Fig 1.
Inhibition of the PCR by genomic DNA.
The primers were amplifying their target sequence in various masses of template DNA. An additional 0.5 μg of non-leukemic genomic DNA was or was not added to each well. In each panel, the 2 solid lines refer to one patient and the 2 broken lines refer to another patient. Panel A shows 2 examples of severe inhibition and panel B shows 2 examples of mild inhibition. A Ct of 50 indicates that the PCR failed and no signal was observed.
Fig 2.
Effect of test primers on amplification of plasmid target in the presence of DNA.
The effect of a primer pair from each of 6 patients on amplification of different numbers of plasmid sequences in the presence of 0.5 μg of genomic DNA was determined. Control reactions, which contained only the plasmid primers, are the solid lines, and the test reactions, which contained both the plasmid primers and a pair of patient primers, are the broken lines.
Fig 3.
Column A shows the results from amplification of patient DNA by patient primer pairs; Column B shows the results from amplification of plasmid DNA by plasmid primers in the presence of patient primer pairs; Column C shows the results from amplification of plasmid DNA by plasmid primers in the presence of widely recommended reverse primers; Column D shows the results from amplification of plasmid DNA by plasmid primers in the presence of HAT-PCR reverse primers and with amplification by the HAT-PCR protocol. The numbers of primer pairs or primers are shown towards the bottom of the Figure and at the top are shown the significance probabilities of the observed results, tested by the Wilcoxon matched-pairs signed ranks test against the null hypothesis of no inhibition.
Table 1.
Frequency of nonspecificity and PCR inhibition produced by conventional PCR and HAT-PCR.
Table 2.
Effect of annealing temperature and Taq concentration on inhibition.
Fig 4.
Effect of the 3’ end of the primer on production of nonspecificity and PCR inhibition.
Experiments were performed with a gradient of annealing temperature. The Ct difference is the Ct for an A/T primer minus the Ct for a G/C primer and the positive value of the median indicated that, at the same annealing temperature, G/C primers evidenced a lower Ct and therefore more nonspecificity. The °C difference is the temperature difference between G/C and A/T primers in the minimum annealing temperature required to prevent inhibition. The positive value of the median indicated that G/C primers required a higher annealing temperature to prevent inhibition. The statistical test was the Wilcoxon matched-pairs signed-rank test.
Fig 5.
Proposed mechanism of inhibition of the PCR by genomic DNA.