Capillary Gel Electrophoresis (CGE)
description
Transcript of Capillary Gel Electrophoresis (CGE)
Capillary Gel Electrophoresis (CGE)Introduction
Replaceable GelDNA separate by size
Sequencing Process AnalysisIntroduction
repeat for 2 batch of 48
Denature Casting PlateCheck Pre-run Load
SampleSeparate/
Detect Tracking Analyze DatabaseInteraction
Beckman Coulter CEQTM 8000 Genetic Analysis System
Denature DetectLoadSample Analyze Database
Interaction
Facilitated ByCapillary Electrophoresis Technology
เดิม : ใชว้ธิเีตรยีมเจลโดยใช้แผ่นกระจก
Advantages of CE in Genetic AnalysisIntroduction
ไมต้่องใช ้Glass Plates Separation gel เป็นแบบพรอ้มใช ้
(Linear polymer) ใชเ้วลาในการแยกน้อย ทำาการวเิคราะห์ได้อยา่งรวดเรว็ ประหยดั DNA samples Dyes ท่ีใชม้คีวามคงตัวสงู ระบบการทำางานเป็นแบบ FULL
AUTOMATION !
Systematic Layout
650nm laser
750nm laser
Waste
Gel Fill
8 Capillary-Array
CEQTM 8000
Detector
Instrument Automatic Process
Manifold Purging Capillary Purging Temperature Ramping Sample Denaturation Optical Alignment Sample Injection Sample Separation Data analyzing
CEQTM 8000
Optical Detection
2 diode lasers : 650 nm & 750 nm• มอีายุการใชง้านยาวนาน > 10,000 hrs• ค่าใชจ้า่ยในการดแูลรกัษาตำ่า• ก่อให้เกิดความรอ้นน้อย
Each laser excite 2 dyes : ลดปัญหาการเหลื่อมซอ้นของสญัญาณและเพิม่ความแมน่ยำาในการอ่านค่า
เลเซอรแ์ต่ละตัวรบัประกัน 5 ปี สามารถทำา Optical alignment ได้โดย
อัตโนมติัก่อนเริม่การวเิคราะห์แต่ละครัง้
CEQTM 8000
Dye Absorbance/Emission Wavelength
ddN Abs (nm) Em (nm)ddT 649 675 (Cy5)ddG 682 708 (DBCy5)ddC 750 782 (Cy7)ddA 784 815 (DBCy7)
CEQTM 8000
Sequencing• De Novo Sequencing• Confirmatory Sequencing
Fragment Analysis• STR / VNTR / SSR / Microsatellite Analysis• AFLP Analysis for Fast Genome Mapping• Multiplex SNP Scoring• Loss of Heterozygosity (LOH) / Microsatellite
Instability• Peak Ratio Calculation / Quantitation
ApplicationsCEQTM 8000
Sequencing Applications
on CEQTM 8000
Applications
CEQTM 8000
Go to be separated into
Capillary Electrophoresis
Sanger Sequencing method
Introduction
A C C G T TA
3’-OH in normal DNA allows elongation.
Dideoxynucleotide cannot be elongated.
TGGCATA
Quality Values
Sequencing
Sequencing
Poly T Poly A
Results
Poly C CA Repeats
SequencingResults
TG Repeats TC Repeats
GA Repeats Complicated Repeats Regions
Automatic Heterozygote Detection===GTAGGTGGACAGATAACAGAAGC===Sequence 1:===GTAGGAGGACAGCTAAAAGAAGC===Sequence 2:
Sequencing
Fragment Analysis on CEQTM 8000
Fragment Analysis• STR / VNTR / Microsatellite Analysis• AFLP Analysis for Fast Genome Mapping• Multiplex SNP Scoring• Loss of Heterozygosity (LOH) /
Microsatellite Instability• Peak Ratio Calculation / Quantitation• Gene Expression Profiling
Fragment AnalysisApplications
STR (VNTR) – What Is It ?Fragment Analysis
Minisatellites: 9 - 80 bp repeats (Variable number tandem repeats -VNTR)Microsatellites: 2 – 7 bp repeats (Short Tandem Repeats – STR)
STR – How it WorkFragment Analysis
Allele 1 - first chromosome Allele 2 - second chromosome
CE CE
PCR PCR
1 2 3 4 5 6 7 8 9101112132 3 4 5 6 7 8 9101
AFLP Fragment DataFragment Analysis
AFLP Fingerprinting
C rea tio n o f D e n d ro g ra m
E x p o rt to C lu s te r A n a ly s is S o ftw a re
D o m in a n t S c o r in g -B in a ry o u tp u t
P e rfo rm C lu s te r in g
A n a lyse - a ss ig n S ize
C o lle c tio n o f R a w D a ta
S e t-u p C E Q
P re p a ra tio n o f S a m p leO p i u m P o p p y A F L P
0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 1 . 0T a s c v - 1 aT a s c v - 1 cT a s c v - 2 aT a s c v - 2 bT a s c v - 2 cN R 9 6 1 3 4 bN R 9 6 1 3 7 aN R 9 6 1 3 7 bT a s c v - 1 bT a s c v - 7 aT a s c v - 7 bN R 9 6 1 3 4 aU N L 1 1 0 aU N L 1 1 0 cU N L 1 1 0 bR u s t i c a " O "V o l u n t e e r - 1V o l u n t e e r - 2P . s e t i g e r u mT a s c v - 3 aT a s c v - 3 bT a s c v - 4 bT a s c v - 3 cT a s c v - 4 aN R 9 6 1 3 3 aN R 9 6 1 3 3 bN R 9 6 1 3 3 cM o r p h i n m a k D D R - 1 aM o r p h i n m a k D D R - 1 bP A P - 0 9 8P A P - 1 9 9G o l u b o j J u b i l e j n y i P A P - 2 5 0P A P - 4 9 8P A P - 5 2 0P A P - 5 1 9P A P - 5 0 1
P A P - 8 0 0T a s c v - 5 aT a s c v - 5 bT a s c v - 6 aT a s c v - 6 bP A P - 8 0 1P A P - 7 2 2P A P - 7 2 5P A P - 7 8 5P A P - 7 9 9A f g h a n 9 6 v 0 1 6 9 aA f g h a n 9 6 v 0 1 6 9 bA f g h a n 9 6 v 0 1 6 3 - 1 aA f g h a n 9 6 v 0 1 6 3 - 1 bS i n j o c a f t j a s t P 3 6 0 P A P - 3 2 7S i n j o c a f t j a s t S 1 8 8 P A P - 3 2 8P A P - 7 1 0U N L - 2 0 8U N L - 2 0 4U N L - 1 3 9P A P - 7 1 1
P . b r a c t e a t u m
P A P - 4 5 4M a d u r o v i c s
Fragment Analysis
Manual Scoring vs CEQ 8000 Dominant Scoring
Fragment Analysis
Using AFLP Analysis in CEQ 5.0 Software
Analyzed AFLP Sample Data
Run CEQ AFLP Analysis
Exportable Binary 1/0 Data
< 2 minutesEstimated
Time Needed to Analyze
100 Samples
40 Hours
Manual Scoring
Analyzed AFLP Sample Data
Record Fragments for Each Sample on Microsoft Excel
Spreadsheet
Clustering of Fragments with the Same Sizes from All
Samples
Convert to Binary 1/0 Data
AFLP Applications
Plant Molecular Genetics : Breeding
• Variety Identification• Germplasm Management• Agronomically Important
Traits Selection Phylogeny & Diversity Study
• Evolution• Geographic Orgins
Animal Molecular Genetics : Gene Mapping (with STR) Diversity
Microbial Genetics : Population Genetic
Analysis Strain Typing Epidemiological Study Evolutionary Study Taxonomy Ecology
Other Applications : Differential Gene
Expression Functional Genomics
Fragment Analysis