DNA Sequencing & Genotyping at AWRI/GVSU

Universal Primer Tag

The genetic analyzer is capable of scoring fragments produced by PCR (e.g. microsatellite, AFLP, etc). To score microsatellites on an automated DNA sequencer, each primer pair requires the addition of a dye on one of the primers. Depending on the dye, this modification to the primer can cost as much as $100. These applications generally involve some optimization at the start of each project and screening new primers can be very expensive.

 

GVSU’s sequencing and genotyping facility can supply you with a fluorescent primer so you can quickly and cheaply screen multiple loci in a single run on the instrument. Employing the methods Boutin-Ganache et al. 2001 and Shimuzu et al 2002, a universal tag can be added during PCR to the 5’ end of a locus specific primer containing the universal tag sequence on the 5’ end. We supply you with the fluorescently labeled universal primer tags (see 1 below), which are normally quite expensive to buy and use only a little. You supply your own locus specific primers: the forward primer with the additional universal tag sequence at the 5’ end where the tag will bind, and the reverse primer (2 and 3 below).

 

The instrument can separate and score multiple colors/size fragments at one time, even within the same capillary. We have three different colored tags (6-FAM, VIC, and NED) that can be combined (three independently run PCR products combined in the same well for a run on the instrument). This means you can explore three loci for as little as $1 per sample for the run on the instrument and even more if you have fragments that are different enough in size to use the same color more than once. Liz-500 (ABI) or MapMarker-1000 (ROX, Bioventures) size standards can be used.

 

The PCR is carried out with three primers:

 

1. Universal Primer Tag (designed by Travis Glenn, Univ. of SC):

 

5’– /DYE/CAGTCGGGCGTCATCA – 3’

 

2. Forward Primer Locus Specific w/ Primer Tag sequence:

 

  Universal Tag Sequence - Locus Specific

5’ – CAGTCGGGCGTCATCAXXXXXXXXXXXXXXXXXXXX – 3’

 

3. Reverse Primer Locus Specific

 

 5’ – XXXXXXXXXXXXXXXXXXXX – 3’

Page last modified May 31, 2011