Abstract

Inhibition of the polymerase chain reaction can jeopardise the success of STR genotyping. A number of studies exist that have looked at the impact of inhibitors on STR profiling; these generally look at the broad impact of inhibition, such as the number of peaks observed, average peak height and profile success. This study investigates inhibition in more detail by comparing patterns of inhibition with known characteristics of an STR kit. By looking at the balance of the loci within STR profiles under different inhibitory conditions and assessing the impact of altering PCR conditions such as annealing temperature on locus performance, hypotheses of inhibitory mechanisms can be formed. The data show that a number of inhibitory mechanisms could be identified. Some of these appeared to be associated with primer annealing, where loci with primers with lower annealing temperatures were observed to be inhibited more than loci with primers with higher annealing temperatures. Other inhibitors appeared to impact on the processivity of the polymerase enzyme affecting high molecular weight loci more than low molecular weight loci; this produces an effect similar to degradation with the classic “ski-slope” pattern. These data can be used to inform what inhibitors may be present within a sample for troubleshooting purposes. Additionally, these data provide information that can be used to improve primer design for improved inhibitor tolerance.

1. Introduction

This study investigates inhibition in more detail by comparing patterns of inhibition with known characteristics of an STR kit. By looking at the balance of the loci within and STR kit under different inhibitory conditions and assessing the impact of altering PCR conditions such as annealing temperature on locus performance; hypotheses on inhibitory mechanisms can be formed.

These data can be used to inform what inhibitors may be present within a sample for troubleshooting purposes. Additionally, these data provide information that can be used to improve primer design to improve inhibitor tolerance.

Following implementation of a new method within the DNA unit laboratory a number of examples of inhibition were observed. Using information obtained from this study the causes of inhibition was quickly determined to be incomplete removal of QSW2 buffer associated with incomplete bead drying during sample purification.

2. Experimental design

The 2800 M control DNA (Promega) was used for all experiments; a 500 pg input of DNA was maintained for all experiments. All samples were amplified using the ESI 17 Fast system as described in the user manual unless otherwise stated.

The experiment was separated into three parts: 1) Understanding the impact of annealing temperature on STR locus success. 2) Assessing the impact of altering the reagent mix in respect to the ratios of reagents used and or the proportion of reagent mix to sample. 3) Spiking samples with known concentrations of inhibitor.

Samples were amplified using six different annealing temperatures ranging from 56 °C to 66 °C at 2 °C intervals using the VeriFlex™ step feature on a Veriti thermal cycler (ThermoFisher). Four replicate samples were performed at each temperature.

The reagent mix experiment was performed in two ways. The first involved systematically increasing or decreasing the amount of buffer or primer by 25 % or 50 % while maintaining the correct volume of the unmodified buffer or primer component. Each condition was tested with two replicates using 7.5 μl of modified mix. The second was to make the reaction mix correctly but to use the wrong volume of reaction mix or sample. Reaction mix volumes were varied between 5 μl and 15 μl (7.5 μl optimal) and sample inputs were varied between 6 μl and 25 μl (17.5 μl optimal) while maintaining a 500 pg DNA input. Full details for all reaction conditions can be found in the supplementary material.

3. ResultsAll results can be found in the supplementary material. Data from the first experiment revealed some interesting trends (Fig. 1). Decreasing the annealing temperature only had a marginal effect on the majority of loci, with the largest affects being observed at the D8S1179 and FGA loci. These loci increased in peak height. At the lowest annealing temperature tested D16S539 showed some reduction in peak height. These trends may be useful in indicating when a thermal cycler is not reaching its intended temperature.

Fig. 1Bar chart showing average normalised peak heights for ESI 17 Fast profiles obtained from different PCR annealing temperatures.

As the annealing temperature is increased a clear pattern of attrition is observed with Amelogenin, D2S441, D8S1179 and FGA markedly reducing in peak height even with just a 2 °C increase in annealing temperature. This suggests that annealing temperatures of the primers associated with these loci may be lower than the other loci in the ESI 17 Fast kit. These data provide estimates of annealing temperature that can be used diagnostically to identify when an inhibitor is present that affects primer binding stability.

The experiments that involved changing the proportion of buffer or primer within the ESI 17 Fast Reagent mix had little impact on inter-locus balance. Increasing the proportion of buffer within the mix decreased peak heights. Decreasing the amount of buffer increased peak heights. Increasing the amount of primer present within the reaction mix increased peak heights, whilst decreasing the amount of primer decreased peak heights.

When the proportion of reaction mix was lowered; by either reducing the volume of reaction mix or increasing the amount of sample/water, then peak heights decreased in a uniform manner with no notable changes to inter-locus balance.

When the proportion of reaction mix was increased; by either increasing the volume of reaction mix or decreasing the amount of sample/water, then some locus specific effects begin to emerge with Amelogenin and D2S441 producing lower peak heights.

This is similar to the pattern observed with increasing annealing temperature without the previously observed impact of D8S1179 and FGA.

A number of different inhibitors were tested to ascertain their impact on the ESI 17 fast kit.

The impact that the various inhibitors had on profile balance was varied. Tannic acid inhibited the samples in a different way to the other inhibitors tested. The impact of tannic acid was very similar to that observed with DNA degradation with high molecular weight loci producing smaller peaks than low molecular weight loci. This may indicate tannic acid degrades DNA or affects the processivity of the polymerase resulting in poorer results from high molecular weight amplicons. QSW1 was highly inhibitory and knocked out most high molecular weight loci even at very low concentrations.

Humic acid, hemin and QSW2 produced results similar to those observed when the annealing temperature was increased, with a negative effect on the Amelogenin, D2S441, D8S1179 and FGA loci. This indicates that these inhibitors affect primer binding stability. Designing primers with higher Tm may improve PCR performance in the presence of these sorts of inhibitor.

These data were used when drop-out of Amelogenin, D2S441 and D8S1179 were sporadically observed after the introduction of a new DNA purification method. The availability of the data presented here allowed for the rapid identification of the most likely cause of these sporadic events. Further investigation confirmed that incomplete drying of the QSW2 reagent from the magnetic beads was the underlying cause. This was removed by improved liquid handling parameters on the robotic platforms used.

4. Conclusions

Increasing PCR annealing temperature causes characteristic patterns of locus drop-out, indicating some primer pairs are more thermally stable than others. Altering PCR reaction conditions can adversely affect those loci that are less thermally stable. Some inhibitors can affect the thermal stability of the primers used within a reaction. Knowledge of primer thermal stability can aid in troubleshooting when unusual results are observed.

Appendix A. Supplementary dataThe following are Supplementary data to this article:Article InfoPublication History

Published online: December 02, 2019

Accepted: November 28, 2019

Received in revised form: November 27, 2019

Received: September 9, 2019

Identification

DOI: https://doi.org/10.1016/j.fsigss.2019.11.018

Copyright

© 2019 Elsevier B.V. All rights reserved.

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