Fragment Analysis


Overview

In addition to SNP detection, we provide capillary electrophoresis services to support the analysis of more complex polymorphisms (e.g., short tandem repeats (STRs)) via the measurement of fragment length polymorphisms (FLPs). We use the ABI 3730xl capillary electrophoresis platform (the same used in our Sanger sequencing services), enabling highly multiplexed FLP detection, and a lower cost per data point. Due to the high-throughput nature of the ABI 3730xl, the unit of submission is a plate of samples.

Researchers can purchase fragment sizing standards themselves, and submit amplicons + standards ready for loading onto the instrument, or may opt to have the UMGC add sizing standards as well as load samples. Although not listed here, the UMGC can also provide a full-service FLP analysis, including amplification. Due to the customized nature of most multiplexed FLP assays, please inquire for the cost of such analysis.

Choose a size standard that has at least two peaks below your smallest basepair size and at least two peaks above your largest basepair size. The table below shows the labeling combinations that correspond to the size standard dye.

Dye Set Labeling Combinations Size Stanard Dye
6-FAM™, HEX™, NED™ MapMarker® or ROX™
6-FAM™, VIC®, NED™ MapMarker® or ROX™
5-FAM™, JOE™, NED™ MapMarker® or ROX™
6-FAM™, VIC®, NED™, PET™ LIZ®

The UMGC offers the following size standard dyes.


LIZ®-120 has the following sizes: 15, 20, 25, 35, 50, 62, 80, 110, and 120.

ROX™-400HD has the following sizes: 50, 60, 90, 100, 120, 150, 160, 180, 190, 200, 220, 240, 260, 280, 290, 300, 320, 340, 360, 380, and 400.

LIZ®-500 has the following sizes: 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and 500.

MapMarker®-1000 has the following sizes: 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000.

Note: PCR products used for fragment analysis don’t need to be purified before separation on the genetic analyzer. There are two general multiplexing strategies:

Multiplex during the PCR reaction by combining more than one pair of primers in the same PCR reaction tube. This strategy significantly decreases the cost per analysis, but requires optimization. Multiplexing primers must not produce products of similar lengths and cannot be labeled with the same fluorescent dyes. Primers cannot contain large regions of complementarity. Primers should have similar melting temperatures. Before performing PCR using this strategy, perform a preliminary check for primer compatibility and test for successful co-amplification.

Pooling PCR product after PCR. This strategy is more simple and flexible, but pooling products from multiple PCR reactions often increases the salt concentration in the loaded samples, which can have unwanted downstream effects.

Questions?


Contact us at umgcseq@umn.edu with any questions.

Pricing

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Standard

Service Scale Unit UMN External
Fragment Analysis.1 1-any plate of 96 $223.16 $256.48
Fragment Analysis. Instrument Run.2 1-any plate of 96 $131.32 $151.09

 

Notes
  1. UMGC provides and adds sizing standards to user-supplied amplicon pools.
  2. Researcher provides amplicon pools containing sizing standards, ready for loading onto capillary electrophoresis instrument.
Guidelines

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Submission

How to Order


University researchers can place an online order by entering the ONLINE ORDERING SYSTEM.

Researchers outside the University can request a form by emailing umgcseq@umn.edu.

Submit 100 ng in 10 ul n any 96-well plate with the Fragment Analysis Request Form. Be sure to cover plate securely. If dropping off samples, please cover plate with a plate seal.

After completing the form, print and submit with your samples. Samples can be dropped-off at one of our laboratory locations or mailed/shipped. Drop-off locations

 

  • 1-202 Nils Hasselmo Hall (Minneapolis campus)
  • 1-210 Cancer & Cardiovascular Research Building (Minneapolis campus)
  • 20 Snyder Hall (St. Paul campus)
If mailing samples through the United States Postal Service

Please secure and protect samples with padding and address to:

University of Minnesota Genomics Center

1475 Gortner Ave.
123 Snyder Hall
St. Paul, MN 55108
612-625-7736

For shipping using carrier, please address shipment to:

University of Minnesota Genomics Center
1475 Gortner Ave.
28 Snyder Hall
St. Paul, MN 55108
612-625-7736

Deliverables

You will receive an email after results from your entire order have been posted. If you have not received an email within 72-hours of sample submission, please contact umgcseq@umn.edu and include your order number.

Results are in one format: sample files (.fsa). There are a few programs to view .fsa files: GeneMapper® and PeakScanner™. To download PeakScanner™ for free, visit https://www.thermofisher.com/order/catalog/product/4381867

For University researchers, data files are uploaded to the order within the online ordering system; "My Account" tab and then the "Result Files" section on the right next to "History".

For researchers outside the University, data files are emailed to the email address provided on the Fragment Analysis Request Form.

FAQ

Can I pool my samples?


Yes, ABI recommends pooling different dyes after your PCR reaction.

What software can be used to view .fsa files?


There are a few programs to view .fsa files: GeneMapper® and PeakScanner™. To download PeakScanner™ for free, visit https://www.thermofisher.com/order/catalog/product/4381867