AFLP® Analysis

Figure 1: AFLP Analysis

Amplified fragment length polymorphism (AFLP®) analysis is a genetic mapping technique that uses selective amplification of a subset of restriction enzyme-digested DNA fragments to generate a unique fingerprint for a particular genome. The power of AFLP analysis derives from its ability to quickly generate large numbers of marker fragments for any organism, without prior knowledge of the genomic sequence.

AFLP analysis on an Applied Biosystems Genetic Analyzer (Figure 1). Plots on the left contain the AFLP peaks (blue) and size standard peaks (red) for the three samples. The plot on the right side shows the bins (gray bars) and allele calls data.

Products
Experimental Workflow: Find the Products You Need for Every Step
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Step 1: DNA Extraction

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 Extraction

NucPrep® DNA Chemistry for Tissues

Starter kit for DNA isolation from animal and plant tissues and cells, etc., using NucPrep® Chemistry.

Step 2: Restriction / Ligation

Cut genomic DNA into fragments with the restriction enzymes Msel and EcoRI and Ligate adaptors.

Step 3: Preselective PCR

Enriches the bulk of EcoRI / MseI tagged genomic fragments.

Products for Step 3: Preselective PCR

AFLP®-based Genotyping Kits and Reagents

Step 4: Selective PCR

Second PCR with selective primer pairs reduces complexity to resolvable and distinct electrophoretic migration patterns.

Products for Step 4: Selective PCR

AFLP®-based Genotyping Kits and Reagents

Step 5: Prepare samples for Fragment Analysis Run

Add aliquot of PCR reaction to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis.

Products for Step 5: Prepare samples for Fragment Analysis Run

Hi-Di™ Formamide

Highly deionized formamide used to resuspend samples before electrokinetic injection in capillary electrophoresis systems.

GeneScan™ Size Standards

GeneScan™ Size Standards are fluorescently-labeled DNA fragments required for performing fragment analysis and genotyping applications on our capillary electrophoresis platforms.

Step 6: Electrophoresis

After performing the selective PCR AFLP reaction, samples are ready for analysis on an Applied Biosystems capillary electrophoresis-based genetic analyzer. During capillary electrophoresis, the products of the cycle sequencing reaction are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries toward the positive electrode.

Products for Step 6: Electrophoresis

3500 Genetic Analyzer

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.

3500xL Genetic Analyzer

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.

3730 DNA Analyzer

The 48-capillary 3730 DNA Analyzer is the Gold Standard in medium-to-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. This is upgradeable to 96 capillaries.

3730xl DNA Analyzer

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.

3130 Genetic Analyzer

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.

3130xl Genetic Analyzer

Applied Biosystems 3130xl Genetic Analyzer is the latest generation of 16-capillary electrophoresis instruments for the medium throughput laboratories.

310 Genetic Analyzer

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 7: Analyze Data

Analyze data with AFLP-specific analysis module in GeneMapper software. Export binary tables of data for subsequent analysis in downstream applications (e.g. phylogenetic or linkage analysis software).

Products for Step 7: Analyze Data

GeneMapper® Software

GeneMapper is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems.
Literature
- Application Note: Amplified Fragment Length Polymorphism (AFLP®) Analysis on Applied Biosystems Capillary Electrophoresis Systems