Information

Fragment analysis is simply the analysis of labeled PCR fragments that takes advantage of the rapidity and accuracy of the capillary electrophoresis capabilities our our DNA sequencing instrument.

Labeled PCR fragments (single or multiplexed) are run along with a labeled DNA ladder of known size, and the precise size of each PCR fragment is determined mathematically based on its elution time relative to the markers.  Data Analysis is conducted using any of several proprietary analytical programs, some of which (e.g. PeakScanner) are available at no cost.

There are several PCR-based labeling methods, but the most efficient and reliable is the use of end-labeled forward primers, which is presented as an option by most oligonucleotide suppliers.

 

Advantages of Fragment Analysis

Have a large population to analyze?  Do you have genetic markers in mind?  Do you wish there was an easy high-throughput way to judge fragment size accurately and consistently?  Fragment Analysis is what you're looking for.

We can run your fragment analyses quickly and efficiently (48 samples per hour) to help you churn out your results in record time.  We can also assist you in developing your strategy and selecting the proper analytical components and in analyzing your data.

We have standard instrument protocols for microsatellite, SNaPshot, AFLP, and other fragment analysis procedures.  Most runs are complete within an hour, and do not require post-reaction cleanup.  Just load your PCR labeling reaction into a plate, mix with your pre-selected labeled DNA ladder, and hand it over to us.

 

Considerations

The size(s) of the fragments to be analyzed must guide the choice of size marker.  You should select a size marker range that encompasses your fragments.

Our instrument can handle all available labeling dyes with the exception of TET.  Dyes come in 5 different emission colors (blue, green, yellow, orange, and red).  The dyes Rox (red), TAMRA (red), and LIZ (orange) are typically reserved for size standards, and should not be used to label your fragments.

When selecting a dye to label your fragments, you should avoid using a dye the same color as your ladder, and if using end-labeled primers, you should select one of the more intense dyes such as 5-FAM, 6-FAM, or VIC, although others work efficiently as well.

 

Sample Submission

To submit your samples, mix your labeling PCR reactions with the appropriate amount of size marker (we can assist in determining the proper ratio).  The aliquot into a 96-well plate, cover with foil to protect from light, and submit them along with a Fragment Analysis Submission Form.  We can help you set up an electronic submission form to eliminate delays due to data entry as well.

Fragment analysis reactions should be run very soon after the reaction terminates, so the best policy is to provide the core facility with a day's warning and then bring the samples directly to the sample refrigerator in 104 HBRC.