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

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts,10.1016/j.fsige

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts   (Citations: 4)
BibTex | RIS | RefWorks Download
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.
Journal: Forensic Science International-genetics - FORENSIC SCI INT-GENET , vol. 2, no. 4, pp. 257-273, 2008
Cumulative Annual
View Publication
The following links allow you to view full publications. These links are maintained by other sources not affiliated with Microsoft Academic Search.
Sort by: