DNA Sequencing Core Laboratory Chang Gung Memorial Hospital
DNA Sequencing Core Laboratory Chang Gung Memorial Hospital, Taoyuan, Taiwan
Sanger DNA Sequencing DNA fragment Denature to separate strains Anneal short end-labeled oligonucleotide to one strain Carry out DNA synthesis primed by the oligonucleotide
Sanger DNA Sequencing A reaction dd. ATP G reaction dd. GTP C reaction T reaction dd. CTP dd. TTP Electrophoresis A G C T
Terminator Sequencing Sample Prep 1 reaction within which each terminator is labeled with a different dye Ampli. Taq FS d. ATP, d. CTP, d. GTP, d. TTP unlabeled primer dd. CTP + template DNA dd. ATP dd. GTP dd. TTP Sample is purified by ethanol precipitation to remove unincorporated dd. NTP’s before loading
Sanger Dideoxy Cycle Sequencing Performed in a thermocycler with a thermostable Taq polymerase
One Lane Sequencing
T 3, T 7 promoter, T 7 terminator M 13 F, M 13 R, SP 6… Dye Terminator Cycle Sequencing Step (1)Template +Primer +Sequencing reagents (with dye terminator) (4) denaturation (2) Cycle Sequencing (5)loading Annealing CA T G T Extension (3) Ethanol + Na. OAc precipitation Denaturation
Cycle Sequencing Program Gene. Amp® PCR system 9700 96℃, 5 min 96℃, 10 sec 50℃, 5 sec 60℃, 4 min 4℃, ∞ 25 cycles
Evolution of DNA Analyzers 3730 1999 Production 3700 2000 373 3100 377 Versatility 1987 One Solution Fits All Variety of solutions for diverse customer needs 310 1995 Mass Market 3100 -Avant (2002)
Polymer Upper Tubing Buffer Jar
Capillary array Buffer reservior
Process of Typical Run 1. Sample Preparation 3. Beginning the Run 2. Software Setup 4. Electrophoresis
5. Excitation and Detection 7. Data Processing: during running 6. Data Collection 8. Automatic Data Extraction and Data Analysis 3730
Sample Plate Stacker Enhanced Automatic Operation Stacker Components: - In-Stack - Out-Stack Accepts Barcoded Plates - work with Data Collection Software can eliminate the need for run scheduling
Capillary Array 48/96 uncoated capillaries Ø 300 runs/array Ø 50 micro ID Ø 36 cm and 50 cm Ø Electrode at the ends of capillaries Ø POP-7 polymer - dynamically coating the capillary during each run
Schematic of “In-capillary” Detection
Dual Side Illumination
9. Viewing the Data: on Sequencing Analysis software
QV= -10 log 10(Pe)
Advantages of Capillary Electrophoresis ¡ More efficient heat dissipation than slab gels higher run voltages faster run times higher sample throughput ¡ Automation elimination of manual operations increased run to run consistency and reliability ¡ Sensitivity less DNA per sample but more strict sample requirement(ex: aware of salt contamination of the seq. product……, etc)
Sequencing production Capacity of the 3730
Overview of ABI PRISM 3730/3730 xl A Fully Integrated System Ø Improved Production Capacity - 48 or 96 capillary array - Simultaneous Injection and Analysis of 48/96 samples - Improved Data Quality Ø Walkaway Automation - Automated Plate Loading from a Stacker that Accommodates up to 16 Plates ( 96 - or 384 well plate) - Automated Polymer Replenishment - Internal Barcode Reader Ø Minimized Operational Cost - In- Capillary Detection Ø Active Temperature Control : 18 - 70 degree
定序送件注意事項 ¡ DNA must be dissolved in dd. H 2 O (Not TE buffer) ¡ If forward primer can’t work Try reverse primer or another forward primer. Ex: M 13 F(-21) M 13 F(-40) ¡ Plasmid: ≧ 400 ng/rxn, PCR product≧ 200 ng/rxn ¡ Your PCR program≠Cycle sequencing program
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