As breakthroughs in reagent chemistries increase the ability to detect trace amounts of DNA, Promega is pushing the limits of available instrumentation. Recently, some of Promega’s PowerPlex 16 and PowerPlex ES System users started to notice a split, or n-1 peak, at the wVA locus, which was affecting the ability of some labs to interpret their data. This only occurred on the 4 and 16 capillary electrophoresis platform, not on single capillary systems and was more prevalent in customer labs during the winter when lab temperatures could dip.
The issue was traced to an insufficiently denaturing environment in the exposed portion of the capillary outside of the heating oven on the multicapillary instruments. This allowed reannealing of the unlabeled unincorporated vWA primer to the 3´ end of the tetramethylrhodomine (TMR)-labeled strand of the vWA amplicon, following electrokinetic injection. The partially double-stranded DNA migrated faster than the single-stranded amplicon and this mobility shift manifested itself as the split or n-1 peak.
“We were in a situation where the split peak artifact was being caused by an instrumentation-related issue, but not being able to resign the instrumentation we had to come up with a work around with the chemistry” said Bob McLaren, Ph.D., senior R & D scientist. Dr. McLaren and his team found that, stretching out the pre-run time would keep this reannealing from occurring, presumably by increasing the temperature in the exposed portion of the capillary array.
“The split would go away, but increasing the pre-run time wasn’t really enough to solve the problem,” Dr. McLaren noted. “What we eventually ended up doing was incorporating into the Internal Lane Standard 600 (ILS600) a sacrificial hybridization sequence oligonucleotide that is complementary to the vWA primer. This preferentially hybridizes to the vWA primer and thus prevents the primer from hybridizing to the 3´ end of the TMR-labeled strand of the vWA amplicon.”
Users also noted an n-10/n-18 artifact at the vWA locus in the same STR multiplexes. Again, the root cause turned out to be an inability for already denatured DNA to be maintained in a denatured state on the exposed portion of the capillary array. In this case, the n-10/n-18 artifact was actually double-stranded vWA amplicon. In a similar fashion to the split peak solution, two complementary oligo targets (COT1 and COT2) were added into the ILS600, which preferentially annealed to the unlabeled strand of the vWA strand, thereby preventing it from annealing to the TMR-labeled strand of the vWA amplicon.
“The presence of these three oligonucleotides effectively eliminates both the split peak and the n-10/n-18 artifacts in both products without affecting sizing of alleles at the vWA locus—or any other locus,” Dr. McLaren explained. “The users found the problem, which turned out to be instrument related, and we went the extra mile to find a solution.”