Novice or expert—learn the basics of fragment analysis

Fragment analysis applications are those in which fluorescent fragments of DNA (produced by PCR using primers designed for a specific interrogation task) are separated using capillary electrophoresis and sized by comparison to a size standard.

What is Fragment Analysis?

Most fragment analysis applications have simplified workflows—straightforward sample preparation (without the purification required for sequencing samples), ability to multiplex, and applications can all be separated using the same capillary electrophoresis (CE) polymer.

The video gives an overview of the fragment analysis workflow and introduces you to some of the applications you can develop on our CE instruments.

The step-by-step guide gives a generalized look at fragment analysis. For application-specific step-by-step guides go here.

Download the New Fragment Analysis Guide

Step-by-Step Guide to General Fragment Analysis

3500 Genetic Analyzer

DNA extraction is a critical first step in the experimental workflow of Fragment analysis. The overall quality of the fragment analyzed 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.

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3500 Genetic Analyzer

Amplify the DNA fragment of interest by PCR using 5' fluorescent labeled primer.

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Fragment Analysis sizing graph image.

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

Recommended Products: Prepare Sample for Analysis

Sizing Information

To learn more about our GeneScan Size standards used in sizing experiments please see our table below.

When DNA fragments are labeled with: Choose a size standards labeled with: Recommended Size Standard Possible Applications Other Kits and Products
dR110, dR6G, dTAMRA™, dROX (Dye Set DS-02 - Filter E5) LIZ®
GeneScan™ 120 LIZ® Size Standard
GeneScan™ 600 LIZ Size Standard v2.0
SNaPshot®
Microsatellite, LOH, ISSR, Chimerism, ISSR, RFLP, T-RFLP, MSMSA
SNaPshot® primer focus kits
Custom Labeled Primer
5-FAM™, HEX™, NED™ (Dye Set DS-30 - Filter D) ROX™ GeneScan™ 500 ROX™ Size Standard Custom Fragment Analysis Custom Labeled Primer
6-FAM™, VIC®, NED™ (Dye Set DS-31 - Filter D**) ROX™ GeneScan™ 500 ROX™ Size Standard

Custom Fragment Analysis

Custom Labeled Primer
5-FAM™, JOE, NED™ (Dye Set DS-32 - Filter F) ROX™ GeneScan™ 500 ROX™ Size Standard
Microsatellite (Forensic)
AFLP
AmpFℓSTR® Kits,
Plant/Microbial AFLP Kits®
6-FAM™, VIC®, NED™, PET® (Dye Set DS-33 - Filter G5) LIZ® GeneScan™ 600 LIZ Size Standard v2.0
Microsatellite (Forensic)
Custom Fragment Analysis
AmpFℓSTR® Kits,
Custom Labeled Primer
3500 Genetic Analyzer

During capillary electrophoresis, the products of the PCR are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the fluorescent DNA fragments are separated by size and are detected by a laser/camera system.

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3500 Genetic Analyzer

Analyze the fragment profile.

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Product Literature

References

SNP Analysis

SNP Quantification

BAC Fingerprinting

Methylation