D21S11 " D8S1179 B
and 9 STR markers in 2.1 (Figure 14.8). There are three STR systems in common between these two kits, namely VWA, TH01, and TPOX, in order to help verify sample concordance and avoid sample shuffling. The STaR Call™ 3.0 genotyping software automatically compares the three overlapping loci between the PowerPlex® 1.1 and 2.1 systems and highlights any non-agreeing alleles as an internal quality control check.
Samples amplified with both PowerPlex® 1.1 and 2.1 are shown in Figure 14.8. The 505 nm scan detects the fluorescein-labeled PCR products while the 585 nm scan detects the tetramethyl rhodamine-labeled PCR products. The PowerPlex® 2.1 STR markers extend to a higher size range with the larger PCR products being almost 500 bp in size. Note that the position of the STR locus impacts its resolution on the gel. Alleles from the D3S1358 STR locus travel further through the gel matrix and are therefore better resolved from one another compared to FGA alleles that are near the top of the gel. Thus, it is more difficult to distinguish off-ladder alleles within the FGA locus compared to the D3S1358 locus because the alleles are not spread apart as well.
The data file size needed to capture the information from a fluorescence scan of a gel is quite large. Due to the different number of alleles and DNA size range, a typical gel scan of PowerPlex® 1.1 is approximately 36 Mb in size while a scan of a full PowerPlex® 2.1 gel takes up approximately 48 Mb for the storage
Rhodamine Red-X Scan Texas Red-X Scan
IPenta E .
450 425 400 375 350 325
of all information on that gel. A computer hard drive can fill up rather quickly with these image files necessitating the use of zip drives or magneto optical disk drives for data storage.
Figure 14.9 shows the spectrally separated gel images from a PowerPlex 16 BIO result on an FMBIO III+ instrument. The FMBIO III+ is more sensitive that the older FMBIO II instruments in part because it uses three lasers with excitation wavelengths of 488 nm, 532 nm, and 635 nm instead of just the 532 nm excitation present in the FMBIO II.
Figure 14.9 FMBIO III+ color-separated STR data collected from four scans of a gel containing PowerPlex® 16 BIO PCR-amplified samples. Each scan detects the PCR products labeled with one of three dyes: fluorescein, JOE, and Rhodamine Red-X. The ILS600 size standard is labeled with the fourth dye Texas Red-X and contains DNA fragments ranging from 100-600 bp in 20 or 25 bp increments (the upper bands are not captured on this gel). The STR loci amplified are indicated above the alleles observed. Allelic ladders are on the far left and right of each scan. The combined color image for the same gel may be seen in Figure 13.11. Figure courtesy of Margaret Kline, NIST.
ISSUES WITH THE FMBIO II APPROACH
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