Abstract
This chapter provides a general workflow for PCR amplification of DNA or cDNA obtained from formalin-fixed and paraffin-embedded samples. The protocol is dedicated to qualitative PCR. Prerequisites for successful PCR in FFPE include the design of optimized primer pairs to select amplicons of proper length, use of appropriate primer concentrations, and optimization of the PCR conditions. The major problem with formalin-fixed and paraffin-embedded tissues is the extent of degradation of the extracted nucleic acids. In routinely fixed samples for PCR analyses, we recommend the design of amplicons no longer than 200–250 bases for DNA and about 60–80 bases for cDNA. Indeed, shorter amplicons increase, the efficiency of the molecular amplification.
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Notes
- 1.
Shorter amplicons are recommended in extracts from autopsy tissues, because of the higher degradation level. For DNA, 90 bases amplicons and for cDNA 70 bases fragments are detectable, on average, in autopsy tissues.
- 2.
Check homology of the sequences by BLAST analysis: http://www.ncbi.nlm.nih.gov/BLAST/.
- 3.
There are commercial PCR premixed solutions for convenient PCR setup, containing PCR buffer and dNTPs. Usually, Taq Polymerase is provided with its dedicated 10× buffer. Check the composition for the presence of MgCl2. Different commercial PCR buffers are MgCl2 free, but a 25 mM or 50 mM MgCl2 solution is supplied with buffer and enzyme.
- 4.
Because of the high temperature dependence of the pKa of Tris, the pH of the reaction will drop to about 7.2 at 72°C.
- 5.
Primer design could be performed using a particular kind of software, please refer to http://molbiol-tools.ca/PCR.htm for on line available software.
- 6.
Clean the pipettes with alcohol or another disinfectant and leave them under the UV lamp for at least 10 min. Alternatively it is possible to autoclave the pipette depending on the provider instructions.
- 7.
Micro titer plates could also be used to run PCR.
- 8.
The amount of target DNA required varies according to the complexity and level of target sequence. Usually, it ranges from 1 pg to 1 μg.
- 9.
The primer and Mg2+ concentration in the PCR buffer and the annealing temperature of the reaction may need to be optimized for each primer pair for a more efficient PCR.
- 10.
In order to prevent artifacts, a hot start PCR using a thermostable Taq Polymerase (e.g., AmpliTaq Gold by Applied Biosystems) is suggested.
- 11.
If the thermal cycler is not fitted with a heated lid, overlay the reaction mixture with one drop (about 20 μl) of light mineral oil to prevent evaporation and cross contamination.
- 12.
Too many PCR cycles could promote aspecific amplifications, and/or PCR product carry-over.
- 13.
EtBr is a potentially carcinogenic compound. Always wear gloves and use it under a laminar hood. Used EtBr solutions must be collected in containers for chemical waste and discharged according to the local hazardous chemical disposal procedures.
References
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Lehmann U, Kreipe H (2001) Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 25(4):409–418
Sambrook J, Russel DW (2001) Molecular cloning. A laboratory manual, 3rd edn. Cold Spring Harbour Laboratory Press, New York
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© 2011 Springer-Verlag Berlin Heidelberg
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Bonin, S., Dotti, I. (2011). General Protocol for End-Point PCR. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_20
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DOI: https://doi.org/10.1007/978-3-642-17890-0_20
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