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