- Internal and RI-EPSCOR user fee: $0.60 per well per run (see table below)
- The Applied Biosystems 3130xl genetic analysis system employs a 16 capillary array.
A single run of 16 wells 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:
|Number of Runs
||Number of Wells
|* Rate charged to URI, RI-INBRE & RI-EPSCoR users. Financial subsidies have been provided by RI-EPSCoR (NSF Award EPS-1004057) and the College of the Environment and Life Sciences at URI.
- The 3130xl system is 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)
and Loss of Heterozygosity (LOH). Following capillary electrophoresis on the
3130xl, the run data is analyzed by users with the ABI GeneMapper v4.0 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. As demand and funding permits, other
licenses may be added. Please inquire with the GSC manager regarding set up
of the GeneMapper client software.
- Multiplexing Capability:
Our 3130xl has 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 standard with fragments upto 1200 bp in size. 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 ABI dye sets can
be found in the following file: ABI Dye Set card.pdf
- 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
Example of the display of microsatellite fragments (red, black and green peaks)
compared to the size standard GS600LIZ (orange peaks) in GeneMapper v4.0.
- 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), 600 LIZ« (P/N 4366589), or 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 (www.idtdna.com). To order primers from Applied Biosystems please read the following document: ABI Primer&Probe.pdf
- 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 10Ál HiDi formamide and 0.5 Ál 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 Ál 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 that 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.
- Supply a data sheet identifying your samples and their plate location. (We can supply you with a simple 96-well matrix).
- 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 host computer to a CD-R 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 acquired a license for the Applied Biosystems GeneMapper v4.0 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. For an additional
overview of microsatellite analysis on the 3130xl please see the following
document: Microsat Anal.pdf.
Applied Biosystems also has Peak Scanner? v1.0 software available for free download.
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 3130xl genetic analyzer.
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. Visit the Applied Biosystems website (www.appliedbiosystems.com)
to obtain a copy of this software.
For additional information, please visit the Applied Biosystems genetic analysis applications page:
This site provides scientific information, published resources and data examples for areas of
study ranging from discovery to validation of genetic information in disease research, population
genetics, diagnostics, or gene discovery.
||Rhode Island Genomics and Sequencing Center,
College of the Environment and Life Sciences
352 Center for Biotechnology & Life Sciences
120 Flagg Rd.
University of Rhode Island, Kingston, RI 02881
Email Chandu Dondeti for questions on website
|Copyright ę 2003.
University of Rhode Island, All rights reserved.