molecular pathology protocols

Humana Press M E T H O D S I N M O L E C U L A R M E D I C I N E TM Molecular Pathology Protocols Edited by Anthony A. Killeen Humana Press Molecular Pathology Protocols Edited by Anthony A. Killeen DNA Extraction from Paraffin-Embedded Tissues 1 1 From: Methods in Molecular Medicine, vol. 49: Molecular Pathology Protocols Edited by: A. A. Killeen © Humana Press Inc., Totowa, NJ 1 DNA Extraction from Paraffin-Embedded Tissues Hongxin Fan and Margaret L. Gulley 1. Introduction In routine histopathology, most tissues are fixed in formalin and embedded in paraffin for long-term preservation. DNA can be extracted from these tissues for subsequent molecular analysis by amplification methods. We describe herein a protocol for DNA preparation from paraffin-embedded tissues based on published procedures (1–3). In brief, tissue sections are placed into microfuge tubes, then deparaffinized with xylene. The xylene is removed with ethanol washes, and the tissue is treated with proteinase K to make DNA available for amplification. This protocol is simple, but there are several factors that influence the suc- cess of subsequent DNA amplification assays, including the type of fixative that is used, the duration of fixation, the age of the paraffin block, and the length of the DNA segment to be amplified (see Note 1). Ethanol fixation pre- serves DNA much better than does formalin. Formalin fixation randomly chops DNA in a duration-dependent manner, resulting in partial degradation. Even more severe DNA degradation occurs in bone samples subjected to acid decal- cification. Because of this degradation, formalin-fixed tissue is not suitable for Southern blot analysis or for amplification of large DNA segments. Neverthe- less, polymerase chain reaction (PCR) amplification of segments ranging up to 1300 bp has been reported (2), and consistent amplification of segments up to 300 bp is commonly achieved from archival fixed tissues. Be aware that partial degradation of DNA may result in sampling bias, and therefore results should be interpreted with caution. For example, amplification of DNA from one cell may produce a PCR product that is not representative of the entire population of cells in the tissue. For this reason, it is wise to run tests in duplicate and always with appropriate controls. 2 Fan and Gulley 2. Materials 2.1. Reagents 1. Xylene. 2. 100% Ethanol. 3. Proteinase K stock solution (20 mg/mL). 4. TEN buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 20 mM NaCl. 2.2. Equipment 1. Microcentrifuge. 2. Heating block or water bath to hold 1.5-mL Eppendorf tubes. 3. Microtome. 3. Methods Preparation of paraffin-embedded tissue for DNA amplification involves several manual manipulations; therefore precautions should be taken to avoid contamination, such as changing gloves frequently. When opening and closing microfuge tubes, do not touch the rim or inside of the cap. (Many laboratory scientists place a fresh gauze square over their thumb when opening a tube, or use a cap-opener device or screw-top lids.) Appropriate negative controls must be used to alert for contamination. 3.1. Cutting and Deparaffinizing Sections 1. Use a microtome to cut five sections, 5–20 µm thick, from a paraffin block, and place these directly into a 1.5-mL microfuge tube. The thickness of the sections depends on the size of the biopsy. For a small biopsy (up to 3 mm), 20-µm thick sections may be required, whereas a large biopsy requires only 5-µm thick sec- tions. Although multiple thin sections can be placed in a single tube, fewer thick sections are more practical for processing. See Note 2 for special precautions against contamination. 2. Add 800 µL of xylene to each tube, close, mix by gentle vortexing, and then incubate at room temperature for 10 min. Pellet the tissue by centrifugation for 3 min in a microfuge at full speed. Carefully remove and discard the supernatant using a pipet; do not disturb the tissue. If any translucent white paraffin remains, repeat the xylene wash one to two more times. 3. Add 800 µL of 100% ethanol to each tube, close the lid, and mix by inverting. Pellet the tissue by centrifugation for 3 min in a microfuge at full speed, and carefully remove and discard the supernatant with a pipet. Repeat the ethanol wash one more time, and remove as much supernatant as possible. 4. Open the tubes and let the residual ethanol evaporate by incubating in a dry heat block at 55°C for 15–30 min or until the sample is completely dry. (Speed- vacuum drying is not recommended because of the risk of contamination.) DNA Extraction from Paraffin-Embedded Tissues 3 3.2. Proteinase K Digestion 1. To the dried tissue samples add 100 µL of TEN buffer containing 200 µg/mL of proteinase K (prepared by mixing 1 µL of proteinase K stock solution in 100 µL of TEN buffer). Large tissue samples should be resuspended in 200 µL or more of this solution. 2. Close the tubes and incubate at 55°C for 3 h. (Large tissues should be incubated overnight.) 3. Spin briefly to remove any liquid from the cap. Cover the caps tightly with cap locks (PGC Scientifics, Gaithersburg, MD) to prevent them from popping open during high-temperature incubation. Incubate in a 95°C heat block for exactly 10 min to inactivate the proteinase K (see Note 3). Pellet the tissue in a microfuge at full speed for 10 min, and then transfer the supernatant to a clean tube and dis- card the pellet. Promptly proceed with PCR amplification. 4. Quantitation of DNA is not recommended; rather, the amount of supernatant required for subsequent DNA amplification is determined empirically. Try 1- and 10-µL vol of the supernatant as a template for a 100-µL PCR amplifica- tion. If PCR products are not generated, then different volumes can be tried (see Note 4). A positive control reaction (e.g., `-globin) should be run to ensure that amplifiable DNA of similar length to the target DNA is present in the sample. (See Note 5 for modification of thermocycling parameters.) 5. Store DNA at –20°C, and avoid thawing and refreezing. (Freshly prepared samples are more efficiently amplified than those stored frozen, perhaps because the freeze-thaw cycle damages DNA.) 4. Notes 1. The most important factor affecting DNA quality is the type of fixative employed and the duration of fixation. Tissues fixed between 12 and 24 h in ethanol, acetone, Omnifix, or 10% buffered formalin usually yield good-quality DNA; but B-5, Zenker’s, or Bouin’s solutions, or duration of fixation longer than 5 d, are poor prognostic factors for PCR productivity (2,4). Prior studies showed that when the length of `-globin amplification product increased (175, 324, and 676 bp), the percentage of fixed tissues containing amplifiable DNA decreased (100, 69, and 45%) (5). And when the age of a block increased, PCR productivity decreased (6,7), although some blocks stored for more than 40 yr were success- fully studied (8). 2. It is important that no tissue be carried over from one case to the next during microtomy. Between each block, shift to a fresh part of the blade. Use smooth- edge rather than toothed forceps for transporting sections into tubes. Do not allow bleach to come in contact with the tissue or the DNA will be destroyed. Ice cubes used to cool a block should be discarded between cases. 3. Longer incubation at 95°C may damage DNA, whereas shorter incubation may not fully inactivate proteinase K. Additional time is needed for volumes >500 µL. 4 Fan and Gulley 4. The optimal amount of template for an amplification reaction depends on numer- ous factors specific to each sample, such as DNA concentration and presence of inhibitors. It is useful to test several concentrations of each template (e.g., 1 and 10 µL of template per 100-µL PCR). Large tissues may necessitate the use of a smaller fraction of the template (e.g., 0.1 µL). The amount of template that is “tolerated” in a PCR may be affected by residual fixation chemicals or paraffin, excessive tissue debris, and other factors. If the first attempt fails, a 10-fold dilu- tion will often reduce inhibitors while still retaining enough DNA to allow amplification. 5. Amplification of DNA prepared from paraffin-embedded tissue is less efficient than amplification of DNA from fresh or frozen tissues. To compensate for this reduced efficiency, consider modifying the thermocycling parameters by increas- ing the number of cycles and lengthening the duration at each temperature within the cycle (1). References 1. Wright, D. K. and Manos, M. M. (1990) Sample preparation from paraffin- embedded tissues, in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Glefand, D. H., and Sninsky, J. J., eds.), Academic, San Diego, pp. 153–158. 2. Greer, C. E., Wheeler, C. M., and Manos, M. M. (1994) Sample preparation and PCR amplification from paraffin-embedded tissues. PCR Methods Appl. 3, S113–S122. 3. Rolfs, A., Schuller, I., Finckh, U., and Weber-Rolfs, I. (1992) PCR: Clinical Diagnostics and Research, Springer-Verlag, Berlin, pp. 85–87. 4. Shibata, D. (1994) Extraction of DNA from paraffin-embedded tissue for analysis by polymerase chain reaction: new tricks from an old friend. Hum. Pathol. 25, 561–563. 5. Liu, J., Johnson, R. M., and Traweek, S. T. (1993) Rearrangement of the BCL-2 gene in follicular lymphoma: detection by PCR in both fresh and fixed tissue samples. Diagn. Mol. Pathol. 2, 241–247. 6. Limpens, J., Beelen, M., Stad, R., Haverkort, M., van Krieken, J. H., van Ommen, G. J., and Kluin, P. M. (1993) Detection of the t(14;18) translocation in frozen and formalin-fixed tissue. Diagn. Mol. Pathol. 2, 99–107. 7. Goelz, S. E., Hamilton, S. R., and Vogelstein, B. (1985) Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem. Biophys. Res. Commun. 130, 118–126. 8. Shibata, D., Martin, W. J., and Arnheim, N. (1988) Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: a bridge between molecu- lar biology and classical histology. Cancer Res. 48, 4564–4566. DNA Extraction from Fresh or Frozen Tissues 5 2 DNA Extraction from Fresh or Frozen Tissues Hongxin Fan and Margaret L. Gulley 1. Introduction The first step in molecular analysis of patient tissues is preparation of puri- fied, high molecular weight DNA. A number of methods and commercial kits are available for DNA isolation. Traditional organic extraction protocols (1,2) are based on the fact that DNA is soluble in water whereas lipids are soluble in phenol. In these protocols, tissues are disaggregated and then treated with detergent to lyse cell membranes followed by proteinase to digest proteins. Phenol, an organic solvent, is added to help separate the lipids and protein remnants from the DNA. Chloroform is then used to facilitate the removal of phenol. DNA is subsequently concentrated and further purified by precipita- tion in a cold mixture of salt and ethanol. Finally, DNA is resolubilized in Tris-EDTA buffer. The traditional organic extraction procedure presented herein is used by many laboratories to obtain abundant high molecular weight DNA. However, in recent years, there has been a trend toward adoption of commercial non- organic protocols that are faster and avoid the toxicity inherent with phenol exposure. A popular nonorganic extraction kit that works particularly well on blood and marrow samples is the Puregene DNA Extraction Kit (Gentra Sys- tems, Minneapolis, MN). This kit can also be adapted for use on solid tissue samples for subsequent polymerase chain reaction (PCR) analysis. 2. Materials 2.1. Reagents 1. Lymphocyte Separation Media (ICN Biomedicals Inc., Aurora, OH). 2. 1X phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 . 5 From: Methods in Molecular Medicine, vol. 49: Molecular Pathology Protocols Edited by: A. A. Killeen © Humana Press Inc., Totowa, NJ 6 Fan and Gulley 3. Liquid nitrogen. 4. DNA extraction buffer: 10 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA. 5. 10% sodium dodecyl sulfate (SDS). 6. Proteinase K solution: 10 mg/mL of proteinase K in 50 mM Tris-HCl, pH 7.5; store at 4°C. 7. Phenol equilibrated with 0.1 M Tris-HCl, pH 8.0. 8. Chloroform: isoamyl alcohol (24Ϻ1). 9. 3 M Sodium acetate, pH 5.2. 10. 100% Ethanol. 11. 70% Ethanol. 12. TE buffer: 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA. 13. Agarose. 14. 1X TAE buffer: 40 mM Tris-acetate, 1 mM EDTA. 15. Ethidium bromide (10 mg/mL). 16. 10X Gel loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol, 15% Ficoll (type 400) in 10X TAE buffer. 17. DNA molecular weight marker. 2.2. Equipment 1. Mortar and pestle. 2. Water baths at 37, 50, and 55°C. 3. Centrifuge. 4. Spectrophotometer. 5. Horizontal gel electrophoresis apparatus. 6. DC power supply. 3. Methods 3.1. Sample Preparation 3.1.1. Tissue Specimen 1. Mince fresh, solid tissue up to 3 mm 3 into small pieces (1–2 mm) with a sterile scalpel blade. Process the tissue within 2 h of collection, or freeze at –20°C or colder until the time of DNA extraction (see Note 1). 2. Place the tissue in a clean mortar filled with liquid nitrogen. (See Note 2 for cleaning instructions.) 3. Using a clean pestle, grind the frozen tissue to a powder while it is submerged in liquid nitrogen. While grinding, cover the mortar with a paper towel to keep tissue fragments inside the mortar, and work under a hood to protect yourself from aerosolized powder. 4. Allow the liquid nitrogen to evaporate, leaving a dry frozen tissue powder in the mortar. DNA Extraction from Fresh or Frozen Tissues 7 3.1.2. Ficoll Separation of Mononuclear Cells from Blood and Marrow Aspirates Prior to DNA extraction, mononuclear cells are isolated from anticoagu- lated blood or bone marrow aspirates by Ficoll centrifugation. (See Note 3 for information about sample stability.) About 10 7 nucleated cells yield 40 µg of DNA for Southern blot analysis, and 3 × 10 6 cells yield sufficient DNA for amplification testing. 1. To a 15-mL conical tube, add 4.5 mL of blood and an equal volume of PBS. For bone marrow aspirates, use 1 mL of marrow and 8 mL of PBS. If less sample volume is available, use all of it. If greater sample volume is desired, split the sample evenly among two or more tubes so that all of it is processed, and then recombine the samples on collection of the mononuclear cell layer. 2. Using a Pasteur pipet, underlay the diluted blood or marrow with 3 mL of Ficoll solution (Lymphocyte Separation Media). 3. Cap the tube and centrifuge at 400g for 30 min at room temperature in a swinging bucket rotor. 4. Use a plastic Pasteur pipet to aspirate the mononuclear cell layer, which is the fuzzy white layer located between the plasma and the separation medium, into a clean 15-mL conical tube. Avoid the red cell layer at the bottom of the tube. (If no mononuclear cell layer is visible, see Note 4.) 5. Resuspend the mononuclear cells in PBS to 12 mL total volume. 6. Centrifuge for 10 min at 1700g at room temperature. Remove and discard the supernatant by pouring it off. 7. Store the cell pellet at –20°C temporarily or at –70°C long term, or proceed directly to DNA or RNA extraction. 3.2. DNA Extraction 3.2.1. Cell Lysis and Digestion The procedure for solid tissue differs from that of blood or marrow mono- nuclear cells only in the first step. 1. For solid tissue, add 920 µL of DNA extraction buffer to the tissue powder in the mortar, and gently mix with the pestle. If the buffer freezes, wait until it thaws before proceeding. Then transfer the fluid to a 15-mL conical tube or microfuge tube by gentle pipeting. For a mononuclear cell pellet (about 10 7 cells), resuspend the cells in 920 µL of DNA extraction buffer and mix well by gentle pipeting. 2. Add 50 µL of 10% SDS to the mixture and mix well; the solution should become viscous. 3. Add 30 µL of proteinase K solution to the viscous mixture. Close the cap tightly and mix vigorously by repeated forceful inversion or vortex. 4. Incubate in a 37°C water bath for at least 6 h or as long as 2 d, or at 55°C for 3 h; gently invert the tube a few times during incubation. 8 Fan and Gulley 5. The lysed sample should be viscous and relatively clear. This sample may be stored at 4°C for up to 1 wk before subjecting it to phenol/chloroform extraction as described in Subheading 3.2.2., or before proceeding with nonorganic extrac- tion as described in Note 5. 3.2.2. Phenol/Chloroform Extraction of DNA 1. Add an equal volume of equilibrated phenol, close the cap tightly, and mix gently by inversion for 1 min. 2. Spin the tube at 1700g in a swinging bucket rotor at room temperature for 10 min. 3. With a plastic pipet, aspirate the upper clear aqueous layer and transfer it to another clean labeled tube. This should be done carefully to avoid carrying over phenol or white proteinaceous material from the interface. 4. Repeat the phenol extraction (steps 1–3) one more time. 5. Next, extract with an equal volume of chloroform instead of phenol, and save the supernatant to another clean tube after centrifugation. 6. Repeat the chloroform extraction; this helps eliminate all of the phenol from the DNA sample. 3.2.3. Purification and Precipitation of DNA 1. To the aqueous DNA solution add 0.1 vol of 3 M sodium acetate (pH 5.2), and mix well by vortexing. 2. Add 2 vol of ice-cold 100% ethanol to the tube. Close the cap tightly and mix by inversion. A white cotton-like precipitate should form. 3. Use a sterile plastic rod to spool the precipitated DNA. (If no precipitate is vis- ible, then microfuge at full speed for 10 min, rinse the pellet with 70% ethanol, air-dry for about 10–15 min, and proceed with step 6.) 4. Rinse the spooled DNA thoroughly in 1 mL of cold 70% ethanol by dipping. 5. Remove the DNA-coated plastic rod and allow the precipitate to air-dry until the white precipitate becomes clear, usually about 5–10 min. 6. Dissolve the precipitate in an appropriate volume of TE buffer (typically about 100–500 µL; targeting an optimal DNA concentration 1 µg/µL), scraping the rod along the wall of the microfuge tube to help detach the viscous DNA. 7. Allow the DNA to dissolve in the TE buffer for at least 4 h at 50°C, gently shak- ing periodically during incubation. Failure to adequately resolubilize the DNA will result in uneven distribution of DNA within the solution. 8. The purified DNA sample may be stored for 4 wk at 4°C prior to analysis, or indefinitely at –20°C. 3.3. DNA Quantitation by Spectrophotometry 1. Mix the DNA sample by gentle vortexing and inversion. 2. Add 5 µL of the DNA sample to 495 µL of sterile water and mix well. 3. Place the diluted sample in a quartz microcuvet and measure the absorbance at 260 and 280 nm against a water blank. (Nucleic acids absorb light maximally at 260 nm whereas proteins absorb strongly at 280 nm.) DNA Extraction from Fresh or Frozen Tissues 9 4. Compute the DNA concentration based on the concept that an OD 260 of 1 corre- sponds to 50 µg/mL of double-stranded DNA, and adjusting for the 100-fold dilution factor, according to the following formula: DNA concentration (µg/µL) = OD 260 × 5 5. The OD 260 ϺOD 280 ratio should be between 1.7 and 2.0. Lower values indicate protein contamination, in which case the DNA can be further purified by addi- tional phenol/chloroform extractions followed by ethanol precipitation. 3.4. Gel Electrophoresis to Analyze DNA Quality Agarose gel electrophoresis can be used to assess the intactness of purified DNA. High molecular weight DNA is needed for Southern blot analysis, whereas partially degraded DNA might be suitable for amplification procedures. 1. Prepare a 0.7% agarose gel in 1X TAE buffer containing 0.5 µg/mL of ethidium bromide. 2. Mix an aliquot of the extracted DNA sample with loading buffer, and load into a submerged well. Control samples representing intact and degraded DNA should be loaded into adjacent wells. 3. Electrophorese in 1X TAE buffer with 0.5 µg/mL ethidium bromide at 2 V/cm, until the dye front reaches the end of the gel. 4. View the gel under UV light. High molecular weight DNA is too large to migrate well under these conditions, whereas degraded DNA contains a spectrum of smaller fragment sizes that appear as a smear across the lane. 4. Notes 1. Solid tissue samples should be processed immediately or else frozen to minimize the activity of endogenous nucleases. If frozen tissue immunohistochemistry is planned, then slice the tissue into pieces no more than 5 mm thick and snap- freeze in liquid nitrogen or in a cryostat. If morphologic preservation is not needed, then place the tissue in a –70°C freezer indefinitely, or at –20°C for up to 3 d until DNA or RNA isolation. 2. After washing with detergent and rinsing well, soak the mortar and pestle in 50% nitric acid or 10% bleach to prevent carryover of DNA to the next case, then rinse well. 3. Peripheral blood or bone marrow aspirate anticoagulated with EDTA or acid citrate dextrose should be stored at room temperature and processed as soon as possible. EDTA beneficially chelates ions to inhibit nucleases from degrading nucleic acid, and consequently DNA and RNA are often stable for up to 48 h at room temperature. Heparin anticoagulant is not recommended because residual heparin may interfere with subsequent restriction enzyme or DNA polymerase activity. [...]... Chloroform Isopropanol Diethylpyrocarbonate (DEPC)-treated H2O (see Note 1) 75% ethanol made with DEPC-H2O Ribonuclease inhibitor (40 U/µL) (Promega, Madison, WI) From: Methods in Molecular Medicine, vol 49: Molecular Pathology Protocols Edited by: A A Killeen © Humana Press Inc., Totowa, NJ 11 12 7 8 9 10 11 12 13 Fan and Gulley 10X DNase I buffer (Gibco-BRL) DNase I, amplification grade (Gibco-BRL) 20... the known polymorphisms of the p53 gene may be involved in the susceptibility to cancers and prognosis of the disease (19,20) Proallele carriers of exon 4 codon From: Methods in Molecular Medicine, vol 49: Molecular Pathology Protocols Edited by: A A Killeen © Humana Press Inc., Totowa, NJ 15 16 Vähäkangas, Castrén, and Welsh 72 polymorphism (CGCArg or CCCPro) were found to be overrepresented among patients... scanning techniques, such as single-strand conformation polymorphism (SSCP) and dideoxy fingerprinting (5,6) Instead of relying on direct observation of these From: Methods in Molecular Medicine, vol 49: Molecular Pathology Protocols Edited by: A A Killeen © Humana Press Inc., Totowa, NJ 29 30 Heisler and Lee Fig 1 Structures recognized by the Cleavase I enzyme The Cleavase I enzyme is a structure-specific... cell lysis steps References 1 Strauss, W M (1995) Preparation of genomic DNA from mammalian tissue, in Current Protocols in Molecular Biology (Ausubel, F M., Brent, R., Kingston, R E., et al., eds.), John Wiley & Sons, New York, pp 2.2.1–2.2.3 2 Sambrook, J., Fritsch, E F., and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY,... clues to cancer etiology and molecular pathogenesis Cancer Res 54, 4855–4878 3 Hainaut, P., Soussi, T., Shomer, B., Hollstein, M., Greenblatt, M., Hovig, E., Harris, C C., and Montesano, R (1997) Database of p53 gene somatic mutations in human tumors and cell lines: updated compilation and future prospects Nucleic Acids Res 25, 151–157 4 Hussain, S P and Harris, C C (1998) Molecular epidemiology of human... Nature 358, 15,16 11 Harris, C C (1996) The 1995 Walter Hubert Lecture molecular epidemiology of human cancer: insights from the mutational analysis of the p53 tumoursuppressor gene Br J Cancer 73, 261–269 12 Perera, F P., Whyatt, R M., Jedrychowski, W., Rauh, V., Manchester, D., Santella, R M., and Ottman, R (1998) Recent developments in molecular epidemiology: a study of the effects of environmental polycyclic... acid guanidinium thiocyanate-phenol-chloroform extraction Analyt Biochem 162, 156–159 2 Kingston, R E., Chomczynski, P., and Sacchi, N (1995) Guanidine methods for total RNA preparation, in Current Protocols in Molecular Biology (Ausubel, F M., Brent, R., Kingston, R E., et al., eds.), John Wiley & Sons, New York, pp 4.2.1–4.2.9 SSCP in Exons 4–8 of the TP53 Gene 15 4 Single-Strand Conformation Polymorphism... supplying up to 2000 V 2.4 Visualization of CFLP Patterns 2.4.1 Fluorescence Detection 1 Hitachi FMBIO®-100 Fluorescent Method Bio-Image Analyzer (Hitachi Software, San Bruno, CA) or Molecular Dynamics 595 FluorImager™ (Molecular Dynamics, Sunnyvale, CA) 2 Lint-free laboratory wipes 3 Lens paper 4 Nonfluorescing detergent, e.g., RBS 35 Detergent Concentrate (Pierce, Rockford, IL) 2.4.2 Chemiluminescence... protein is putatively the protection of the genome (9,10), implicate the mutations of the TP53 gene in environmentally induced carcinogenesis in humans and the possible use of TP53-related markers in molecular epidemiology (11–13) Mutations of the p53 gene with the loss of wild-type function also seem to have clinical importance The reported tumor types, in which either a TP53 gene mutation or aberrant... Steinarsdottir, M., Olafsdottir, K., Jonasdottir, S., Jonasson, J G., Ögmundsdottir, H M., and Eyfjörd, J E (1997) Genomic instability and poor prognosis associated with abnormal TP53 in breast carcinomas: molecular and immunohistochemical analysis APMIS 105, 121–130 Soini, Y., Turpeenniemi-Hujanen, T., Kamel, D., Autio-Harmainen, H., Risteli, J., Risteli, L., Nuorva, K., Pääkkö, P., and Vähäkangas, K (1993) . H O D S I N M O L E C U L A R M E D I C I N E TM Molecular Pathology Protocols Edited by Anthony A. Killeen Humana Press Molecular Pathology Protocols Edited by Anthony A. Killeen DNA Extraction. Killeen DNA Extraction from Paraffin-Embedded Tissues 1 1 From: Methods in Molecular Medicine, vol. 49: Molecular Pathology Protocols Edited by: A. A. Killeen © Humana Press Inc., Totowa, NJ 1 DNA. NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 . 5 From: Methods in Molecular Medicine, vol. 49: Molecular Pathology Protocols Edited by: A. A. Killeen © Humana Press Inc., Totowa, NJ 6 Fan