DNA isolation is a critical step in the ³²P-postlabeling method. Care in the isolation and handling of the DNA samples improves the chromatogram quality and substantially lowers the background radioactivity on the chromatograms.

Important DNA isolation points to emphasize are

• All tissue samples are frozen immediately after collection in liquid nitrogen or on dry ice.
• All tissue and purified DNA are stored at -80°C; use of a -20°C freezer for any length of time can yield DNA with breakdown products that co-chromatograph and interfere with the quantitation of DNA adducts derived from hydrophobic compounds, such as PAHs (Fig. 3). After several months at -20°C, the yield of DNA from tissue samples starts to decline and eventually DNA is not recoverable, whereas, ³²P-postlabeling grade DNA can be extracted successfully from tissues stored at -80°C for at least 2 years.
• If the DNA samples are stored for an extended period before being postlabeled, storage of whole tissue is preferable to storage of extracted DNA. DNA appears to be stable in whole tissue for at least 2 years and extracted double-stranded DNA is stable in a Tris/EDTA buffer (pH 7.4) for at least 2 months at -80°C.
• High quality chemicals must be used for the extraction of DNA. Phenol and m-cresol must be redistilled before use, saturated with nitrogen, and stored at -20°C. However, Boehringer Mannheim sells a molecular biology grade of phenol (No. 100300) that has been redistilled and stored under argon which is suitable for DNA purification. Oxidation of phenol yields quinones that crosslink nucleotides resulting in chromatograms with high backgrounds that are difficult to quantitate.

1. Thaw tissue and keep on ice.
2. Place 125-250 mg of tissue and 1.7 mL of 10 mM Tris/100 mM EDTA, pH 7.4 in either a glass culture tube (10 mm x 75 mm) and homogenize using a Polytron 1200C at setting 2 for 5-10 seconds or in a 7 mL glass Dounce homogenizer and homogenize using 5-10 slow strokes.
   Note: If homogenization is not performed gently, the nuclear membrane may be ruptured resulting in the loss of DNA during the nuclei precipitation step.
3. Transfer the homogenate to a labeled 1.9 mL microcentrifuge tube and keep on ice.
4. Centrifuge at 4°C for 10 minutes at 6,000 rpm to pellet the crude nuclei. If large amounts of lipid are present, centrifugation at 14,000 rpm for 3 to 5 minutes may be necessary to pellet the nuclei.
5. Decant and discard supernatant. If there is a ring of lipid left on the tube wall, use a cotton swab to remove it.
6. Resuspend pellet in 850 µL of 1% SDS/20 mM EDTA (pH 7.4) solution.
7. Add 4 µL of solution containing RNAses and alpha-amylase and incubate for 30 minutes at 37°C. The RNAses and alpha-amylase solution is prepared as follows: An appropriate aliquot of alpha-amylase stock solution is taken (20 µg of alpha-amylase per sample) for the total number of samples to be processed and placed in a 1.5 mL microcentrifuge tube. The alpha-amylase is included in the RNAse mixture for hydrolysis of glycogen which will interfere with the UV absorbence readings. Centrifuge at 14,000 rpm to precipitate the alpha-amylase and remove the supernatant with a pipet. To the alpha-amylase precipitate add 2 µL per sample of 50 mM Tris-HCl, pH 7.4 and 2 µL per sample of a RNAse (footnote 4) solution which contains heat-treated RNAse A (10 µg/µL) and RNAse T₁ (10 units/µL). If the ³²P-DNA base analysis indicates that substantial amounts of RNA remain after RNAse treatment, then add 1 unit of RNAse T₂ per sample.
8. At the end of the RNAse incubation, the proteins are digested by the addition of 0.4 mg of proteinase K in 40 µL of 1 M Tris-HCl (pH 7.4) per sample. The samples are incubated for 30 minutes at 37°C with occasional mixing.
9. Proteins are removed by sequential organic solvent extractions as follows: Add one volume of the appropriate solvent (see Reagents for DNA isolation section), mix by inverting the tube repeatedly for 1 minute, centrifuge at 14,000 rpm for the recommended time length and remove the organic layer with a pipette. Use the solvent extraction sequence outlined below.
   

   Extraction	Reagent	Centrifuge (min)
   1	phenol	7
   2	phenol:chloroform:isoamyl alcohol, 25:24:1 (CIP)	5
   3	chloroform:isoamyl alcohol, 24:1 (CIA)	3

   
   Caution: Microcentrifuge tubes sometimes fail with the chloroform solvent systems and leakage can occur during centrifugation. To avoid this problem, the DNA solution (aqueous phase) is transferred to a fresh tube after the CIP and CIA extraction steps.
   When handling phenol solutions wear gloves, a longsleeve labcoat, and protective glasses. Small amounts of phenol (<50 µL) can cause severe chemical burns.
10. Add 0.1 volumes of 5 M NaCl to the aqueous phase and invert the tube repeatedly for 30 seconds.
11. Add 1 volume of cold (-20°C) 100% ethanol and gently invert the tube repeatedly for 2 minutes to mix. The DNA will precipitate out of solution as a stringy gelatinous clump unless it is sheared. If the DNA is sheared, then try precipitating the DNA by placing the tube in a -20°C freezer from 20 minutes to overnight.
12. Centrifuge for 5 minutes at 14,000 rpm to pellet the DNA.
13. Wash the DNA pellet with 1 mL of 70% ethanol by mixing, centrifuging for 5 minutes at 14,000 rpm, and decanting the supernatant. Remove residual 70% ethanol by centrifuging for a few seconds and then using a pipette.
14. Resuspend DNA in 35-50 µL of 10 mM Tris/1 mM EDTA, pH 7.4 (TE) buffer depending on the size of the pellet recovered.
    Caution: The DNA precipitate often can be difficult to dissolve. Sometimes after attempting dissolution, the DNA can be a large, clear, and colorless aggregate in solution that can be either inadvertently pipetted leading to an abnormally high UV absorbence reading or the aggregate may be missed by the pipette leading to a low UV absorbence value and an erroneous amount of DNA being used for the enzyme hydrolysis step. If this is a persistent problem, and heat and vortexing do not take care of it, then pass the DNA solution through a 26-gauge needle repeatedly to obtain a homogeneous solution.
    
15. Take 5 µL of DNA solution and dilute to 1 mL with TE and measure the absorbances against a TE blank at 280, 260 and 230 nm.
16. Determine the absorbence ratios of 260/230 and 260/280. They should be:
    

    A260/A230 > 2.3

    A260/A280 > 1.8

    
    These ratios will yield information about protein contamination but not RNA contamination. The chromatograms from the ³²P-DNA base analysis will give an estimate of RNA contamination (Fig. 4).
17. Calculate the concentration of DNA as follows:
    Concentration (mg DNA/mL) = (A₂₆₀)(dilution factor)/(22.9 mL/mg DNA)
    Example:
    dilution factor = 1000 µL final volume/5 µL original volume = 200
    absorbence (A₂₆₀) = 0.262 OD units
    Concentration = 0.262 x 200/22.9 = 2.28 mg/mL

1. Weigh out 125-250 mg of tissue and place it in a 7 mL glass Dounce homogenizer.
   Note: If homogenization is not performed gently, the nuclear membrane may be ruptured resulting in the loss of DNA during the nuclei precipitation step.
2. Add 1.7 mL of 10 mM Tris/100 mM EDTA, pH 7.4 and gently homogenize. The homogenate is then transferred to a 1.9 mL microcentrifuge tube and placed on ice.
3. Centrifuge at 6,000 rpm for 10 minutes at 4°C to precipitate the nuclei. If large amounts of lipid are present, centrifuge at 14,000 rpm for 3 to 5 minutes to pellet the nuclei.
4. Decant and discard the supernatant. If there is a ring of lipid left on the tube wall, use a cotton swab to remove it. Add 1.5 mL of 1% SDS/20 mM EDTA, pH 7.4 and resuspend the pellet using a glass Pasteur pipet.
5. Add 8 µL of solution containing RNAses and alpha-amylase and incubate for 30 minutes at 37°C. The RNAses and alpha-amylase solution is prepared as follows: An appropriate aliquot of alpha-amylase stock solution is taken (20 mg of alpha-amylase per sample) for the total number of samples to be processed and placed in a 1.5 mL microcentrifuge tube. Centrifuge at 14,000 rpm to precipitate the alpha-amylase and remove supernatant with a pipet. To the alpha-amylase precipitate add 2 µL per sample of 50 mM Tris-HCl, pH 7.4 and 6 µL per sample of a RNAse solution which contains heat-treated RNAse A (10 µg/µL) and RNAse T₁ (10 units/µL).
6. Turn on the ABI 341 Nucleic Acid Purification System, check helium pressure gauges, reagent and waste bottle levels.
7. Prompt the machine to perform a self-test of its systems, then pressurize all bottles.
8. Applied Biosystems Inc.'s DNA isolation method No. 1 (the program commands DDig, DEx1, DEx1, DEx2, DPpt are used in this method) is used with the following modifications:
   • The proteinase K digestion is done at 37°C for 45 minutes.
   • All extractions are done at room temperature with function No. 66 (slow educe) separating the phases.
   • After the DNA has been precipitated onto the filter, only one wash with 80% ethanol is performed.
9. Remove filter paper with the DNA from the precipitette cartridge with forceps, place it in a 1.5 mL microcentrifuge tube, and add 600 µL TE. Remember! Clean forceps between samples to prevent cross-contamination.
10. The microcentrifuge tube is gently agitated on an Eppendorf Thermomixer for 15 minutes at 37°C to dissolve the DNA and then the filter paper is removed with forceps.
11. Add 0.1 volumes of 5 M NaCl to the aqueous phase and invert the tube repeatedly for 30 seconds.
12. Add 1 volume of cold 100% ethanol (stored at -20°C) and gently invert the tube for at least 2 minutes repeatedly to mix. The DNA will precipitate out of solution as a stringy gelatinous clump unless it is sheared. If the DNA is sheared, then try precipitating the DNA by placing the tube in a -20°C freezer from 20 minutes to overnight.
13. Centrifuge for 5 minutes at 14,000 rpm to pellet the DNA.
14. Wash the DNA pellet with 1 mL of 70% ethanol by mixing, centrifuging for 3 minutes at 14,000 rpm and decanting the supernatant. Remove residual 70% ethanol by centrifuging for a few seconds and then using a pipette.
15. Resuspend DNA in 35 to 75 µL of 10 mM Tris/1 mM EDTA, pH 7.4 (TE) buffer depending on the size of the pellet recovered. Heat and agitate DNA on Thermomixer at 37°C for 5 minutes and then mix the solution with a pipette to dissolve DNA pellet.
16. Please see Manual Procedure section on how to measure DNA concentrations.

Some of the reagents used with the 341 Nucleic Acid Purification System were purchased from Applied Biosystems Inc. If you choose to prepare your own reagents for use with the DNA extractor, these reagents must be filtered through a 22 µm filter before use. Please see DNA extractor manual for instructions on the preparation of reagents for use on the DNA extractor:

Following is a list of reagents for DNA isolation:

Phenol Reagent

• Prepare all organic based reagents in a fume hood.
• Place phenol, distilled water, 100 mL of 2 M Tris buffer (pH 7.4) and 8-hydroxyquinoline in a separatory funnel and shake vigorously for 20 seconds.
• Allow the phenol layer to settle out and separate the phases.
• To the phenol phase, add 100 mL of 2 M Tris buffer, 25 mL of m-cresol and 2 mL of ß-mercaptoethanol.
• Bubble inert gas through the solution for 5 minutes to remove dissolved oxygen.
• Store aliquots of aqueous and phenol phases together at -20°C.

phenol:chloroform:isoamyl alcohol, 25:24:1, v/v/v (CIP)

250 mL of phenol reagent 240 mL of chloroform
10 mL of isoamyl alcohol

• Combine components and bubble inert gas through the solution for 5 minutes to remove dissolved oxygen.
• Store at -20°C.

chloroform:isoamyl alcohol, 24:1, v/v (CIA) To make 250 mL combine:

240 mL of chloroform
10 mL of isoamyl alcohol

• Combine components and bubble inert gas through the solution for 5 minutes to remove the dissolved oxygen.
• Store at 4°C.

70% ethanol (v/v):

• Add 300 mL of distilled water to 700 mL of 100% ethanol and store at room temperature.

5 M NaCl:

• Dissolve 292.2 g of NaCl in distilled water and bring to a final volume of 1 L. Store at room temperature.

1% SDS, 20 mM EDTA:

• Add 8 mL of 500 mM EDTA (pH 7.4) to 180 mL of distilled water.
  
• Bubble inert gas through the solution for 5 minutes to remove dissolved oxygen.
  
• Add 2 g of sodium dodecyl sulfate, and mix with a stir bar.
  
• Check pH and adjust to 7.4. Bring to a final volume of 200 mL and store at room temperature.

10 mM Tris/100 mM EDTA, pH 7.4 (ultra TE):

1.211 g of Tris base
38.0 g of EDTA (tetrasodium salt)

• Dissolve the Tris base and EDTA in 800 mL of distilled water.
• Adjust pH to 7.4 with 6 M HCl.
• Bring to a final volume of 1 liter with distilled water.
• Bubble inert gas through the solution for 5 minutes to remove dissolved oxygen.
• Store at 4°C.

2 M Tris, pH 7.4:

For 1 liter of buffer combine:
850 mL of distilled water
242.2 g of Tris base
75 mL of concentrated HCl

• Dissolve Tris base in water.
• Slowly add HCl to the solution.
• Add additional 6 M HCl to adjust pH to 7.4.
• Bring to a final volume of 1 liter with distilled water.
• Store at 4°C.

500 mM EDTA, pH 7.4:

• Place 190 g of EDTA (tetrasodium salt) in 800 mL of distilled water.
• Adjust pH to 7.4 with 6 M HCl.
• Bring to a final volume of 1 liter with distilled water. Store at 4°C.

50 mM Tris, pH 7.4:

• Place 25 mL of 2 M Tris-HCl, pH 7.4 into 800 mL of distilled water.
• Adjust pH to 7.4 with 1 M HCl.
• Bring to a final volume of 1 liter with distilled water. Store at 4°C.

10 mM Tris/1 mM EDTA, pH 7.4 (TE):
1.211 g of Tris base
0.380 g of EDTA (tetrasodium salt)

• Dissolve the Tris base and EDTA in 800 mL of distilled water.
• Adjust pH to 7.4 with 1 M HCl.
• Bring to a final volume of 1 liter with distilled water.
• Bubble inert gas through the solution for 5 minutes to remove dissolved oxygen.
• Store at 4°C.

Enzymatic digestion of xenobiotic-modified DNA to 3'-mononucleotides is a crucial step in the ³²P-postlabeling process. The levels of micrococcal nuclease (MN) and spleen phosphodiesterase (SPD) now being used are lower than what was recommended in the original procedure (Gupta et al. 1982). Excessive amounts of MN can lower the recovery of some DNA adducts (Beach and Gupta 1992). The levels of MN present in the DNA digestion solution should be in the range of 0.15-0.35 mg MN/mg DNA. However, SPD concentrations do not appear to have a negative effect on adduct recovery and a concentration of 1 µg SPD/µg DNA works well (Beach and Gupta 1992). For samples containing DNA adducts derived from PAHs, incubation of DNA with MN and SPD for 3-6 hours at 37°C is sufficient. Overnight digestion of the samples can result in the loss of some aromatic amine adducts and a lower recovery of some PAHs. Moreover, normal nucleotides can degrade during a lengthy sample hydrolysis and yield breakdown products that may substantially raise the background radioactivity levels on chromatograms or interfere with the detection of some adducts (Gupta 1989). The choice of MN and SPD concentrations and length of DNA digestion is adduct-dependent, and digestion conditions should be optimized for specific needs.

The following enzyme hydrolysis protocol is for generating a 1-10 mg DNA sample for postlabeling.
All tubes used in this procedure must be rinsed with methanol, then with distilled water and thoroughly dried to remove any residual plasticizers, fungicides or other chemicals that may interfere with enzyme activities.

1. For each sample, place 25 µg of DNA in TE buffer into a 1.5 mL microcentrifuge tube and bring to a final volume of 15 µL with distilled water. If the DNA concentration is too low, then use less DNA or concentrate the DNA solution, if the situation permits, by adding 0.1 volume of 5 M NaCl and precipitate the DNA by adding 1 volume of cold 100% ethanol. Dissolve the precipitate in the appropriate volume of TE to give a 1.6 µg/µL DNA solution.
2. Add 10 µL of the MN/SPD in buffer to each sample.
   Preparation of MN/SPD in buffer:
   Based on the number of samples, place an appropriate volume (10 µL x number of samples) of SPD solution (2 µg SPD/µL) in a microcentrifuge tube and centrifuge at 14,000 rpm for 2 minutes. Remove the ammonium sulfate supernatant with a pipettor. Then add an appropriate volume (5 µL x number of samples) of dialyzed MN (1 µg MN/µL H₂O) solution and a volume (5 µL x number of samples) of buffer (20 mM sodium succinate, 10 mM calcium chloride, pH 6.0), respectively, to the SPD residue. Preparation of dialyzed MN:
   Dissolve MN in distilled water to give a concentration of 1 µg/µL. Place MN solution in a 10 mm wide dialysis tube and dialyze overnight (4°C) against distilled water (2,000 mL volume).
3. Briefly centrifuge the samples to bring the enzyme solution to the tube bottom and then mix with a pipettor.
4. Incubate samples for 3-6 hours at 37°C and vortex occasionally.
5. Remove samples from the waterbath and briefly centrifuge to bring the tube contents to the bottom. The enzyme hydrolysate can be stored for several days at -80°C without loss of adducts.
6. A 5 µL aliquot of each hydrolysate is placed in a 1.5 mL tube along with 495 µL of ddH₂O and vortexed. This sample is used for determination of the total amount of DNA hydrolyzed.
7. A 10 µL aliquot of the enzyme hydrolysate will be taken for the DNA adduct postlabeling assay.

The normal ³²P-postlabeling method can detect DNA adducts in the range of 1 modification per 10⁶ to 10⁷ nucleotides. However, sensitivity for large, hydrophobic DNA adducts can be substantially improved to detect DNA adducts in the range of 1 modification per 10⁹ to 10¹⁰ nucleotides with the use of adduct enhancement techniques. The two enhancement methods most commonly used are 1) extraction of the DNA-adducts into water-saturated n-butanol (Gupta 1985) and 2) selective enzymatic degradation of normal mononucleotides to nucleosides using nuclease P1 (Reddy and Randerath 1986). For PAHs, nuclease P1 generally gives better recoveries than the butanol method and is easier to perform (Gupta and Earley 1988). However, some aromatic amine adducts (e.g., dG-C8 derivatives of 2-acetylaminofluorene, 2-aminophenanthrene and 4-aminobiphenyl) are substrates for nuclease P1 and may be lost; whereas, the butanol extraction method gives better recoveries for these adducts (Gupta and Earley 1988). For DNA from fish exposed to complex mixtures, the samples should be processed by both procedures to determine adduct levels and profiles. If both methods give similar results, then the less labor-intensive nuclease P1 procedure would be the method of choice.

The butanol adduct extraction procedure is based on the observation that mononucleotides modified with hydrophobic structures will preferentially partition into water-saturated n-butanol. The presence of the phase transfer agent, tetrabutylammonium bromide, can enhance the extraction of some structures (Gupta 1985). This method can process up to 100 mg hydrolyzed DNA (Beach and Gupta 1992). However, some of the benefits gained by using larger quantities of DNA will be offset by increasing background radioactivity on the chromatograms.

We normally run duplicate samples of the 3'-BaPDE-dG standard through the butanol extraction process as an extraction efficiency standard to assess procedural losses. If you are using this method for a specific adduct, it would be wise to run a standard of that specific adduct through the butanol method for a recovery estimate.

Samples which have been stored at -20°C for an extended time period or tissue that is partially decomposed may contain DNA breakdown products that interfere with the detection of DNA adducts when the normal lithium choride/Tris/urea (LTU) solvent systems are used for development in the D4 direction (see chapter on Chromatography for description of solvent systems and Fig. 3). The isopropanol/4N ammonia solvent system should be used instead of LTU system for D4 as these breakdown products will migrate with the solvent front and not interfere with the autoradiographic detection of adducts. Another alternative is to use the nuclease P1 method which removes most of these breakdown products before ³²P-labeling.

Procedure

1. Place an aliquot of the DNA enzyme hydrolysate (up to 40 µg of hydrolyzed DNA), 20 µL of 10 mM tetrabutylammonium bromide, 20 µL of 100 mM ammonium formate, pH 3.5, in a 1.5 mL microcentrifuge tube and add sufficient distilled water to bring the volume to 200 µL. The microcentrifuge tubes should be labeled in two places (i.e., top and side) to avoid loss of sample identification from an inadvertent butanol leak.
2. Extract the enzyme hydrolysate solution twice with 180 µL water-saturated double distilled n-butanol (footnote 5) by vortexing for 15 seconds and centrifuging for 2 minutes at 14,000 rpm. Transfer the butanol layer containing the DNA adducts (top layer) to a clean 1.5 mL microcentrifuge tube using a pipettor.
   Do not transfer any water with the butanol phase. Also, be sure to change pipette tips after each sample to prevent cross-contamination.
3. Backextract the butanol fraction with 180 µL of n-butanol-saturated distilled deionized water by vortexing for 15 seconds and then centrifuging for 2 minutes at 14,000 rpm. This step removes any normal nucleotides which may have transferred into the butanol phase. For 1-2 µg DNA do 1 backextraction, 3-10 µg DNA do 2 back extractions and for 15-40 µg DNA do 3 backextractions. Discard the water extracts (the lower phase). Remove the butanol phase after the last water backextraction and place in a methanol rinsed 0.5 mL microcentrifuge tube.
4. The samples are placed in a Savant concentrator/evaporator (heater on the unit is set at 45°C) for 30 to 60 minutes until all the butanol has evaporated. A 1.5 mL microcentrifuge tube is used as a sleeve to hold the 0.5 mL microcentrifuge tubes in the Savant rotor. Add 100 µL of ddH₂O to each tube and vortex vigorously. Use the Savant concentrator/evaporator to remove the water in the tubes. Add 15 µl ddH₂O and flick the bottom of the tube vigorously with your finger to dissolve adduct residue. The samples are ready for postlabeling.

The nuclease P1 enhancement method is easy to use and based on the observation that chemically-modified DNA is resistant to the 3'-phosphatase activity of nuclease P1; whereas, normal nucleotides are hydrolyzed to nucleosides, which are not substrates for phosphorylation by T4-polynucleotide kinase. However, the method has several potential pitfalls. The degree of resistance of DNA adducts to nuclease P1 hydrolysis is dependent on the adduct type and the physical size of the adduct (Gupta and Earley, 1988). For some DNA adducts, a variable percentage of the adducts will be hydrolyzed during nuclease P1 treatment depending on the amount of nuclease P1 used and how long the samples were hydrolyzed. If one is targeting a specific DNA-adduct, it is necessary to assess its resistance to hydrolysis by nuclease P1 and adjust the procedure accordingly to maximize recovery.

It is important that both RNAse A and RNAse T1 are used in the RNA removal steps of DNA isolation, otherwise the autoradiograms from the nuclease P1 enhancement method may have substantial smears due to RNA contamination.

Procedure

1. Place 7 µL of a nuclease P1 solution containing 2 µL of 4 µg nuclease P1/µL, 0.9 µL of 1 M sodium acetate (pH 5.0), and 4.1 µL of 1 mM zinc chloride at the bottom of a 0.5 mL microcentrifuge tube. Then add 10 µL of DNA enzyme hydrolysate directly into the nuclease P1 solution by submerging the pipet tip containing the DNA into the nuclease P1 solution, and pumping with the pipettor to mix the solution.
   This is a critical step because if any of the hydrolysate escapes nuclease P1 digestion poor quality autoradiograms will be generated with a high background.

   The final concentration of the nuclease P1 in the microcentrifuge tube is 0.5 µg/µL.

2. Incubate samples for 45 minutes at 37°C. Add either 3 µL of 0.5 M CHES buffer (pH 9.6) or 0.5 M Tris base to the hydrolysate, briefly centrifuge the tube contents to the bottom, and mix with a pipettor. This step raises the pH of the hydrolysate. The samples are now ready for the addition of the [gamma-³²P]ATP solution for postlabeling.

Xenobiotic-DNA adducts and DNA bases are postlabeled using T4-polynucleotide kinase (PNK) to enzymatically transfer ³²P from [gamma-³²P]ATP to 3'-mononucleotides to form [5'-³²P]deoxyribonucleoside-3',5'-bisphosphates. Important points for successful labeling are 1) [gamma-³²P]ATP is in excess of the DNA, 2) sufficient PNK is present to carry out the reaction, and 3) the [gamma-³²P]ATP used has a high specific activity. We normally use 100 µCi of [gamma-³²P]ATP with a specific activity of 2,000 to 3,000 Ci per mmol when labeling 10 µg of DNA that has gone through either the butanol or nuclease P1 enhancement procedure. It is important to have the concentration of [gamma-³²P]ATP in the range of 0.8 to 1.6 mM to be on a labeling plateau for PAHs (Segerbeck and Vodicka 1993); however, for other types of DNA adducts the concentration of [gamma-³²P]ATP used may need to be higher (Beach and Gupta 1992).

The sensitivity of adduct detection increases with the specific activity of [gamma-³²P]ATP used. However, 100 µCi of [gamma-³²P]ATP with a specific activity of 6000 Ci per mmol contains approximately 16 pmol of ([gamma-³²P]ATP+ATP) versus 33 pmol when the specific activity is 3,000 Ci per mmol (the lower specific activity is due to increased presence of nonradioactive ATP). This means that 200 µCi of [gamma-³²P]ATP with a specific activity of 6,000 Ci per mmol is needed per sample to have the same starting molar concentration of ATP that 100 µCi of 3,000 Ci per mmol [gamma-³²P]ATP would yield. We have found that a specific activity for [gamma-³²P]ATP in the range of 2,000 to 3,000 Ci per mmol is satisfactory.

Labeling efficiency is also dependent on the amount of PNK used and the pH of the labeling medium. In a review by Beach and Gupta (1992), they report that the amount of PNK per sample used by various laboratories ranged from 2 to 68 units and was dependent on the compounds that were labeled. Segerback and Vodicka (1993) found that a labeling plateau for individual PAHs and PAH mixtures was reached when the PNK concentration in the samples exceeds 0.3 units/µl. At pH 8.0, PNK has some residual 3'-phosphatase activity, which can affect labeling efficiency. However, at higher pH levels (>pH 8.5) this phosphatase activity is almost nonexistent. Generally, in most procedures the labeling buffer pH is 9.5 before addition to a sample. However, the actual pH in the sample during labeling is usually lower because of the buffering capacity of components present in the enzyme hydrolysate. With BaPDE-dG adducts, maximum labeling occurred when the actual labeling pH in solution was approximately 8.8, and there was a substantially lower labeling level of adducts when the actual pH at the time of labeling was 9.5 or 8.0 (unpublished data).

This procedure is for 1-10 µg DNA samples that have gone through an enhancement step to remove normal nucleotides (i.e., nuclease P1 or butanol extraction).

1. Make sufficient [gamma-³²P]ATP labeling solution for the number of samples being processed and then add 10 µL of this solution to each sample. Each 10 µL of [gamma-³²P]ATP labeling solution will contain:
   • 100 µCi of 2000 to 3000 Ci per mmol of [gamma-³²P]ATP
   • 8 units of PNK in a 50% glycerol solution
   • 5 µL of labeling buffer (0.1 M bicine, 0.1 M MgCl2, 0.1 M dithiothreitol, 10 mM spermidine, pH 9.0)
   • Additional labeling buffer to bring to a 10 µl volume
   • Keep solution on ice until added to samples.
2. Place the samples (be sure to cut off the caps of the 0.5 mL tubes) in a preheated (37°C) Plexiglas carousel and add 10 µL of [gamma-³²P]ATP labeling mix to each tube using a micropipettor that has a Plexiglas shield (Reddy and Blackburn 1990).
3. Centrifuge the carousel briefly to concentrate the solutions at the bottom of the microcentrifuge tubes. Mix the contents of each tube using a pipettor. Note: Pipet slowly to avoid forming ³²P aerosols that will contaminate the inside of the pipettor. Place the carousel in a 37°C waterbath for 45 minutes. Optional Apyrase Treatment: Potato apyrase can be used to degrade any unreacted [gamma-³²P]ATP in solution. However, this step can be omitted when labeling large bulky hydrophobic adducts, such as PAHs (the solvent system used for D1 will remove all of the unreacted [gamma-³²P]ATP). After incubation with [gamma-³²P]ATP labeling mix, add 4 µL of potato apyrase solution (2.5 µg apyrase/µL) to each tube. Centrifuge to concentrate solution at the bottom of the tubes and mix using a pipettor. Incubate solution at 37°C for 30 minutes.
4. After incubation, centrifuge the carousel briefly to concentrate solution at the bottom of the tubes. After mixing with a pipettor, spot 5 to 20 µL of each hydrolysate slowly on the origin of a premarked PEI cellulose sheet that has a filter paper wick stapled to it (Fig. 5). Keep the spot as small as possible by applying the sample slowly to the sheet. During chromatography, the spots will expand and the smaller the spot size on application to the PEI-cellulose sheet the smaller and more intense the spot will be on the autoradiogram.
5. Please go to Chromatography of Xenobiotic DNA Adducts section for specific instructions on the chromatography procedures.
   Note: Spot a small aliquot (<1 µL) on a separate PEI-cellulose sheet for development in 0.3 M ammonium sulfate and 10 mM sodium phosphate, pH 7.4. This step is necessary to verify that an excess of [gamma-³²P]ATP was present in the sample at the end of the postlabeling. The absence of [gamma-³²P]ATP at reaction completion usually means that the enhancement step (butanol or nuclease P1) was not successful in removing the normal nucleotides which consume [gamma-³²P]ATP and that ³²P-labeling of samples may have not have gone to completion. If an apyrase step is included in the procedure, then consider taking an aliquot of the reaction solution just prior to the addition of the apyrase; otherwise the apyrase will degrade all of the [gamma-³²P]ATP remaining in the sample.

1. Make enough [gamma-³²P]ATP labeling solution for the number of samples being processed and then add 10 µL of this solution to 10 µL of each diluted sample of hydrolyzed DNA (5 µL of each DNA enzyme hydrolysate is diluted to 500 µL with ddH₂O for base analysis). Each 10 µL of [gamma-³²P]ATP labeling solution will contain:
   • 8 units of PNK in a 50% glycerol solution
   • 7.3 µL of labeling buffer (0.1 M bicine, 0.1 M MgCl₂, 0.1 M dithiothreitol, 10 mM spermidine, pH 9.0)
   • 0.5 µCi of [gamma-³²P]ATP (footnote 6)
   • 2.4 µL of nonradioactive ATP solution (0.5 mg ATP/mL) (footnote 7)
   • additional labeling buffer to volume
   • keep solution on ice until added to samples
2. To determine the specific activity of the [gamma-³²P]ATP bases labeling mix:
   • Take two 5 µL aliquots and count using a liquid scintillation counter
   • Two 0.5 µL aliquots are chromatographed on a PEI-cellulose sheet using 1 M LiCl as an eluant. If decomposition of the [gamma-³²P]ATP to ³²Pi and ADP has occurred, then two spots will appear in the autoradiogram. The lower spot (Rf 0.4) will be [gamma-³²P]ATP and the upper spot (Rf 0.9) will be ³²Pi. Excise both spots and count them by liquid scintillation spectrometry (LSS). The percentage of [gamma-³²P]ATP in the sample is defined as: % [gamma-³²P]ATP = {[gamma-³²P]ATP dpm}/{[gamma-³²P]ATP dpm + ³²Pi dpm}
   Note: This step for determining the % of [gamma-³²P]ATP will not account for the breakdown of nonradioactive ATP.

   Specific activity of bases labeling solution =
   

   [(% radioactivity due to [gamma-³²P]ATP) x (average dpm in a 5 µL aliquot of the bases labeling mix)]/ [concentration of ATP in the bases labeling mix aliquot]

3. Place 10 µL of the diluted enzyme hydrolysate (from the enzyme hydrolysis section) in a 0.5 mL microcentrifuge tube for DNA base analysis and place tube in a preheated (37°C) Plexiglas carousel (be sure to trim the cap off of the tube).
4. Add 10 µL of the [gamma-³²P]ATP labeling solution to each tube. Briefly centrifuge the carousel to bring the contents to the tube bottom and then mix the samples using a pipettor. Samples are incubated for 45 minutes at 37°C.
5. After incubation, add 4 µL of potato apyrase solution (3-5 mg potato apyrase in 2 mL of distilled water) to each sample and then centrifuge briefly to concentrate the solutions at the bottom. The potato apyrase hydrolyzes unreacted [gamma-³²P]ATP. Mix the samples with a pipettor and incubate for 45 minutes at 37°C. At the end of this incubation, centrifuge the samples briefly to bring the liquid to the bottom of the tubes.
6. Mix with a pipettor and then spot 10 µL from each sample 2 cm above the bottom of a PEI-cellulose sheet and space them about 2 cm apart across the sheet.
7. The PEI-cellulose sheet is developed in 0.3 M ammonium sulfate and 10 mM sodium phosphate, pH 7.4 (dilute the solvent to 80% strength if you are using laboratory prepared PEI-cellulose sheets).
8. At the end of the development dry the sheets and expose the chromatograms to film for 2 hours at room temperature in an autoradiography cassette. The sequence of spots from the origin in this solvent system are dG, dA, dC, dT and the spot at the top is ³²Pi (see Fig. 4). The approximate Rf values are 0.26, 0.46, 0.52, 0.69 and 0.84, respectively (values are for laboratory prepared 0.5% PEI-cellulose sheets). If other spots are present and adjacent to these four DNA base spots, this would indicate the presence of RNA contamination (see Fig. 4). Use the developed autoradiogram to locate the [5'-³²P]dpGp spot by laying the film over the PEI-cellulose sheet and mark the position of the [5'-³²P]dpGp spot on the chromatogram with a marking pen.
9. Cut out the marked spots using a razor blade and place the piece of the PEI-cellulose sheet containing the [5'-³²P]dpGp spot in a 20 mL scintillation vial, add scintillation cocktail and count on the liquid scintillation counter. The 5 µL aliquot of the [gamma-³²P]ATP bases labeling mix for specific activity determination should be counted at the same time as the dG spots. This way, all of the samples are counted at the same time and a decay correction does not have to be made.

   If an imaging system for radioactivity is available, then image the chromatograms and process directly. The 5 µL aliquot of the [gamma-³²P]ATP bases labeling mix for specific activity determination and the excised spots from the calibration strip used for calibrating the imaging system should be counted at the same time on the liquid scintillation spectrometer.

   Please see section on autoradiography and storage phosphor imaging for further information.