Ruth H. Ikeda
Forensic Science Service, Orange County Sheriff-Coroner, Santa Ana, CA
Artifactual "stutter" or "shadow" bands are produced during PCR amplification of many, if not al all, STR loci. Although some theories have been advanced as to the cause of this phenomenon, to date, no method has been described in the literature which reduces or eliminates these artifactual bands.
"Stutter" or "shadow" bands can pose problems for forensic DNA typing, especially if they are one or multiple repeat unit(s) less than the allelic band. Forensic samples are often of unknown origin and may contain mixtures from two or more sources. Consequently, STR genetic typing results can be difficult to interpret if shadow bands are particularly pronounced or the mixture consists of two donors who have contributed unequal amounts of DNA.
In our laboratory's efforts to validate the GenePrint™ CTT Triplex System (Promega) for forensic casework, I have found the stutter bands were quantitated and compared to their respective allelic bands using FluorImager and Image QuaNT software (Molecular Dynamics), the amount (peak height) of stutter bands averaged 7% ± 4% (maximum =18%) of the allelic band. In order to decrease the amount of shadow bands, many different PCR amplification conditions were tried. The one condition that had a profound effect was replacing Taq DNA polymerase with a "proof-reading" DNA polymerase from Pyrococcus woesei called Pwo (Boehringer-Mannheim). When Pwo DNA polymerase was used instead of Taq, stutter bands were virtually eliminated at the CSF1PO locus with no difference in the total amount of amplified products. Effects at the TPOX locus were unchanged. However, at the TH01 locus, shadow bands became more apparent.
Pwo DNA polymerase cannot be a substitute for Taq for the CTT triplex because of the increased stuttering at the TH01 locus. Therefore, we plan to continue using Taq for the CTT triplex. For samples with pronounced stutter bands, we will use Pwo to amplify the CSF1PO locus alone. This should enable us to differentiate between an artifactual "stutter" and a mixed sample.
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