Ann M. Lins, Paul Otto, and James W. Schumm
Promega Corporation, Madison, WI

× Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø × Ø

With the increasing use of STR loci for forensic and paternity analyses, fluorescent detection of amplified fragments has become quite commonplace using the Hitachi FMBIO^® Fluorescent Scanner, the Molecular Dynamics FluorImager^tm SI Fluorescent Scanner, or the Applied Biosystems 373 and Prism^tm 377 DNA Sequencers. Current marker systems have been limited, first by the uneven spacing of fragments contained within the different fragments of the marker set. These limitations have potential to cause imprecision in defining sizes of unknowns, especially in regions which have a paucity of fragments, and may cause deviation from a linear sizing plot.

We have developed a Fluorescent Size Marker which contains evenly spaced fragments to overcome the limitations mentioned above. This marker contains 16 DNA fragments of 60, 80, 100, 120, 225, 275, 300, 325, 350, 375 and 400 bases, respectively. Each fragment contains the entire sequence of all smaller fragments in the set. That is to say, the small fragments are subsets of each larger fragment. This characteristic limits deviation from linearity in the measurements, while the even spacing provides increased precision.

Each fragment of the Fluorescent Size Marker, 60-400 base, is labeled with carboxy-X-tetramethylrhodamine (TMR). This fluorescent tag is readily visualized using either the Hitachi FMBIO^® Fluorescent Scanner, or one of the Applied Biosystems DNA Sequencers.

This marker has been used to increase precision in analysis of the GenePrint^tm multiplex systems. When included in each lane of a gel, gel artifacts which may result in lane to lane variability of fragment migration, can be corrected with the use of instrument software. By including one or two lanes of allelic ladder along with the lanes containing amplified samples, it is possible to generate very high throughput.

This approach has been used with the fluorescein-labeled GenePrint^tm CTTv, GenePrint^tm FFFL, and GenePrint^tm GammaSTR^tm quadriplex systems. Of course, the highest throughput has been achieved by combining the use of the Fluorescent Size Marker with the two-color, eight locus GenePrint^tm Powerplex^tm System.