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Capillary Gel Electrophoresis (CGE) PowerPoint PPT Presentation


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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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Capillary gel electrophoresis cge

Replaceable Gel

DNA separate by size

Introduction

Capillary Gel Electrophoresis (CGE)


Capillary gel electrophoresis cge

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


Capillary gel electrophoresis cge

Introduction

Advantages of CE in Genetic Analysis

  • ไม่ต้องใช้ Glass Plates

  • Separation gel เป็นแบบพร้อมใช้ (Linear polymer)

  • ใช้เวลาในการแยกน้อย

  • ทำการวิเคราะห์ได้อย่างรวดเร็ว

  • ประหยัด DNA samples

  • Dyes ที่ใช้มีความคงตัวสูง

  • ระบบการทำงานเป็นแบบFULL AUTOMATION !


Capillary gel electrophoresis cge

8 Capillary-Array

650nm laser

750nm laser

Gel Fill

Waste

CEQTM 8000

Systematic Layout

Detector


Capillary gel electrophoresis cge

Manifold Purging

Capillary Purging

Temperature Ramping

Sample Denaturation

Optical Alignment

Sample Injection

Sample Separation

Data analyzing

CEQTM 8000

Instrument Automatic Process


Capillary gel electrophoresis cge

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


Capillary gel electrophoresis cge

CEQTM 8000

Dye Absorbance/Emission Wavelength

ddNAbs (nm)Em (nm)

ddT 649675 (Cy5)

ddG 682708 (DBCy5)

ddC 750782 (Cy7)

ddA 784815 (DBCy7)


Capillary gel electrophoresis cge

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


Capillary gel electrophoresis cge

CEQTM 8000

Applications

Sequencing Applications

on CEQTM 8000


Sanger sequencing method

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


Capillary gel electrophoresis cge

Sequencing

Quality Values


Capillary gel electrophoresis cge

Sequencing

Results

Poly A

Poly T

Poly C

CA Repeats


Capillary gel electrophoresis cge

Sequencing

Results

TG Repeats

TC Repeats

GA Repeats

Complicated Repeats Regions


Capillary gel electrophoresis cge

Sequence 1:

===GTAGGTGGACAGATAACAGAAGC===

Sequence 2:

===GTAGGAGGACAGCTAAAAGAAGC===

Sequencing

Automatic Heterozygote Detection


Capillary gel electrophoresis cge

Fragment Analysis

on CEQTM 8000


Capillary gel electrophoresis cge

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


Capillary gel electrophoresis cge

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)


Capillary gel electrophoresis cge

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


Capillary gel electrophoresis cge

Fragment Analysis

AFLP Fragment Data


Capillary gel electrophoresis cge

Fragment Analysis

AFLP Fingerprinting


Capillary gel electrophoresis cge

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


Capillary gel electrophoresis cge

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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