Ramping up concentrations of polymerase and dNTPs, and lengthening reaction time in PCR on formalin-fixed paraffin-embedded samples overcomes common barriers to detection, according to recently published research.
Scientists from Bonn University Hospital in Germany implemented these steps after surmising that FFPE-derived DNA debris is at least partly responsible for directly inhibiting PCR. They found that their method minimized this inhibition, even faring well on bisulfite-converted DNA. The researchers anticipate their protocol will benefit molecular diagnostics from FFPE biopsies as well as basic research on banked samples.
Formalin fixation — a routine processing step for biopsies — can glue DNA to proteins, making it brittle and blocking enzyme access. In addition, formic acid (an oxidation product of formalin) causes DNA strand breaks and depurination. Thus, the longer the DNA sequence one would like to examine, the more likely it will be too damaged to even amplify from FFPE — or so the common lab wisdom went.
The new study, published in October in PLOS One, suggests shards of DNA debris competitively inhibit polymerase in PCR through unknown mechanisms, and this inhibition can be overcome by brute force through raising concentrations of reactants.
The findings stemmed from an incidental observation made by lead author Dimo Dietrich about six years ago, while he was working on his PhD. He told PCR Insider that he noticed adding a super-abundance of template DNA to real-time PCR did not lead to threshold lowering.
At the time, Dietrich posited that some component was dragging his system down, and could not let it rest. "I'm basically an engineer, so I thought that inhibition of the enzyme is maybe one of the mechanisms that leads to this effect, and inhibition of enzymatic reactions can be improved by increasing catalyst" which, he reasoned, is the polymerase in the case of PCR.
According to Dietrich, past research took for granted that there were no long strands of template DNA left in FFPE samples, and achieved modest improvements attempting to re-ligate bits of DNA.
Dietrich and his colleagues, however, demonstrated the presence of a small fraction of long strands of sufficient integrity in FFPE tissues, which allowed for robust PCR once the PCR inhibition was alleviated.
For their proof-of-principle paper, they targeted two loci — PITX2 and ACTB — for PCR amplification of DNA derived from archived FFPE prostate and placenta specimens, some of which were nearly 30 years old. These they compared to high molecular weight DNA from fresh placental tissue.
They showed a sharp rise in Cq value at high DNA template amounts, indicating strong PCR inhibition by DNA from FFPE tissues. This inhibition occurred for genomic as well as bisulfite-converted template DNA, such as is needed for epigenetic methylation analyses. The effect could be compensated for by very simple means such as increasing annealing and elongation times, as well as dNTP and polymerase concentrations. Notably, the technique applied both to Taq and Pfu DNA polymerases, suggesting that the findings are applicable to DNA polymerases in general.
Dietrich said that he thinks there is more to the problem than debris size, since in unpublished results he observed sonicated DNA by itself did not show the inhibitory effect. The DNA from FFPE is "fragmented on the one hand, but on the other hand there are base adducts; there are covalent modifications of the single bases; there are abasic sites; there are cross-links to proteins which might not be completely removed; and so on," Dietrich said. "So there are many obstacles on this template for a polymerase which can lead the polymerase to dissociate from the strand, for example."
The improvements demonstrated by Dietrich's group could be useful to various fields. "What I think is very cool is with very simple means you can improve PCR performance dramatically, not only in the research area but also in the routine diagnostic field" which demands "absolutely reliable results," he said.
For Dietrich, a small trade-off in cost — he now uses about $.50 of polymerase per reaction, versus $.15 cents in the past — is made up for by reliability and the ability to amplify larger fragments since, as he put it, "a study which is more expensive is better than a study which is cheap but failed."
On the issue of why increasing polymerase concentration for FFPE hasn't been shown previously, Dietrich mused "maybe because it is so simple, it's too simple," and suggested perhaps because "it was an accidental finding and I was probably the only weird nerd who continued working on it." He noted his troubleshooting study is "definitely not something which you can use for a Nature publication," but added "I think it will help many people."