(医学PPT课件)pyrosequencing焦磷酸测序
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19
Quantitative peak heights
1,000
Relative
0,900
peak height 0,800
0,700
75 %
0,600
0,500 0,400
30 %
0,300 0,200
15 %
0,100
0,000 0
10 20 30 40 50 60 70 80 90 100
Allele frequency (%)
2
Principle of Pyrosequencing
双磷酸酶
APS＋PPi Luciferin＋ATP
硫酸化酶 荧光素酶
oxyluciferin
3
Detection of the light
light
time
4
每次向反应体系中加入一种dNTP 相应位置的峰代表该种dNTP的掺入情况 峰高与掺入的核苷酸数量成正比 多余的dNTP在加入下一种dNTP前就被降解
PyroMarkTM ID
Complete solution for Clinical Microbiology
Assay Design SW Sample Prep
Pyrosequencing
IdentiFireTM SW
Featuring Pyro Gold chemistry
7
Pyrosequencing 技术的流程
3
ref
ref
2,5
2
1,5
1
0,5
0 TA GCT G CA CG AT G
18
User creates a SNP/ mutation sequence database
Software calculates theoretical genotyping results
Each DNA fragment is assigned a color
C
G TT CC A CC T
5
Pyrosequencing 系统平台
A complete solution for accurate genetic determination
Sample Prep Workstation
Reagent Kits
Software
Instrumentation
6
Pyrosequencing 系统平台
Single nucleotide InDels (e.g. [C]) Multiple nucleotide InDels (e.g. [CGACGGT])
11
CYP2D6 - A2637del (Allele 3)
T/T
Sequence (reverse) : TCC[ T ]GTG
T/-
Fast
Simple
Dedicated Software
Accuracy
Sequence
Dedicated Reagent Kit
Flexibility
Efficient
Scalable
Multiplexing
Short Optimization Time
实验设计
PCR
试样预处理， 获得单链模板
1-2h /96 samples
15 min/96 samples
结果分析
Pyrosequencing 序列分析
SNP: 10min/96 samples SQA:35 min/96 samples
8
Peak height is proportional to the number of incorporated nucleotides
4
7 8 (9) (10) (11) (12)
8/12
14
Tri- and tetra-allelic SNPs
Genotyping of tri-allelic SNP (C/T/G)
C/C
C/T
T/T
C/G
G/G
T/G
15
Multiple polymorphisms
5’…AACATTCGGCTTACAT/GGAATGAA/C/G/TCGGTACGAACGATTTTAGA...
Relative peak height =
peak height
=
1
peak1 height + peak2 height
Allele frequency =
= relative peak height x 100 (%)
20
Advantage and Benefit of Pyeosequencing
焦磷酸测序法的原理及应用
（Pyrosequencing遗传分析技术）
1
Pyrosequencing遗传分析技术
pyro=pyrophosphate焦磷酸
一种全新的基于酶级联反应的 新型遗传分析技术;
一套完整的实验方案； 诸多领域的广泛应用……
Pyrosequencing这项专利技术完全由Biotage公司拥有
3’TAAGCCGAATG
16
Multiplexing
SNPs located on different fragments…... …..or on the same
17
Principle of multiplexing
Design of sequencing primers and dispensation order
杂合子标本:
Allele 1: ACTG Allele 2: GCTG
9
Genotypes are clearly distinguished
ACTGCCT GCTGCCT
杂合子 A/GCTGCCT
10
Insertions/deleБайду номын сангаасions
The software supports analysis of insertions/deletions (InDels)
-/-
12
Large deletion
5’...ACGTACGCTTACACCGTAAGGTTCTAAAGGTG[CACCATGACTGGGGTTA]CAGTCATC... 3’ATGTGGCATTCCAA
13
Microsatellites: TG[AATG]nTTTGGGCAAAT
4
78
8/8
2
1,5
1 TTGAG[A/G]CTGCCTG 1
0,5
0 T A G C T G CA CG AT G
2
1,5
2 TGGACCACC[A/G]TGG 1
0,5
Combined [A/G]CCTGCCACC[A/G]TTGGG
Relative light units
0
T A G C T G CA CG AT G 3,5
Quantitative peak heights
1,000
Relative
0,900
peak height 0,800
0,700
75 %
0,600
0,500 0,400
30 %
0,300 0,200
15 %
0,100
0,000 0
10 20 30 40 50 60 70 80 90 100
Allele frequency (%)
2
Principle of Pyrosequencing
双磷酸酶
APS＋PPi Luciferin＋ATP
硫酸化酶 荧光素酶
oxyluciferin
3
Detection of the light
light
time
4
每次向反应体系中加入一种dNTP 相应位置的峰代表该种dNTP的掺入情况 峰高与掺入的核苷酸数量成正比 多余的dNTP在加入下一种dNTP前就被降解
PyroMarkTM ID
Complete solution for Clinical Microbiology
Assay Design SW Sample Prep
Pyrosequencing
IdentiFireTM SW
Featuring Pyro Gold chemistry
7
Pyrosequencing 技术的流程
3
ref
ref
2,5
2
1,5
1
0,5
0 TA GCT G CA CG AT G
18
User creates a SNP/ mutation sequence database
Software calculates theoretical genotyping results
Each DNA fragment is assigned a color
C
G TT CC A CC T
5
Pyrosequencing 系统平台
A complete solution for accurate genetic determination
Sample Prep Workstation
Reagent Kits
Software
Instrumentation
6
Pyrosequencing 系统平台
Single nucleotide InDels (e.g. [C]) Multiple nucleotide InDels (e.g. [CGACGGT])
11
CYP2D6 - A2637del (Allele 3)
T/T
Sequence (reverse) : TCC[ T ]GTG
T/-
Fast
Simple
Dedicated Software
Accuracy
Sequence
Dedicated Reagent Kit
Flexibility
Efficient
Scalable
Multiplexing
Short Optimization Time
实验设计
PCR
试样预处理， 获得单链模板
1-2h /96 samples
15 min/96 samples
结果分析
Pyrosequencing 序列分析
SNP: 10min/96 samples SQA:35 min/96 samples
8
Peak height is proportional to the number of incorporated nucleotides
4
7 8 (9) (10) (11) (12)
8/12
14
Tri- and tetra-allelic SNPs
Genotyping of tri-allelic SNP (C/T/G)
C/C
C/T
T/T
C/G
G/G
T/G
15
Multiple polymorphisms
5’…AACATTCGGCTTACAT/GGAATGAA/C/G/TCGGTACGAACGATTTTAGA...
Relative peak height =
peak height
=
1
peak1 height + peak2 height
Allele frequency =
= relative peak height x 100 (%)
20
Advantage and Benefit of Pyeosequencing
焦磷酸测序法的原理及应用
（Pyrosequencing遗传分析技术）
1
Pyrosequencing遗传分析技术
pyro=pyrophosphate焦磷酸
一种全新的基于酶级联反应的 新型遗传分析技术;
一套完整的实验方案； 诸多领域的广泛应用……
Pyrosequencing这项专利技术完全由Biotage公司拥有
3’TAAGCCGAATG
16
Multiplexing
SNPs located on different fragments…... …..or on the same
17
Principle of multiplexing
Design of sequencing primers and dispensation order
杂合子标本:
Allele 1: ACTG Allele 2: GCTG
9
Genotypes are clearly distinguished
ACTGCCT GCTGCCT
杂合子 A/GCTGCCT
10
Insertions/deleБайду номын сангаасions
The software supports analysis of insertions/deletions (InDels)
-/-
12
Large deletion
5’...ACGTACGCTTACACCGTAAGGTTCTAAAGGTG[CACCATGACTGGGGTTA]CAGTCATC... 3’ATGTGGCATTCCAA
13
Microsatellites: TG[AATG]nTTTGGGCAAAT
4
78
8/8
2
1,5
1 TTGAG[A/G]CTGCCTG 1
0,5
0 T A G C T G CA CG AT G
2
1,5
2 TGGACCACC[A/G]TGG 1
0,5
Combined [A/G]CCTGCCACC[A/G]TTGGG
Relative light units
0
T A G C T G CA CG AT G 3,5