Post-Injection Hybridization of Complementary DNA Strands on Capillary Electrophoresis Platforms: A Novel Solution for dsDNA Artifacts

• NCJ Number: 225042
• Journal: Forensic Science International: Genetics Volume: 2 Issue: 4 Dated: September 2008 Pages: 257-273
• Author(s): Robert S. McLaren; Martin G. Ensenberger; Bruce Budowle; Dawn Rabbach; Patricia M. Fulmer; Cindy J. Sprecher; Joseph Bessetti; Terri M. Sundquist; Douglas R. Storts
• Date Published: September 2008
• Length: 17 pages
• Publication Format: Document
• Publication Type: Report (Study/Research)
• Language: English
• Country: Ireland

Annotation

After describing two artifacts that have been observed at the vWA locus in the PowerPlex 16 System, this article presents a simple solution for addressing both artifacts that involves the addition of three, unlabeled oligonucleotides to the Internal Lane Standard 600 (ILS600).

Abstract

The two artifacts are due to double-stranded DNA products formed by reannealing of complementary DNA strands while residing in the capillary containing POP-4TM polymer. This reannealing occurs in the capillary post-electrokinetic injection. In the proposed solution to these artifacts, the oligonucleotides compete with the tetramethylrhodamine (TMR)-labeled vWA amplification fragment for hybridization to the causative complementary DNA strands, minimizing the formation of these artifacts. The oligonucleotides used are two Complementary Oligo Targets (COT1 and COT2) and the sacrificial hybridization sequence (SHS) oligonucleotide. These 3 oligonucleotides are complementary to the 33 bases at the 5’-end of the unlabeled vWA amplicon strand and the 60 bases at its 3’-end. They therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminates both the split peak and n - 10/n - 18 artifact in PowerPlex 16 and PowerPlex ES amplification products without affecting sizing of alleles at the vWA locus or any locus in the PowerPlex 16, PowerPlex Y, PowerPlex ES, AmpFISTR Profiler Plus ID, AmpFISTR Cofiler, and Amp/FISTR SGM Plus kits. The addition of the three oligonucleotides to the loading cocktail involves only a minor modification of the overall assay. The descriptions of materials and methods addresses DNA samples for amplification and for concordance, PCR amplifications, electrophoresis on the Applied Biosystems 3130 genetic analyzer and the ABI PRISM 310 genetic analyzer, and the oligonucleotides. 7 figures, 1 table, and 12 references