Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts

Robert S. McLaren, Martin G. Ensenberger, Bruce Budowle, Dawn Rabbach, Patricia M. Fulmer, Cindy J. Sprecher, Joseph Bessetti, Terri M. Sundquist, Douglas R. Storts

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Several laboratories have reported the occurrence of a split or n - 1 peak at the vWA locus in PowerPlex® 16 and PowerPlex® ES amplification products separated on 4- and 16-capillary electrophoresis instruments. The root cause of this artifact is post-PCR reannealing of the unlabeled, unincorporated vWA primer to the 3′-end of the tetramethylrhodamine (TMR)-labeled strand of the vWA amplicon. This reannealing occurs in the capillary post-electrokinetic injection. The split peak is eliminated by incorporation into the loading cocktail of a sacrificial hybridization sequence (SHS) oligonucleotide that is complementary to the vWA primer. The SHS preferentially anneals to the primer instead of the TMR-labeled strand of the vWA amplicon. In addition, the n - 10/n - 18 artifact that may be seen at the vWA locus was determined to be due to double-stranded amplicon formed post-electrokinetic injection into the capillary. This was also eliminated by adding in two Complementary Oligo Targets (COT1 and COT2) in addition to the SHS oligonucleotide into the loading cocktail. These three 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 and therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminate 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, AmpFlSTR® Profiler Plus® ID, AmpFlSTR® Cofiler®, and AmpFlSTR® SGM Plus® kits.

Original languageEnglish
Pages (from-to)257-273
Number of pages17
JournalForensic Science International: Genetics
Volume2
Issue number4
DOIs
Publication statusPublished - 1 Sep 2008

    Fingerprint

Keywords

  • Artifacts
  • Capillary electrophoresis
  • Denaturation
  • PowerPlex
  • Secondary structure
  • Short tandem repeat (STR)
  • Split peak
  • vWA

Cite this