Microsatellite, SNP, MLVA, VNTR, MIRU tRFLP, ARISA
Our Laboratory provides automated multicolour fluorescent analysis of PCR-based DNA fragments.
Examples of the types of projects that may be undertaken include:
- gene mapping using amplified fragment length polymorphisms (AFLP)
- tRFLP (Restriction Fragment Length Polymorphism)
- screening for single nucleotide polymorphisms (SNP)
Also applications to Microorganism Typing are proposed by our lab like : MLVA (Multilocus Length Variable alleles), MLST (Multilocus Sequence Typing), VNTR (Variable Number Repeats).
Up to four different fluorescent dyes may be used for labeling, allowing for a greater amount of multiplexing in a single lane for higher throughput. An internal size standard, labeled with a fifth dye, provides accurate and reproducible size determination and quantification.
The dye mostly used to label the PCR product is 6-FAM (it works well and is cheaper than others), although our sequencer is calibrated to use all the following combinations:
– When Size standardd is ROX, eligible dyes are 6-FAM, VIC or HEX and NED.
– When Size standard is LIZ, eligible dyes are 6-FAM, VIC, NED and PET.
|LIZ (Size Standard)||ORANGE|
Fragment analysis kits may be purchased through Applied Biosystems. Alternately, custom synthesized fluorescently labeled primers may be obtained directly from primer synthesis Providers.
Purchase of the appropriate kit and primers, sample set-up and amplification, and (optional) multiplexing of samples into 96-well sample plates is done prior to submitting the samples for analysis.
The DNA Sequencing Laboratory will acquire the data using our Applied Biosystems 96 capillary 3730XL analyzer.
Due to the complexity of assigning genotypes, the automatically called data analysis is not done by our laboratory staff.