Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: a novel solution for dsDNA artifacts.
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.
McLaren
Robert S
RS
Promega Corporation, Madison, WI 53711, USA. bob.mclaren@promega.com
Ensenberger
Martin G
MG
Budowle
Bruce
B
Rabbach
Dawn
D
Fulmer
Patricia M
PM
Sprecher
Cindy J
CJ
Bessetti
Joseph
J
Sundquist
Terri M
TM
Storts
Douglas R
DR
D016477:Artifacts; D001483:Base Sequence; D004247:DNA / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes; D005784:Gene Amplification; D015894:Genome, Human; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D012235:Rhodamines
D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D012235:Rhodamines; C005358:tetramethylrhodamine; D004247:DNA