Questions? Call us at +45 25 32 17 73


PCR Problems

Efficacy is the overall "performance" of a PCR assay – the final outcome of the chosen cycling time, Ta, primer concentrations polymerase function and buffer stringency. High efficacy of a PCR assay implies that the assay has been optimized to short cycling times, high annealing temperatures, low primer concentrations and high stringency of the buffer system – while still maintaining high sensitivity. Each – and all - of these variables require perfect match and optimal performance of the PCR primers.

Unsatisfactory efficacy can be considered as primer dependent or primer independent. Primer independent causes for loss of efficacy may e.g. be suboptimal sample extraction (may leave PCR inhibitors - e.g. salts, proteins - in the sample), suboptimal buffer, suboptimal Mg++ or polymerase concentration or suboptimal cycling profile. Primer dependant causes for loss of efficacy may e.g. be primer mismatch, suboptimal primer concentration, and suboptimal annealing temperature.

False negative results

If the extraction procedure applied does not remove inhibitory factors present in the clinical material, even a high copy number of the target gene will not produce a positive signal. In theory, the PCR reaction can ensure a positive signal from just one copy of the target gene hidden in an infinite amount of unspecific DNA. In practical terms, however, 3–10 copies of the specific target gene sequence are needed to reproducibly give a positive signal, and more than 0.5–1 μg unspecific genomic DNA may inhibit the analysis.

If the primers are not specific, the primer annealing temperature is not optimized, or the concentration of the components of the reaction is not optimized, a false negative result can occur because of inefficient or unspecific amplification. Products only consisting of primer sequences can arise if the two primers have complementary sequences, but can also be seen if the primer and/or enzyme concentration is too high—even if the primers are not complementary. These primer–dimer artifacts will dramatically reduce the efficiency of the specific amplification and will likely result in a false negative result.

False positive results

If the primers are homologous to other sequences than the target gene or if aerosol products from previous PCR analysis with identical primer/target combination are contaminating the reaction, a false positive signal will be the result. Primers cross reacting with other sequences can be a problem when conserved sequences (e.g., the bacterial ribosomal RNA gene) are amplified.

The problem can be avoided by a homology search in GeneBank or EMBL combined with a screening test using DNA from a number of related as well as, unrelated targets. Contamination has in the past been considered the major problem of the PCR technology, but this problem can be minimized by rigorous personnel training, designing the PCR laboratory according to the specific needs of this technology and application of the dUTP/UNG carryover prevention system already included in commercial PCR kits. Likewise, random off-target amplification has been limited by the introduction of hot-start polymerases and heat-activated nucleotides.

This system substitutes uracil for thymine in the PCR, and if the following PCR analyses are initiated with an incubation with a uracil-degrading enzyme such as uracil-N-glycosylase, contaminating–but not wild-type–DNA will be degraded. Implementation of this technology in the PCR analysis has reduced the problem of contamination in most routine PCR laboratories.