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Capillary Gel Electrophoresis (CGE)

Replaceable Gel. DNA separate by size. Introduction. Capillary Gel Electrophoresis (CGE). Denature. Casting. Plate Check. Pre-run. Load Sample. Separate/ Detect. Tracking. Analyze. Database Interaction. repeat for 2 batch of 48.

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Capillary Gel Electrophoresis (CGE)

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  1. Replaceable Gel DNA separate by size Introduction Capillary Gel Electrophoresis (CGE)

  2. Denature Casting Plate Check Pre-run Load Sample Separate/ Detect Tracking Analyze Database Interaction repeat for 2 batch of 48 Beckman Coulter CEQTM 8000 Genetic Analysis System Database Interaction Load Sample Denature Detect Analyze Facilitated By Capillary Electrophoresis Technology Introduction Sequencing Process Analysis เดิม : ใช้วิธีเตรียมเจลโดยใช้แผ่นกระจก

  3. Introduction Advantages of CE in Genetic Analysis • ไม่ต้องใช้ Glass Plates • Separation gel เป็นแบบพร้อมใช้ (Linear polymer) • ใช้เวลาในการแยกน้อย • ทำการวิเคราะห์ได้อย่างรวดเร็ว • ประหยัด DNA samples • Dyes ที่ใช้มีความคงตัวสูง • ระบบการทำงานเป็นแบบFULL AUTOMATION !

  4. 8 Capillary-Array 650nm laser 750nm laser Gel Fill Waste CEQTM 8000 Systematic Layout Detector

  5. Manifold Purging Capillary Purging Temperature Ramping Sample Denaturation Optical Alignment Sample Injection Sample Separation Data analyzing CEQTM 8000 Instrument Automatic Process

  6. 2 diode lasers: 650 nm & 750 nm มีอายุการใช้งานยาวนาน > 10,000 hrs ค่าใช้จ่ายในการดูแลรักษาต่ำ ก่อให้เกิดความร้อนน้อย Each laser excite 2 dyes : ลดปัญหาการเหลื่อมซ้อนของสัญญาณและเพิ่มความแม่นยำในการอ่านค่า เลเซอร์แต่ละตัวรับประกัน 5 ปี สามารถทำ Optical alignmentได้โดยอัตโนมัติก่อนเริ่มการวิเคราะห์แต่ละครั้ง CEQTM 8000 Optical Detection

  7. CEQTM 8000 Dye Absorbance/Emission Wavelength ddNAbs (nm)Em (nm) ddT 649 675 (Cy5) ddG 682 708 (DBCy5) ddC 750 782 (Cy7) ddA 784 815 (DBCy7)

  8. 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

  9. CEQTM 8000 Applications Sequencing Applications on CEQTM 8000

  10. Introduction Sanger Sequencing method 3’-OH in normal DNA allows elongation. Dideoxynucleotide cannot be elongated. TGGCATA A C C G T A T Go to be separated into Capillary Electrophoresis

  11. Sequencing Quality Values

  12. Sequencing Results Poly A Poly T Poly C CA Repeats

  13. Sequencing Results TG Repeats TC Repeats GA Repeats Complicated Repeats Regions

  14. Sequence 1: ===GTAGGTGGACAGATAACAGAAGC=== Sequence 2: ===GTAGGAGGACAGCTAAAAGAAGC=== Sequencing Automatic Heterozygote Detection

  15. Fragment Analysis on CEQTM 8000

  16. 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

  17. 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)

  18. 6 3 4 5 7 8 9 10 3 4 5 6 7 10 11 13 2 1 2 8 9 12 1 Fragment Analysis STR – How it Work Allele 1 - first chromosome Allele 2 - second chromosome PCR PCR CE CE

  19. Fragment Analysis AFLP Fragment Data

  20. Fragment Analysis AFLP Fingerprinting

  21. Using AFLP Analysis in CEQ 5.0 Software Manual Scoring Analyzed AFLP Sample Data 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 < 2 minutes 40 Hours Fragment Analysis Manual Scoring vs CEQ 8000 Dominant Scoring

  22. Fragment Analysis 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 • EpidemiologicalStudy • EvolutionaryStudy • Taxonomy • Ecology • Other Applications : • Differential Gene Expression • Functional Genomics

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