De Novo Sequencing
Figure 1: Sequencing Run
Example of a typical sequencing run (Figure 1) using BigDye® Terminator Sequencing Standard v3.1 on an Applied Biosystems Genetic Analyzer.
Experimental Workflow: Find the Products You Need for Every Step
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Step 1:
DNA Purification -
Step 2:
PCR Amplification & Cleanup -
Step 3:
Cycle Sequencing -
Step 4:
Sequencing Reaction Cleanup -
Step 5:
Capillary Electrophoresis -
Step 6:
Data Analysis
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Step 1: DNA Purification
DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.
Step 2: PCR Amplification & Cleanup
Sonication or Enzymatic Digestion of Genomic DNA
Step 3: Cycle Sequencing
Cycle sequencing is a simple method in which successive rounds of denaturation, annealing, and extension in a thermal cycler result in linear amplification of extension products. The products are then injected into a capillary. All current Applied Biosystems DNA sequencing kits use cycle sequencing protocols. For de novo sequencing we recommend Dye Terminator chemistry.
Products for Step 3: Cycle Sequencing
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BigDye® Terminator v1.1 Cycle Sequencing Kits
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BigDye® Terminator v1.1 Cycle Sequencing Kits are designed for specialty applications that require optimal basecalling adjacent to the primer and for sequencing short PCR product templates with rapid electrophoresis run modules.
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BigDye® Terminator v3.1 Cycle Sequencing Kits
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The BigDye Terminator v3.1 Cycle Sequencing Kit's robust, highly flexible chemistry is ideal for de novo, resequencing, and finishing with PCR product, Plasmid, Fosmid, and BAC templates.
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dRhodamine Terminator Cycle Sequencing Kits
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The dRhodamine Terminator Cycle Sequencing Kit provides optimized dRhodamine dideoxy terminators chemistries for decreased background noise in a wide array sequencing applications.
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dGTP BigDye® Terminator Cycle Sequencing Kits
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The dGTP BigDye® Terminator Cycle Sequencing Kit's fluorescent dideoxy terminator chemistry is optimized for sequencing through GT- and G-rich templates.
Step 4: Sequencing Reaction Cleanup
After the sequencing reaction, it is important to remove unincorporated dye terminators and salts that may compete for capillary electrophoretic injection. Unincorporated terminators can co-migrate with the sequencing template, resulting in basecalling errors, and excess salt translates to poor signal-to-noise ratios.
Products for Step 4: Sequencing Reaction Cleanup
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Centri-Sep™ 96-Well Plates
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Centri-Sep™ 96's hydrated matrix enables effective and reliable high-throughput removal of excess BigDye® terminators from DNA sequencing reactions, giving you clean sequencing reads with uniform signal intensity starting with the first base.
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BigDye XTerminator® Purification Kit
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The BigDye XTerminator® Purification Kit is a fast, simple purification method for DNA sequencing reactions that removes unincorporated BigDye® terminators. No more dye blobs! Cleanup is complete in under 40 minutes and requires less than 10 minutes of labor.
Step 5: Capillary Electrophoresis
After performing the post-sequencing reaction purification, samples are ready for analysis on an Applied Biosystems capillary electrophoresis-based genetic analyzer. During capillary electrophoresis, the products of the cycle sequencing reaction migrate through capillaries filled with polymer. The negatively charged DNA fragments are size separated as they move through the polymer in the capillaries toward the positive electrode.
Products for Step 5: Capillary Electrophoresis
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3500xL Genetic Analyzer
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The 3500xL 24-capillary platform can run a wide variety of applications, including de novo sequencing and resequencing (mutational profiling), as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.
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3500/3500xL Genetic Analyzer Polymers
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POP-4™, POP-6™, and POP-7™ are separation matrices for the 3500/3500xL Genetic Analyzers.
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3730 DNA Analyzer
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The 48-capillary 3730 DNA Analyzer is the Gold Standard in medium-to-high throughput genetic analysis.
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3730xl DNA Analyzer
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The 96-capillary 3730xl DNA Analyzer is the Gold Standard for high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample.
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3730/3730xl DNA Analyzer Polymers
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POP-7™ and Conformation Analysis Polymers are separation matrices for the 3730/3730xl Genetic Analyzers.
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3130xl Genetic Analyzer
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Applied Biosystems 3130xl Genetic Analyzer is the latest generation of 16-capillary electrophoresis instruments for the medium throughput laboratories. The system offers industry-leading performance, plus sophisticated automation capabilities allowing researchers to save time, reduce costs and increase productivity.
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3130/3130xl Genetic Analyzer Polymers
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POP-4™, POP-6™, POP-7™ and Conformation Analysis Polymers are separation matrices for the 3130/3130xl-Avant™ Genetic Analyzers.
Step 6: Data Analysis
After electrophoresis, Data Collection software creates a Sample File of the raw data. Using downstream software applications, further data analysis is required to translate the raw data into the corresponding electropherogram.
Products for Step 6: Data Analysis
- Sequencing Analysis Software
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This software enables you to basecall, trim, display, edit, and print data from our entire line of capillary DNA sequencing instruments for data analysis and quality control.
Application Note
- Accurate Long-Read DNA Sequencing on the Applied Biosystems 3130 Series Genetic Analyzers with the 80 cm Capillary Array
- KRAS Variant Identification
Publications
- Energy Crops Fuel a Better Environment Publication: Innovations - Issue 09, Oct 2008
- Shark CSI: Genetic Tracking of the Global Trade in Shark Fins Publication: Innovations - Issue 09, Oct 2008
Training Courses
North American training: Europen training:
