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Effect of Internal Control Sequence and Length on the Response to PCR Inhibition in Real-Time PCR Quantitation

NCJ Number
Date Published
March 2014
6 pages
This study examined the effects of commonly encountered PCR (polymerase chain reaction) inhibitors on internal controls of varying amplicon length and sequence structure, hypothesizing that the sequences with greater guanine and cytosine content and smaller length would be the least prone to inhibitory interactions.
The results indicate that although amplicon sequence has minor effects on amplification efficiency and melt curves, amplicon length has a more dramatic effect, regardless of inhibitor type. The changes in efficiency between the 80 bp amplicon and the 230 bp for any sequence composition show dramatic differences; the largest amplicon fails in many inhibitor concentrations where the smallest amplicon amplifies rather well. This is presumably because the larger sequences require a longer interaction with the polymerase and provide a wider range of DNA sequence variations with which to bind. This is an expected finding, since inhibitory events commonly cause large STR loci to drop out first. When comparing across a range of amplicon sizes, the short amplicon sizes are more capable of amplifying in the presence of an inhibitor. This is a significant finding, since current real time PCR kits vary in the size of their internal PCR control length and sequence, thus producing disparate responses to inhibition. It should be possible to tailor the composition of internal PCR controls in quantification kits to a desired level of sensitivity by altering the length of the internal control sequences. 6 figures, 5 tables and 16 references
Date Published: March 1, 2014