Resequencing Heterozygote Detection
Accurate heterozygous indel mutation detection 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.
Products for Step 1: DNA Purification
Step 2: PCR Amplification & Cleanup
Design high quality PCR primers to cover regions of interest using in silico primer design tools. Perform PCR to amplify region of interest. To amplify your DNA sample, you need DNA polymerase (such as AmpliTaq® Gold DNA Polymerase), nucleotides (dNTPs), reaction buffer, primers, and a thermal cycler.
Products for Step 2: PCR Amplification & Cleanup
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. Perform cycle sequencing on PCR products using gene specific primers or universal primers commonly tailed to PCR amplification primers.
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.
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
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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3500 Genetic Analyzer
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The 3500 8-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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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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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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3130 Genetic Analyzer
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Applied Biosystems 3130 Genetic Analyzer is the latest generation of 4-capillary electrophoresis instruments for the low to medium throughput laboratories. The system offers industry-leading performance, plus sophisticated automation capabilities allowing you to save time, reduce costs and increase productivity.
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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.
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310 Genetic Analyzer
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The ABI PRISM® 310 Genetic Analyzer is an automated single-capillary genetic analyzer designed for a wide range of sequencing and fragment analysis applications.
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 collected color-data images into the corresponding nucleotide bases.
Products for Step 6: Data Analysis
- Variant Reporter™ Software
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The Variant Reporter™ Software performs comparative sequencing, also known as direct sequencing, medical sequencing, PCR sequencing and resequencing with DNA sequencing files. The software is designed for reference based and non-reference based analysis such as mutation detection and analysis, SNP discovery and validation and sequence confirmation.
- 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.
- Sequence Scanner Software
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The free Sequence Scanner Software enables you to view, edit, print and export sequence data generated using the Applied Biosystems Genetic Analyzers. The software generates graphically expressive reports on results.
- SeqScape® Software
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Applied Biosystems SeqScape® software is designed for reference based analysis such as mutation detection and analysis, SNP discovery and validation, pathogen sub-typing, allele identification, and sequence confirmation. This product is also integrated for use with VariantSEQr™ Resequencing Set.
- TargetSeq™ Resequencing System
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The TargetSeq™ Resequencing System consists of a new software module that optimizes the Applied Biosystems 3730 and 3730xl DNA Analyzers for high-throughput resequencing or medical research sequencing of small exons and short DNA fragments.
Application Notes
- A Workflow for Obtaining High Quality Sequencing Data from Bacterial Artificial Chromosome (BAC) DNA
- DNA Resequencing Analysis Using the Applied Biosystems 3500xL Genetic Analyzer
- mitoSEQr™ Resequencing System
Product Bulletin
Protocol Summary
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