Direct PCR from FFPE tissue using
Phusion® DNA polymerase

Single prostate tissue section (10 µm) was treated with proteinase K (0.2 mg/ml) for one hour. Fragments of 221 and 313 bp were amplified with Phusion DNA Polymerase from different volumes of tissue supernatant (40 cycles). Piko® Thermal Cycler and UTW® reaction vessels were used in PCR.

 
 

Comparing Phusion® DNA Polymerase to Taq DNA polymerase

Single FFPE prostate tissue sections (10 µm) were incubated o/n with proteinase K (0.2 mg/ml). A 221 bp DNA fragment was amplified with Phusion DNA Polymerasefrom different concentrations of each tissue supernatant (40 cycles; arrow indicates increasing template amount). Piko Thermal Cycler and UTW reaction vessels were used in PCR.

 
 

Phusion® DNA Polymerase performs equally well from disrupted tissue samples as from DNA extracted with a commercial kit
A 261 bp fragment was amplified with Phusion DNA Polymerase either directly from FFPE breast tissue sections treated with a simple disruption protocol (o/n proteinase K treatment) or from DNA purified with a commercial kit (paraffin removal step included). Piko Thermal Cycler and UTW reaction vessels were used in PCR.
 
Complete pipetting and cycling instructions can be found in the application note "PCR from FFPE tissues without DNA extraction using Phusion® DNA Polymerase".
 

General notes

  • As in all direct PCR applications, it is highly recommended to include a positive control (purified DNA) to ensure that the PCR reaction conditions are optimal.
  • This starting material has been tested only with end point PCR protocols.