Abstract
The aim of this chapter is to provide a reliable method of obtaining DNA from formalin-fixed and paraffin-embedded (FFPE) tissue specimens. The application of this method allows the extraction of DNA from specimens of both biopsy and autopsy origin. The method described here is based on proteolytic digestion, phenol purification, and alcohol precipitation, using a point-to-point protocol with notes and references to guide the researcher into the laboratory practice. The DNA extracts obtained by the application of this method are suitable for most of the PCR analyses used in molecular pathology laboratories, ranging from single to complex multiplexed PCRs or B-/T-cell clonality PCR tests, as well as for PCR sequencing.
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Notes
- 1.
The solubilization of Proteinase K in 50% sterile glycerol maintains the solution fluid at −20°C with a better preservation of the enzymatic activity.
- 2.
It is possible to digest the proteins using the following buffer: PCR buffer 1× final (10 mM Tris–HCI pH 8.3, 50 mM KCl) and Proteinase K, 1 mg/ml final. The use of this buffer, without EDTA and detergent, is suggested to avoid the possible inhibition of PCR reaction by the omitted reagents.
- 3.
We strongly recommend purchase of saturated phenol pH 8 from a commercial manufacturer.
- 4.
Clean the pipettes with alcohol or another disinfectant and leave them under the UV lamp for 10 min. Alternatively, it is possible to autoclave the pipette depending on the provider instructions.
- 5.
Clean the microtome with xylene.
- 6.
Deparaffinization step could be completely skipped; alternaÂtively, it could be performed by adding 300 μl of mineral oil to the tube containing the section and incubating at 90°C for 20 min to dissolve the wax [ 8 ].
- 7.
When working with xylene, avoid breathing fumes. It is better to perform the deparaffinization step under a fume hood.
- 8.
Wear gloves when isolating and handling DNA to minimize the contamination with exogenous nucleases. Use autoclaved pipette tips and 1.5 ml microcentrifuge tubes.
- 9.
Xylene is harmful; the wasted xylene must be collected in a chemical waste container and discharged according to the local hazardous chemical disposal procedures.
- 10.
If the pellet is firmly lodged at the bottom of the tube, it is possible to dislodge it in the digestion buffer using a sterile toothpick.
- 11.
Longer digestion time (at least 48 h) increases the yield of the DNA.
- 12.
Phenol is very toxic and should be handled in a fume hood; the wasted phenol must be collected with hazardous chemical waste.
- 13.
The extraction can also be performed with 1volume of phenol (Tris saturated)-chloroform-(50:50, v/v). Phenol is an inhibitor of PCR reaction, because of Taq Polymerase inactivation. A single chloroform-isoamyl alcohol (24:1, v/v) extraction could be performed after the phenol (Tris saturated)-chloroform-isoamyl alcohol extraction in order to completely remove phenol traces.
- 14.
The concentration of dsDNA expressed in μg/μl is obtained as follows: [DNA]  =  A260 × dilution factor × 50 × 10−3 (see Chap. 16 ). A clean DNA preparation should have a A260/A280 ratio of 1.5–2. This ratio is decreased by the presÂence of proteins, oligo-, and polysaccharides. Concentration estimation can also be affected by phenol contamination, as phenol absorbs strongly at 260 nm and therefore can mimic higher DNA yield and purity.
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Bonin, S., Groenen, P.J.T.A., Halbwedl, I., Popper, H.H. (2011). DNA Extraction from Formalin-Fixed Paraffin-Embedded (FFPE) Tissues. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_7
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DOI: https://doi.org/10.1007/978-3-642-17890-0_7
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