Abstract
This chapter provides the general guidelines with a specific protocol, as an example of the application of nested-PCR to DNA or cDNA obtained from formalin-fixed and paraffin-embedded (FFPE) samples. The described method presents the standard workflow for nested amplification, along with a non-isotopical system for PCR product detection. Nested-PCR is related to the reamplification of a first product of PCR by using a second set of primers within the first amplification product. The utilized primers should be different in both reactions. This methodology increases both the sensitivity and specificity of the assay. On the other hand, the risk of contamination increases significantly because of the greater amount of amplification products and working steps involved. Particular considerations and suggestions are given along the chapter for the preparation of samples and PCR runs in order to avoid contaminations.
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Notes
- 1.
There are commercial PCR premixed solutions for convenient PCR setup, containing PCR buffer and dNTPs. Taq Polymerase is usually 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.
- 2.
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.
- 3.
Primer design could be performed using a specific software; please refer to http://molbiol-tools.ca/PCR.htm for online available software.
- 4.
EtBr is a potentially carcinogenic compound. Always wear gloves and work under a chemical hood. Used EtBr solutions must be collected in containers for chemical waste and discharged according to the local hazardous chemical disposal procedures.
- 5.
Weigh the proper amount of SDS powder under a fume hood, because it is harmful.
- 6.
Clean the pipettes with a disinfectant (e.g., Meliseptol®rapid) and leave them under the UV lamp for almost 10 min. Alternatively it is possible to autoclave the pipette depending on the provider instructions.
- 7.
Microtiter plates could also be used to run PCR.
- 8.
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.
- 9.
Special precautions should be taken in this step for the possibility of carry-over (false-positive results due to contamination among different tubes). It is recommended to work in a flow hood with samples cooled in ice and with anti-aerosol pipette tips.
- 10.
Bubbles can be removed by rolling a Pasteur pipette on the surface.
- 11.
Alternatively to Southern blot, it is possible to transfer directly the nested PCR products onto the membrane by dot-blot apparatus without electrophoretic separation (See Chap. 23 for details). In this case, after the dot-blot transfer, go directly to the UV cross linking of the membrane and follow the protocol for membrane hybridization.
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Bonin, S., Dotti, I. (2011). Nested-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_22
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DOI: https://doi.org/10.1007/978-3-642-17890-0_22
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