Fragment Analysis

  • Internal and RI-EPSCOR user fee for FY2016: $2.10 per well per run (see table below)
  • Please see our Fees page for instructions regarding payment for services as well as our rate for External Academic Users.

Our Life Technologies / Applied Biosystems 3130xl and 3500xl genetic analysis systems employ a 16- or 24-capillary array, respectively. A single run of 16 or 24 wells in a 96-well sample plate therefore uses the same amount of consumables in all capillaries regardless of the number of wells analyzed during the run. As a result, pricing for all Fragment Analysis methods is set on a per run basis:

3130xl 3500xl
Number of Runs Number of Wells Cost* Number of Runs Number of Wells Cost*
1 1-16 $33.60 1 1-24 $50.40
2 17-32 $67.20 2 25-48 $100.80
3 33-48 $100.80 3 48-72 $151.20
4 49-64 $134.40 4 72-96 $201.60
5 65-80 $168.00
6 81-96 $201.60

* Rate charged to URI, RI-INBRE & RI-EPSCoR users. Financial assistance has been provided by RI-EPSCoR (NSF Award EPS-1004057) and the College of the Environment and Life Sciences at URI.

The 3130xl and 3500xl systems are capable of performing many different genotyping methods based on the sizing of fluorescently labeled DNA fragments. These methods include the analysis of Short Tandem Repeats (STR) or Microsatellites, Single Nucleotide Polymorphism (SNP), Amplified Fragment Length Polymorphism (AFLP), Terminal-Restriction Fragment Length Polymorphism (T-RFLP), Loss of Heterozygosity (LOH) and Multiplex Ligation-dependent Probe Amplification (MLPA®). Following capillary electrophoresis, the run data is analyzed by users with the ABI GeneMapper v5.0 software that is set up on a dedicated server PC in room 352 CBLS. This analytical software may be used in the GSC or on the URI LAN. We currently have 1 network client license for off-site analysis. Please inquire with the GSC manager regarding set up of the GeneMapper client software.

Example
Example of the display of microsatellite fragments (red, black and green peaks) compared to the size standard GS600LIZ (orange peaks) in GeneMapper

Multiplexing Capability: The 3130xl and 3500xl have been calibrated for use with Applied Biosystems five-dye chemistry, the DS-33 Dye Set (G5 filter set). The DS-33 dyes used for specific labeling of fragment primers are 6-FAM™, VIC®, NED™, and PET®. A fifth dye, LIZ®, is used for the size standard with either 500, 600 or 1,200 bp ladders. This five-dye chemistry permits the analysis of multiple fragments in the same reaction well. The increased multiplexing capacity over four-dye systems is advantageous in improving sample throughput and in reducing user costs. Additional information on dye sets can be found in the ABI Dye Set card

Microsatellite Analysis

Short Tandem Repeats (STRs) or microsatellites are polymorphic DNA loci that contain a repeat sequence of 2-7 nucleotides. Microsatellites can be used to detect genetic variations or polymorphisms in the alleles of most organisms. Genome specific polymorphisms can then be identified with a locus tag or marker and applied to population studies of genotypic diversity.

User supplied consumables:

DNA template
Primers – one unlabeled & one labeled at the 5′-end with a DS-33 dye
HiDi formamide (ABI P/N 4311320)
Standard PCR reagents
ABI Size Standard: GeneScan 500 LIZ® (P/N 4322682), GeneScan 600 LIZ® v2.0 (P/N 4366589), or GeneScan 1200 LIZ® (P/N 4379950)

When ordering primers, note that Applied Biosystems is the sole vendor for the VIC, NED, and PET dye labels. 6-FAM labeled primers may be ordered from other vendors such as Integrated DNA Technologies.  To order primers from Life Technologies please visit their website.

Sample Setup:

We strongly suggest that you use the GS600-LIZ size standard. When multiplexing, primer pairs should be designed to allow sufficient separation (>100 bp) between the expected amplified fragments. To setup the analysis mixture, a master mix should be prepared that contains 10µl HiDi formamide and 0.5 µl of GS600LIZ standard for each reaction well to be analyzed. At the beginning, the first runs will be trial and error to see how well the PCR products were amplified. Add 1 µl of each of the PCR products to the master mix in a strip tube or wells in a 96-well plate. The goal is to achieve intensities between 1,000 to 4,000 counts in the raw fluorescent signal. PCR products beyond these limits will need to be either diluted (in formamide) or augmented in the volume added. Adjustments may also need to be made for the intensity of the specific dyes used for labeling. FAM labeled primers are the brightest and most stable and will therefore require less sample volume than the other dyes.

Sample submission guidelines:

  • Samples must be submitted in strip tubes or a 96-well plate (sealed or capped).
  • We strongly recommend that you include one well with size standard ONLY.
  • Submit your sample IDs using the FA Sample Setup Form.
  • Label your samples with the following code: Your initials followed by the number 1 to 999 (i.e.: PJ1). You should increment the number with each submission. This code enables easy file distribution.
  • Indicate the expected sizes of your fragments and which dyes were used.

Data Distribution:

The data files are saved in the fsa format. Raw data will be copied from the 3130xl/3500xl host computer and then distributed by email as well as transferred to the GeneMapper server computer. The D-drive of this PC is accessible for access by users on the URI LAN. Please see the GSC manager for details.

Data Analysis:

Fragment analysis files can be analyzed with a variety of software. As described above, the GSC has a license for the Life Technologies GeneMapper v5.0 software as well as one client-server license for use on the URI LAN. GeneMapper is a flexible genotyping software package that provides quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems and can perform many applications including amplified fragment length polymorphism (AFLP), loss of heterozygosity (LOH), microsatellite, and SNP genotyping analysis. The latest version of the software also offers new methods for analyzing microsatellite markers that contain mono-, penta- and hexa-nucleotide repeats as well as the existing di and tetra-nucleotide repeat functionality.

Applied Biosystems also has Peak Scanner™ v1.0 software available for free download (Windows PCs only). Use this 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. The software allows you to view, edit, analyze, print, and export fragment analysis data generated using the Applied Biosystems genetic analyzers. Peak Scanner™ enables the simultaneous viewing of raw and analyzed data as well as the ability to size large fragments such as 1200 bp sized fragments. This flexible software tool has the potential to be used in many novel applications and is an easy way to perform DNA fragment analysis.