The sizing of DNA fragments with internal standards is performed as illustrated in Figure 15.2. The most common algorithm used for determining the DNA fragment size is known as the Local Southern method. This method uses the size of two peaks on either side of the unknown one being measured in order to make the calculations (Elder and Southern 1983). Using the example in Figure 15.2, the '165.05 bp' peak size is determined with Local Southern sizing by the position of the 150 and 160 bp peaks on the lower side and the position of the 200 and 250 bp peaks on the upper side.
The Local Southern method works very well for accurate sizing of DNA fragments over the 100-450 bp size range necessary for STR alleles. However, there are some caveats that should be kept in mind that depend upon the internal size standard used. Within the GS500-R0X and GS500-LIZ size standard, the 250 bp peak (and sometimes the 340 bp peak as well) does not size reproducibly especially when there are temperature fluctuations across or between runs. Therefore, the 250 bp peak is typically left out of analyses by not designating it as a standard peak (Moretti et al. 2001, Klein et al. 2003).
It is important to realize that unknown DNA fragment peaks cannot be accurately determined which are larger than the peaks present (or designated by the GeneScan software) in the internal sizing standard. Nor can peaks that fall near the edge of the region defined by the internal sizing standard. This is due to the fact that two peaks from the size standard are needed on either side of the unknown peak with Local Southern sizing. Therefore, with the GS500-ROX internal standard commonly used in conjunction with the AmpFlSTR kits, any unknown peaks falling above 490 bp or below 50 bp will not be sized with the Local Southern method. Likewise, if the signal intensity for any of the calibration peaks in the internal sizing standard is too weak, then unknown peaks in that region will not be sized accurately. For this reason it is important to check that all peaks in the internal sizing standard are above the relative fluorescence threshold to be called as peaks and that these peaks are accurately designated by the software.
Two studies have found that a different sizing algorithm called the Global Southern method works well and maintains a better precision than Local Southern sizing in situations when temperature fluctuations can occur (Hartzell et al. 2003, Klein et al. 2003). Global Southern involves fitting all of the peaks in the size standard to form a best fit size calibration line rather than just using the two peaks above and below the peak of interest as is done with Local Southern. Regardless of which method is used it must be consistently applied to both the allelic ladders and samples being typed so that equivalent size comparisons may be made.
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