Overview of Fragment Analysis
Fragment analysis generates a size estimate for DNA fragments relative to a size standard of DNA fragments with known lengths. The size standard is combined with the sample of interest and co-injected on the capillary electrophoresis system.
Experimental Workflow: Find the Products You Need for Every Step
Primer Design & PCR Amplification
Sample Prep for Fragment Analysis Run
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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
- MELT™ Total Nucleic Acid Isolation System
Stop homogenizing tissue samples. With the MELT Total RNA Isolation System, tissue samples are quickly and easily lysed in MELT solution.
- BloodPrep® DNA Chemistry for Cultured Cells and Blood
BloodPrep® DNA Chemistry is designed to rapidly isolate DNA from fresh or frozen blood from any animal species, tissue-cultured cells, or buccal swabs.
- RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues
Extract total RNA or DNA from formalin or paraformalin-fixed, paraffin-embedded (FFPE) tissues. This kit enables the molecular analysis of archived tissue samples at both the genomic and gene expression levels.
Step 2: Primer Design & PCR Amplification
The DNA region of interest is amplified using a primer labeled with Fluorescent tag using a PCR instrument. This is a critical part of the workflow as it ensures that DNA fragments become dye-labeled during PCR. For higher throughput, you can parallel experiments with multiple dyes.
To amplify your DNA sample, you will also need DNA polymerase (such as AmpliTaq Gold® DNA Polymerase), nucleotides (dNTPs), reaction buffer, primers, and a thermal cycler.
Products for Step 2: Primer Design & PCR Amplification
- Veriti® 96-Well Fast Thermal Cycler
The Veriti® 96-Well Fast Thermal Cycler delivers the proven reliability of Applied Biosystems 9600 and 9700 GeneAmp® PCR instruments. With the added control of VeriFlex™ Blocks, you have six independent temperature blocks providing precise control over your PCR optimization.
- Veriti® 384-Well Thermal Cycler
The Veriti® 384-Well Thermal Cycler (0.02 mL) delivers the proven reliability of Applied Biosystems 9700 GeneAmp® instruments with the throughput you need for large experiments.
- AmpliTaq Gold® 360 DNA Polymerase
AmpliTaq Gold® 360 DNA Polymerase, when used with improved AmpliTaq Gold 360 Buffer, and the optional 360 GC Enhancer, amplifies a vast range of DNA sequence contexts.
Step 3: Sample Prep for Fragment Analysis Run
Amplified DNA fragments are mixed with size standards. Depending on the anticipated DNA fragment sizes, appropriate size standards should be used that cover corresponding size range. Depending on the dye label used for DNA fragment samples, size standards with a different dye label than the samples should be selected. (Please see our table below for selection of dye sets for different applications): DNA fragments and standards and mixed in a size standard buffer which contains a denaturing agent (such as Hi-Di™ Formamide). Afterwards, mixed samples and standards are loaded on the instrument. Learn more about sizing.
Products for Step 3: Sample Prep for Fragment Analysis Run
- GeneScan™ Size Standards
GeneScan™ Size Standards are fluorescently-labeled DNA fragments required for performing fragment analysis and genotyping applications on our capillary electrophoresis platforms.
To learn more about our GeneScan Size standards used in sizing experiments please see our table below.
When DNA sample fragments are labeled with: Choose a size standards labeled with: AB Dye Set: Possible Applications Other AB kits and products: dR110, dR6G, dTAMRA™, dROX LIZ DS-02 SNaPshot® SNaPshot® primer focus kits, SNaPshot® Multiplex kits 5-FAM™, HEX™, NED™ ROX™ DS-30 Custom Oligos 6-FAM™, VIC™, NED™ ROX™ DS-31 Microsatellites (linkage mapping sets) LMS v2.5, Mouse Mapping Primers, Custom Oligos 5-FAM™, JOE, NED™ ROX™ DS-32 Microsatellite (Forensic) AmpFISTR Profiler Plus®, Cofiler, SGM Plus Kits, StockMarks Kits, Plant/Microbial AFLP Kits® 6-FAM™, VIC™, NED™, PET® LIZ DS-33 Microsatellite (Forensic) AmpFISTR Identifier Kits, AmpFISTR Yflir Amplification Kits, Custom Oligos, LMS v2.5, Mouse Mapping Primers, StockMarks Kits 6-FAM™, TET, HEX™ TAMRA DS-34 6-FAM™, dR6G LIZ DS-40 SNP Genotyping &SNPlex Genotyping System
Step 4: Capillary Electrophoresis
Amplified fragments, along with appropriate size standards, move through a capillary filled with polymer and are detected using Capillary Electrophoresis Genetic Analyzer.
During an electrophoresis run, the AB Genetic Analyzer records the fluoresence intensity as a function of time and wavelength from regions on a CCD camera that correspond to different detection wavelength ranges.
Products for Step 4: Capillary Electrophoresis
- 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.
- 3500/3500xL Genetic Analyzer Polymers
POP-4™, POP-6™, and POP-7™ are separation matrices for the 3500/3500xL Genetic Analyzers.
- 3730 DNA Analyzer
The 48-capillary 3730 DNA Analyzer is the Gold Standard in medium-to-high throughput genetic analysis.
- 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
- 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 5: Data Analysis
As part of the primary data analysis performed by the AB genetic analyzer, multicomponent matrix is applied to the fluoresence intensity data to correct for spectral overlap between the dyes. After correction, the fluoresence intensities are color-coded and displayed as peaks in the electropherogram (pictured below.)
As part of the secondary data analysis, the analysis software will generate a sizing curve based on the known size standard fragments, and the unknown fragment sizes are determined. Learn more about secondary data analysis.
Products for Step 5: Data Analysis
- GeneMapper® Software v4.1
GeneMapper is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems.
- Peak Scanner™ Software v1.0
Use this free software to perform DNA fragment analysis; separate a mixture of DNA fragments according to their sizes, provide a profile of the separation, and precisely calculate the sizes of the fragments.
|Key technical applications of Fragment Sizing Analysis include:|
|Amplified Fragment Length Polymorphism (AFLP) Mapping|| |
|Microsatellite Analysis Application || |
|Single Stranded Conformation Polymorphism (SSCP) Analysis|