉴定全基因组拷贝数变异（Copy number variation，CNV）
Integrative genomic analyses identify MITF as a lineage survival
oncogene amplified in malignant melanoma.
Garraway LA, Widlund HR, et al.
Nature. 436(7047):117-22, July 7, 2005.
Nature. 436(7047):117-22, July 7, 2005一文利用AFFYMETRIX 100K
SNP芯片,扫描58个NCI60细胞系,在恶性黑色素瘤细胞系中发现3号
染色体3p13–3p14区域(约3.5M)扩增.结合AFFYMETRIX表达谱芯片
技术,发现这段染色体内的转录因子MITF基因表达异常高.进一步的
功能实验确认MITF基因DNA拷贝数增加和随后mRNA表达量增加
与黑色素瘤有关.
Nat Genet. 2005 Oct;37(10):1099-103一文利用100K SNP芯片证明了传代
培养的人胚胎干细胞发生基因组/染色体突变包括染色体的扩增和缺失,
这些变化类似癌症细胞中DNA的突变.这些突变包括大的染色体区域的
扩增或缺失(如整个17q臂的扩增)和离散的变化(如含MYC癌基因的一段
2M染色体区域的扩增).
Cultured human embryonic stem cell (hESC) lines are an invaluable
resource because they provide a uniform and stable genetic system for
functional analyses and therapeutic applications. Nevertheless, these
dividing cells, like other cells, probably undergo spontaneous mutation
at a rate of 10(-9) per nucleotide. Because each mutant has only a few
progeny, the overall biological properties of the cell culture are not
altered unless a mutation provides a survival or growth advantage.
Clonal evolution that leads to emergence of a dominant mutant
genotype may potentially affect cellular phenotype as well. We
assessed the genomic fidelity of paired early- and late-passage hESC
lines in the course of tissue culture. Relative to early-passage lines,
eight of nine late-passage hESC lines had one or more genomic
alterations commonly observed in human cancers, including
aberrations in copy number (45%), mitochondrial DNA sequence (22%)
and gene promoter methylation (90%), although the latter was
essentially restricted to 2 of 14 promoters examined. The observation
that hESC lines maintained in vitro develop genetic and epigenetic
alterations implies that periodic monitoring of these lines will be
required before they are used in in vivo applications and that some
late-passage hESC lines may be unusable for therapeutic purposes.
Nat Genet. 2005 Oct;37(10):1099-103一文除利用100K SNP芯片证明了传代培
养的人胚胎干细胞发生基因组/染色体突变外,也利用AFFYMETRIX 线粒体
DNA再测序芯片鉴定了干细胞传代培养引发的线粒体DNA发生的一些突变.
基因组/染色体突变分析的经典方法
核型分析(Karyotyping)
比较基因组杂交(CGH)
以上方法发现染色体畸变
荧光原位杂交(FISH)
畸变染色体区域DNA制备的探针作FISH可以
对个体进行染色体畸变分析
• 1 genome mutations基因组突变: changes in
chromosome number染色体数目变化
• 2 chromosome mutations染色体突变: changes in
chromosome structure染色体结构变化
large, microscopically visible chromosome
anomalies
基因组突变: 染色体数目变化
• Trisomies eg Trisomy 21
Monosomies eg Monosomy 14, 16
Sex Chromosome number changes eg 47, XXY
Gross hyperdiploidy, near haploidy, triploidy eg in
leukemias, abnormal fetuses
• Techniques: Karyotyping, conventional cytogenetics
• Functional consequence: some are not viable, eg
monosomy 21, 14 and trisomy 14.
Phenotypic changes in others depend on the
particular genes involved.
Downs’ Syndrome
47,XY+21
染色体突变: 染色体结构变化
• Translocations [易位], Large Deletions，Insertions [大片
段DNA缺失，插入], Inversions [倒位],
Duplications/Amplifications [复制/扩增]
• Techniques:
Conventional cytogenetics and molecular cytogenetics:
FISH, M-FISH, CGH
Molecular: PFGE, Southern blotting, Northern Blotting
Fluorescence Dosage analysis
• Functional consequences: depend on the extent and
location of change and the particular genes involved
易位
can have several effects. The effects depend on the genes involved,
precise breakpoints and whether balanced or not. Unbalanced
rearrangements result in loss of chromosome material.
• position effects: due to acquisition of a novel transcriptional promoter
or enhancer sequences. eg the gene for a T-cell receptor or an
immunoglobin protein comes to lie near a proto-oncogene thereby
activating it eg c-myc in Burkitt’s lymphoma t(8:14).
• structural change: eg break and rejoin creates fusion gene encoding a
chimeric protein eg t(9:22) the Philadelphia chromosome in chronic
myelogenous leukemia giving rise to bcr-abl with tyrosine kinase
activity and transforming ability.
• rearrangements involving the X chromosome: the spreading of
‘inactivation’ from the translocated X chromosome allows genes at
some distance form the breakpoint to become inactivated.
易位
t(7;11)
倒位
• could give rise to fusion genes eg inverted
16 in leukemia (AML M-4)
大片段DNA缺失,插入
• eg loss of tumour suppressor genes eg
13q14 RB1, insertions eg in leukemias
giving rise to chimeric genes depending on
breakpoints
大片段DNA缺失Del 10q
扩增
• eg overexpression of a proto-oncogene eg
N-myc in neuroblastoma due to
amplification of region 2p24
基因组/染色体突变分析经典方法
Cytogenetics
Starting
material
Technique
Basis
Living cells
G-Banding
Differential
staining:
AT rich
FISH
Hybridisation
CGH
Global;
hybridisation
基因组/染色体突变分析经典方法
DNA Time
Resolution Probe
Karyotyping ++++ slow
5-10 Mb
No
FISH
++
slow
1-5 Mb
Yes
BACCGH
++
medium 3 Mb（使用 No
高端分析软件
如AI才能达到
该分辨率）
About AI
• 全英国的细胞遗传及基因研究中心
• 通过 FDA 认证，唯一一个可发临床报告的公司
• 拥有大于80% 的市场占有量
• 相关的应用文献居世界第一
系统的组成：
• 显微镜
• CCD
• 分析软件
• 电脑平台
可用于分析的图像类型
•
•
•
•
染色体核型分析 (Karyotyping)
荧光原位杂交（FISH）
24色荧光原位杂交（M-FISH）
高分辨率比较基因组杂交（ HR-CGH ）
为什么选择AI遗传工作站？
1 品牌优势
• 通过 FDA 认证，唯一一个可发临床报告的公司
• 拥有大于80% 的市场占有量
• 相关的应用文献居世界第一
2 界面优势
1）操作界面整体性强，易于学习，操作方便，利于
升级。
2）Gallery的功能。
3）同一窗口下6幅图像平铺显示及处理功能。
4）有显示图像来源的标签，避免打开多个Case时的
混淆。
5）能在同一界面下打开不同软件模块的图像。
3 核型分析软件
• FDA认证
• 自动/手动分割染色体重叠、交叉和连接；
• 应用不同的数阵显示格式使条带明晰；
• 染色体放大、缩小、旋转、翻转、镜向翻转、拉直；
• 可以创建染色体分类体系，并对已有分类器进行训练，分类更准确；
• 可编辑定义染色体组型条带；
• 可识别G、R、Q、C、DAPI的显带分型；
• G带有400、550、850条带的分类标准、R带有400、550条带的标准；
• 可自动或人工干预识别着丝粒位置；
• 可显示染色体二维和三维图像
4 荧光原位杂交软件
• 多达9通道颜色采集
• 可对目标探针明暗度单独或全部进行调节修饰
• 可计算荧光信号的数目、强度、面积、距离等
• 可做FISH图
• 可测量FLPTER值，对信号进行定位
• 可对采集图像重新扣除背景
4，24色荧光原位杂交
软件开放，支持多种M-FISH试剂
自动提示染色体易位条带来源
自动和人工干预修正染色体杂交着色均衡度
对每一条染色体的多种杂交探针进行着色的独
立定位显示和综合的多彩色剖面图显示
• 新增Cleanup功能，单键修整假色差
• P/Q臂的独立标注及分类方法
•
•
•
•
5 高分辨率比较基因组杂交 HR-CGH
•
•
•
•
AI的专利产品，最小分辨率3Mb，较传统高3-4倍
仅需要传统用量的20%-30%
染色体分析同时适用于着丝粒和端粒
对于不同的染色体区段有更高的灵敏度，可修正 1P、
16、17、22号染色体
• 仅用99.5%的标准即可得到结果
6 试剂、技术支持、售后服务和持续性发展
• AI专业性，持续性发展 3.8中文版软件
• 基因公司的产品售后
• 试剂及实验技术的支持
对特定染色体区域进行精细扫描鉴定特定疾病相关基因
a,特定染色体区域加密STR分析
b,特定染色体区域中通量SNP分析: Pyrosequencing技术
定量PCR技术
c,特定染色体区域高通量SNP分析:
Affymetrix MegAllele技术(1500-20000 SNP)
Pyrosequencing™
PPi
ATP
Detection of the light
light
time
Nucleotides dispensed sequentially
A
GG
C
A
G
C
A
G
11
10
9
8
7
6
5
4
3
2
1
0
-1
T
A
G
The sequence in this pyrogram™ is AGGCAG
Clearly distinguish heterozygotes vs
homozygotes
ACTGCCT
Heterozygote
A/GCTGCCT
GCTGCCT
Pyrosequencing
不依赖电泳、不依赖DNA荧光标记/激发/检测技术的高通量、快速、
自动化DNA序列分析技术。比常规序列分析技术更适合分子临床
诊断。
适合SNPs基因型鉴定、频率分析和DNA甲基化程度分析的技术。
Pyrosequencing™ for SNP
analysis
SNP
Discovery
SNP
Confirmation
Allele
Frequency
SNP
Relevance
SNP
Diagnostics
New SNPs
• Sanger
• dHPLC
• SSCP
• In silico
Verify
SNP as
true SNP
Frequency
of SNP in
populations
Validate SNP
as marker
for phenotype
Utilization of
SNP markers
Pools of genomic DNA versus
PCR products
Pooled PCR products
Allele frequency (%)
(5 replicates/pool)
30
25
20
15
10
5
0
n=90
0
5
10
15
20
25
30
Expected allele frequency (%)
Pooled genomic DNA
Allele frequency (%)
(5 replicates/pool)
35
30
25
20
15
10
5
0
n=90
0
5
10
15
20
Expected allele frequency (%)
25
30
Pools of PCR products
compared to pools of
genomic DNA mixed
before PCR showed
excellent agreement with
expected allele frequency
ratios
Pooling DNA samples
can have dramatic impact on efficiency and
cost of SNP studies
– Reduction in number of analyses
– Reduced costs, reagents, and labor
– Less genomic material required
Allele Frequency
Karolinska Institute, Stockholm Sweden
85.3%
14.7%
SNP 1:
SNP AQ:
Expected:
1126 individuals
G: 14.7% T: 85.3%
G: 15% T: 85%
 Verification of SNPs
 Association/Linkage studies
 Analysis of mutations associated with mixed populations
 Presence of normal cells in tumor samples give mixed genotypes
 Bacterial and viral mixed populations
Allele Frequency Assessment
0%
20%
45%
70%
2%
25%
50%
75%
5%
30%
55%
80%
10%
35%
60%
85%
15%
40%
65%
90%
Allele frequency accuracy = 98-99%
Allele frequency – pooled samples
Relative response
1.0
0.9
Excellent correlation of
peak heights and SNP
allele frequencies
0.8
0.7
0.6
0.5
Allele frequencies down
to 5% with high
accuracy
0.4
0.3
0.2
0.1
0.0
0
10
20
30
40
50
60
70
80
90
Allele ratio
100
PyroMarkTM ID System
PyroMarkTM ID Instrument（主机）
Vacuum Prep Workstation（试样预处理平台）
Assay Design Software（引物设计软件）
专为微生物鉴定分型而设计的新软件IdentiFire Software
How to analyze CpG methylation
1. Bisulfite treatment of denatured DNA
mC
mC
C
U
在重硫酸盐的作用下，所有未甲基化
的胞嘧啶发生脱氨基反映转变成了尿
嘧啶，但是5-甲基胞嘧啶不发生转变。
2. PCR amplification
mC
C
U
T
3. Analysis: sequencing, MS-PCR, primer extension, restriction
enzyme digestion, hybridization…
or best of all: Pyrosequencing!
定量PCR先锋：
the world’s only rotary real-time
thermo-optical analyzer
独特的HRM技术：SNP基因型分析
What is the Rotor-Gene 6000?
• An integrated device that:
– Maintains identical well-to-well conditions to monitor micro reaction
tubes
– Illuminates and collects a wide range of optical signals
– Enables exquisite control of thermal conditions
– Provides an open platform for all chemistries
– Features a very fast data acquisition rate
• Capable of the broadest application set:
–
–
–
–
–
–
Real-time analysis (e.g. quantitative PCR)
End-point analysis (e.g. SNP genotyping)
SYBR melt analysis
HRM (high resolution melt) analysis
Concentration analysis
+ future applications…
Channel Excite/Detect (nm)
U
V
IR
Example fluorophores detected
Blue
365/470
BiosearchBlue™, Marina Blue®, Bothell Blue®, Alexa Fluor® 350
Green
470/510
FAM™, SYBR® Green 1, Fluorescein, EvaGreen™, Alexa Fluor® 488
Yellow
530/555
JOE™, VIC™, HEX™, TET™, Yakima Yellow®, Cal Flour® Gold 540
Orange
585/610
ROX™, Cy3.5®, Redmond Red®, Alexa Fluor® 568
Red
625/660
Cy5®, Quasar670™, LCRed640®, Texas Red®, CAL Fluor™ Red
Crimson
670/710
Quasar705™, LCRed705®, Alexa Fluor® 680
HRM
460/510
SYTO®9, LC Green, LC Green™Plus+, EvaGreen™
5 Models to suit different needs and budgets:
1. 2-Plex
2. 2-Plex HRM
Green/Yellow
3. 5-Plex
4. 5-Plex HRM
Green/Yellow/Orange/Red/Crimson
5. 6-Plex
Blue/Green/Yellow/Orange/Red/Crimson
Green/Yellow + High Resolution Melt channel
Green/Yellow/Orange/Red/Crimson + High Resolution Melt channel
NOTE: ROX™ normalization is not needed so all channels can be “plexed” for separate reactions
Thermal Uniformity in a Block-based Cycler
± 0.50 ºC (or more) across the 96 well block
(thus > 1.00 ºC variation is typical)
Corner and Edge wells most affected
NOTE: Fluorescence 
1/
Temp
Localized “hotspots” as Peltier device junctions
begin to fail
The Rotor-Gene does not use Peltier devices—
because they fail unpredictably and are expensive to
repair
NOTE
Rotor-Gene 6000 specifications: Uniformity: ±0.01°C, Resolution: ±0.02°C
Tube Formats
1. 36  0.2 mL PCR tubes
Attached flat or domed caps
NOTE: optical caps are not required since
detection is through the base of the tube
2.
72  0.1 mL tubes
- allow small reaction volumes (5–10 µL)
- in strips of 4 for ease of use
- Frosted cap extensions allow
write-on labelling + easy handling
™
Gene-Disc
3.
Gene-Disc™ 72
4.
Gene-Disc™ 100
- 0.1 mL tubes in a rotary “plate” design
- Tubes oriented vertically (not angled)
- Heat-sealed in seconds
- 30 µL wells in a rotary “plate” design
- Wells oriented vertically (not angled)
- Heat-sealed in seconds
Plates
SYBR Melt Analysis and
™
HRM (high resolution melt)
a new application for
a new type of instrument
SYBR™ Green I
Generic dsDNA intercalation dye
Inexpensive & simple
Used for real-time PCR product detection
Used for DNA dissociation (melt) analysis
Widely used
SYBR melt analysis of a DNA
fragment
Raw data plot
Fluorescence drops as DNA
melts and SYBR is released
Derivative data plot
This “rate” curve peaks at
maximum dissociation rate which
equals the Tm (temperature of
melting)
SYBR™ melts can reflect product
size
500 bp
fragment
250 bp
fragment
250 bp
fragment
Raw data plot:
fluorescence vs. temp.
Derivative data plot:
dF/dT vs. temp
500 bp
fragment
SYBR™ Green I melts can reflect
sequence
detection of alleles
Low primer conc
(50 nM)
High primer conc
(900 nM)
Single band
contains two
species
Primer-dimer
appears as a third
species
Melt comparison: LCGreen I vs.
SYBR Green I
Derivative melting curve
plots of a molecular size
ladder (inset) using either
SYBR Green I (gray line) or
LCGreen (black line)
The DNA ladder consisted of
six DNA fragments (100 bp to
2 kb). All six fragments were
clearly observed when
LCGreen was used (the highest
melting product appears to split
into two melting domains).
With SYBR Green I, the lowtemperature peaks were
minimized, with the peaks
increasing in size with
temperature.
From: High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen
Carl T. Wittwer, Gudrun H. Reed, Cameron N. Gundry, Joshua G. Vandersteen, and
Robert J. Pryor. Clinical Chemistry 49:6, 853–860 (2003)
HRM on the Rotor-Gene 6000
– high-intensity + high sensitivity
optics
– high-speed data capture
– very precise temperature control
– extreme temperature resolution
(C a lle le )
Fluorescence
• To support HRM an instrument
requires:
Wild typ es
Muta nts
(T a lle le )
Heterozyg otes
82
83
84
85
86
87
Temperature (°C)
• To support multiple wells:
– Superlative thermal and optical
well-to-well uniformity
•Example SNP genotyping using HRM analysis.
ACTN3 (R577X) SNP genotypes (C—T).
•Ten replicates each genotype are shown.
•Fragment pre-amplified using a 40 cycle fast
protocol (<40 min).
88
Comparison of HRM
genotyping data on the
Rotor-Gene vs. the HR-1
Rotor-Gene 6000
HR-1 High Resolution Melter
Homozygotes
Homozygotes
Heterozygotes
Heterozygotes
The HR-1 Melter normalizes both data axes & aligns all curves according to “shape”
The shape of homozygote plots is so similar they cannot be reliably resolved on the HR-1
The Rotor-Gene does not need to normalize the Temp. axis so homozygotes are easily resolved
HRM workflow on the Idaho
HR-1
Setup PCR in
capillaries
Setup PCR in
tubes or plates
Run PCR
or
Run PCR in a capillary cycler
Single
capilary
Manually transfer PCR
product to capillary
Load capillaries into HR-1
for melt analysis
Manual Data analysis on individual
samples
1 sample at a time
HRM workflow on the RotorGene 6000
0.2 mL tubes
0.1 mL tubes
Gene-Disc™ 72
Run PCR and HRM
Autocall genotypes
Up to 100 at a time
Gene-Disc™ 100
Choose preferred tube
Available HRM Instrumentation
Instrument
Rotor-Gene 6000
Idaho HR-1
Idaho LightScanner
Applications
•Thermal cycling
•Real-time + end-point
•HRM
•Conc. measurement
•Post-PCR HRM only
•Post-PCR HRM only
Resolution
0.02°C
0.05°C (½ of Rotor-Gene)
0.20°C (1/10th of Rotor-Gene)
Genotyping
Autocall
Manual call, one-by-one
Autocall
Tube Format
1.
2.
3.
4.
Single glass capillary
96-well or 384-well plates
white well, black skirted plate
oil overlay required
Aust. Price
0.2 mL tube
0.1 mL tube
Gene-Disc 72
Gene-Disc 100
(plate type not interchangeable)
HRM Applications
Many applications are envisioned for HRM. Some currently under investigation include:
1. Mutation Discovery/Screening/Scanning
2. SNP genotyping/ Allelic discrimination
- Identify Candidate Predisposition Genes
- Association (case/control) Studies comparing genotype to phenotype
- Prevalence; within population or different sub groups
- Loss of Heterozygosity
- DNA fingerprinting
- Haplotype blocks
3. Predicative Testing
- Penetrance/Linkage studies; tracking variants for disease within a family
4. Species Identification
5. Epigenetic/DNA Methylation (bisulfate treatment required)
6. Microsatellite Analysis (tandem repeats)
- linkage
- fingerprinting
- instability
- loss of hetrozygosity
Affymetrix的适合特定染色体区域SNP分析的MegAllele技术
MegAllele相关产品
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standard SNP panels for disease-related studies
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