Fragment Analysis and Microsatellite Genotyping

AGRF uses state-of-the-art technologies and high throughput processes for microsatellite genotyping.  This service has been applied to a wide variety of species and can accommodate any species for which microsatellite markers are available. Microsatellites, due to their highly informative nature, provide a powerful tool to any linkage mapping or population genetics study.

Microsatellite Genotyping Applications include:

Fragment Analysis

Microsatellites

Cell Line Identification

Fragment Analysis

The fragment analysis service offered by the AGRF provides both capillary separation and analysis services for fluorescently labelled DNA fragments. Capillary electrophoresis is used for fragment separation and data is analysed using AB GeneMapper software.  Marker types applied to the service include microsatellites, AFLP (amplified fragment length polymorphisms), T-RFLP (terminal restriction fragment length polymorphisms) and MLPA (multiplex ligation-dependent probe amplification), for which analysis can be customised to your marker type and project requirements. 

For this service the customer performs PCR and provides the AGRF with PCR product for electrophoresis.  The DNA Analyser platform can accommodate up to four different coloured fluorescent dyes: 6-FAM, VIC, NED or PET.  This allows multiple markers to be amplified (for the same individual), pooled and then run in a single capillary.  Where multiple markers of the same colour are pooled, it is important to ensure that the fragment size range of markers does not overlap.  A maximum fragment length of 1,000 base pairs can be analysed.

Microsatellites

AGRF has extensive experience in providing support for the selection and design of microsatellite markers for a wide range of plant and animal species.  AGRF offers a full service which includes assistance in designing and ordering fluorescently labelled primer pairs for the microsatellite markers of your choice.  In cases where multiple markers are to be genotyped, this can be done in a cost-effective manner whereby AGRF can panel markers for capillary separation.  Genotyping data is analysed using AB GeneMapper software for the calling of alleles. 

AGRF can assist with microsatellite discovery by using the Illumina MiSeq. A genome can be scanned at reasonably low coverage and still provide microsatellite sequence information and the surrounding sequence data allowing for marker design.

Cell Line Identification

The AGRF human cell line identification service is a method useful in genotyping and is particularly useful in tissue culture strain identification. Two different kits are available multiplex STR systems are available

• Promega PowerPlex 16HS, uses 16 loci markers*
• Promega GenePrint 10 System, uses 10 loci markers*

*Please note there is a minimum number of eight samples per submission and this service is available for research purposes only.

There are two access options for this service:
Full data service

• Users submit DNA for amplification with the PowerPlex assay reagents.

Fragment Analysis Service

• Users submit a PCR sample already amplified by the client using the PowerPlex assay reagents.

Both options include electrophoresis and analysis using the Genemapper ID software to provide called alleles for each of the markers. Final data is supplied to the client for interpretation.
Data interpretation can be performed by matching the provided STR profile to the ATCC database www.atcc.org/STR_Database.aspx