Competitive primers were synthesized on basis of identical sequence and blocked by amination at the 3' end to allow annealing, but avoid elongation during the PCR process.
A six-point 4-fold serial dilution series made from reference human genomic DNA (Roche), starting from 64 ng/µl down to 0.0625 ng/µl, was created in 10 ng/µl yeast tRNA as carrier. The same dilution of the carrier was used to create a NTC sample.
qPCR amplifications were performed in 7.5 µl total reaction volume containing:
3.75 µl 2x custom made qPCR SYBR green I Mastermix (Eurogentec)
0.375 µl forward primer (5 µM) and 0.375 µl reverse primer (5 µM)
1 µl of a mixture of nuclease-free water and equal amounts of both forward and reverse competitive (aminated) primers
2 µl diluted standard
A total of 7 competitive mixes were prepared for each dilution point, containing 0%, 5%, 10%, 20%, 30%, 40%, and 50% (of the total amount of primer) competitive (aminated) forward and reverse primers. Each reaction was run in triplicate.
The qPCR cycling was performed on the LightCycler480 (Roche) using white LightCycler480 384-multiwell plates with Light-Cycler480 sealing foils (Roche).
The cycling conditions were com prised of 10 min polymerase activation at 95 °C, and 45 cycles of 15 s at 95 °C, 30 s at 60 °C, followed by a dissociation curve analysis from 60 to 95 °C.
Format
ds_competimer
A data frame with 6,615 rows and 10 columns:
wellWell identifier.
replicateReplicate identifier.
dyeIn all reactions the SYBR Green I master mix (Roche) was used, so the value is always
"SYBR".pctPercentage of competitive (aminated) primers in the mix.
concConcentration of reference human genomic DNA (Roche): from 64 ng/µl down to 0.0625 ng/µl.
targetTarget identifier: AluSx.
sample_typeSample type:
"ntc"(no template control) or"std"(standard).dilutionDilution factor (the reciprocal of
conc, i.e.64 / conc). Higher number means greater dilution.cyclePCR cycle.
fluorRaw fluorescence values.