Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

Abstract

Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.

Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis

“…The deparaffinization process and the different phases of extraction can also cause fragmentation, loss of DNA, and inhibition of the reaction (Dietrich et al, 2013). The efficiency of the DNA extraction technique is also related to the method used (Fernandes et al, 2004).…”

“…However, the process of PCR inhibition can be reversed with adaptations of the technique, such as the use of a higher concentration of polymerase and dNTP, and increased extension time (Dietrich et al, 2013).…”

Enzymatic detection of formalin-fixed museum specimens for DNA analysis and enzymatic maceration of formalin-fixed specimens

“…As pressure on wild populations has increased over the past 200 years, the rare and extinct species found in museums have become increasingly valued as samples for genetic studies and representations of biological history (Austin andMelville 2006, Burrell et al 2015). However, DNA extraction from archival specimens depends on their storage history, and it is widely known that formalin-preserved specimens give limited results for various reasons (Schander and Kenneth 2003, Austin and Melville 2006, Gilbert et al 2007, Dietrich et al 2013, Burrell et al 2015. Treatment with formalin-containing solutions results in crosslinks not only between proteins, but also between proteins and nucleic acids (Gilbert et al 2007, Dietrich et al 2013, Burrell et al 2015.…”

“…However, DNA extraction from archival specimens depends on their storage history, and it is widely known that formalin-preserved specimens give limited results for various reasons (Schander and Kenneth 2003, Austin and Melville 2006, Gilbert et al 2007, Dietrich et al 2013, Burrell et al 2015. Treatment with formalin-containing solutions results in crosslinks not only between proteins, but also between proteins and nucleic acids (Gilbert et al 2007, Dietrich et al 2013, Burrell et al 2015. Additionally, cross-linking complicates isolation of DNA and introduces polymerase "blocks" during PCR (Gilbert et al 2007).…”

“…Additionally, cross-linking complicates isolation of DNA and introduces polymerase "blocks" during PCR (Gilbert et al 2007). Formalin treatment also results in fragmentation of DNA, which can cause inhibition of the DNA polymerase, resulting in poor PCR amplification (Dietrich et al 2013).…”