Introduction Capillary Gel Electrophoresis CGE Replaceable Gel DNA
Introduction Capillary Gel Electrophoresis (CGE) Replaceable Gel DNA separate by size
Introduction Sequencing Process Analysis Denature Casting Plate Check Pre-run เดม : ใชวธเตร ยมเจลโดยใชแผนกร Load Separate/ Database Tracking Analyze ะจก Detect Sample Interaction repeat for 2 batch of 48 Beckman Coulter CEQTM 8000 Genetic Analysis System Load Sample Denature Detect Analyze Facilitated By Capillary Electrophoresis Technology Database Interaction
CEQTM 8000 Systematic Layout 8 Capillary-Array Detector 650 nm laser 750 nm laser Gel Fill Waste
CEQTM 8000 Instrument Automatic Process § Manifold Purging § Capillary Purging § Temperature Ramping § Sample Denaturation § Optical Alignment § Sample Injection § Sample Separation § Data analyzing
CEQTM 8000 Dye Absorbance/Emission Wavelength dd. N Abs (nm) Em (nm) dd. T 649 675 (Cy 5) dd. G 682 708 (DBCy 5) dd. C 750 782 (Cy 7) dd. A 784 815 (DBCy 7)
CEQTM 8000 Applications § 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
CEQTM 8000 Applications Sequencing Applications TM on CEQ 8000
Introduction Sanger Sequencing method 3’-OH in normal DNA allows elongation. Dideoxynucleotide cannot be elongated. TGGCATA A CCGTA T Go to be separated into Capillary Electrophoresis
Sequencing Quality Values
Sequencing Results Poly T Poly A Poly C CA Repeats
Sequencing Results TG Repeats GA Repeats TC Repeats Complicated Repeats Regions
Sequencing Automatic Heterozygote Detection Sequence 1: ===GTAGGTGGACAGATAACAGAAGC=== Sequence 2: ===GTAGGAGGACAGCTAAAAGAAGC===
Fragment Analysis TM on CEQ 8000
Fragment Analysis Applications § 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 Analysis STR (VNTR) – What Is It ? Minisatellites: 9 - 80 bp repeats (Variable number tandem repeats -VNTR) Microsatellites: 2 – 7 bp repeats (Short Tandem Repeats – STR)
Fragment Analysis STR – How it Work Allele 1 - first chromosome 1 2 3 4 5 6 7 8 9 10 Allele 2 - second chromosome 1 2 3 4 5 6 7 8 9 10111213 PCR CE CE
Fragment Analysis AFLP Fragment Data
Fragment Analysis AFLP Fingerprinting
Fragment Analysis Manual Scoring vs CEQ 8000 Dominant Scoring Manual Scoring Using AFLP Analysis in CEQ 5. 0 Software Analyzed AFLP Sample Data Record Fragments for Each Sample on Microsoft Excel Spreadsheet Run CEQ AFLP Analysis Clustering of Fragments with the Same Sizes from All Samples Exportable Binary 1/0 Data Convert to Binary 1/0 Data Estimated Time Needed to Analyze 100 Samples 40 Hours < 2 minutes
Fragment Analysis AFLP Applications Plant Molecular Genetics : § Breeding • Variety Identification • Germplasm Management • Agronomically Important Traits Selection § Phylogeny & Diversity Study • Evolution • Geographic Orgins Microbial Genetics : Animal Molecular Genetics : Other Applications : § Gene Mapping (with STR) § Differential Gene Expression § Functional Genomics § Diversity § § § Population Genetic Analysis Strain Typing Epidemiological Study Evolutionary Study Taxonomy Ecology
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