Preparation of DNA Probes

A typical QDFM experiment uses several different probes simultaneously.

One probe is needed to counterstain the DNA fibers. This probe is usually prepared by labeling DNA from the same batch that was used to prepare the fibers. Probes for sequences to be mapped along the DNA fibers are made such that they can be detected in a different color. Furthermore, it is recommended to include landmark probes that provide reference points by binding specifically to the vector part or the ends of DNA molecules (60).

3.3.1. Alkaline Lysis Protocol and Purification of DNA from P1, PAC, or BAC Clones

1. The Pl/PAC/BAC clones typically show far fewer deletions than YACs, so that it often suffices to pick two to three colonies from a plate, grow them overnight in AHC, and extract the DNA using an alkaline lysis protocol. The DNA can then be loaded directly onto the PFGE gel using a common gel loading dye. This protocol describes the isolation of DNA from approx 20-mL overnight cultures using 40-mL Oak Ridge centrifugation tubes (Nalgene). The protocol can be scaled down to accommodate smaller volumes.

2. Grow cultures overnight in 25 to 30 mL LB medium containing the recommended amount of antibiotic.

3. Prepare Oak Ridge tubes. Write the clone ID on a small piece of tape stuck to the cap. Spin 18.5 mL of culture at 2000g for 10 min at 4°C and discard the supernatant.

4. Resuspend the pellet in 2340 ^L of AL solution I, then add 100 ^L of lysozyme stock to each tube. Incubate tubes for 5 min at room temperature. Then place the tubes on ice.

5. Add 5.2 mL of AL solution II. The mixture should now become clear. Mix gently by inverting the tubes several times. Incubate for 5 min on ice.

6. Add 3.8 mL of AL solution III and mix gently by inverting the tubes several times. Incubate for 10 min on ice.

7. Spin for 15 min at 14,000g.

8. Transfer 10.4 mL of supernatant into a new Oak Ridge tube, add 5.8 mL of isopro-panol, and mix gently by inverting tubes several times. Use the old cap (with the ID sticker) on the new tube.

9. Spin for 5 min at approx 10,000g and discard the supernatant. Watch the pellet!

10. Wash the pellet in cold 70% ethanol. Let the pellets dry briefly, i.e., at approx 20 to 40 min at 20°C to 37°C.

11. Resuspend the pellet in 0.8 mL of TE buffer and split the volume into two 1.5-mL microcentrifuge tubes.

12. Add 400 ^L phenol/chloroform/isoamyl alcohol (Invitrogen) to each tube. All centrifugations during the following phenol/chloroform extraction are done at 12,000g.

13. Vortex for 15 s and spin down for 3 min.

14. Remove most of the bottom layer and spin again for 3 min.

15. Transfer the top layer to new microcentrifuge tubes and add 400 ^L chloroform/ isoamyl alcohol (24:1, v/v; Invitrogen).

16. Vortex well for 15 s, spin down for 3 min, and remove most of the bottom layer followed by a second centrifugation for 3 min.

17. Transfer top layer to a new microcentrifuge tube, add 2.5 vol, i.e., 1 mL of 100% ethanol, and let the DNA precipitate for 30 min at -20°C.

18. Spin down for 15 min, discard the supernatant, wash the pellet in ice-cold 70% ethanol, spin again briefly, remove supernatant, and air-dry the pellet.

19. Resuspend the pellet in 20 to 40 ^L TE, pH 7.4, containing 10 ^g/mL RNAse (Roche) made DNase-free by boiling at 100°C for 10 min and store in aliquots at -20°C.

20. Incubate for 30 min at 37°C (in water bath) and store at -20°C until use.

3.3.2. Preparation of DNA From Yeast Artificial Chromosome Clones

1. Retrieve the desired yeast clone containing the YAC from the library and grow it on AHC agar for 2 to 3 d at 30°C. Pick colonies from the plate and culture the clones in up to 35 mL AHC media at 30°C for 2 to 3 d.

2. Centrifuge cells in AHC media at 2000g at 4°C for 5 min.

3. Decant the supernatant and resuspend cells in 3 mL total of 0.9 M sorbitol, 0.1 M EDTA, pH 7.5, containing 4 ^L P-mercaptoethanol, followed by addition of 100 ^L of zymolase (2.5 mg/mL), and incubate at 37°C for 60 min.

4. Pellet the cells at 2000g and 4°C for 5 min and decant supernatant.

5. Resuspend pellet in 5 mL of 50 mM Tris-HCl, pH 7.4, 20 mM EDTA. Add 0.5 mL of 10% SDS and mix gently. Incubate at 65°C for 30 min.

6. Add 1.5 mL of 5 M potassium acetate and place on ice for 60 min.

7. Spin at 12,000g for 15 min at 4°C, and transfer the supernatant to a new tube.

8. Mix the supernatant gently with 2 vol of 100% ethanol by inverting the tube a few times. Spin at 2000g for 15 min at room temperature.

9. Prepare sets of four 1.5-mL microcentrifuge tubes for each clone.

10. Decant supernatant and air-dry the pellet. Resuspend pellet in 3 mL of 1X TE, pH 7.5.

11. Transfer 750 ^L of the DNA solution to each of the four 1.5-mL microcentrifuge tubes.

12. Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, pH 8.0), vortex well, and spin at 10,000g for 3 min.

13. Transfer the top layer to new 1.5-mL microcentrifuge tubes and add an equal volume of chloroform/isoamyl alcohol (24:1). Vortex well and centrifuge at high speed (10,000g) for 3 min.

14. Transfer the top layer to new 1.5-mL microcentrifuge tubes. Add 40 ^L of RNAse (1 mg/mL, DNAse free) to each of the four tubes and incubate at 37°C for 30 min.

15. Add 1 vol of isopropanol and gently mix by inversion. Centrifuge at high speed (10,000g) for 20 min.

16. Decant supernatant, wash pellet with 1 vol of cold 70% ethanol, and centrifuge at 10,000g for 3 min.

17. Decant the 70% ethanol, air-dry the pellet, and resuspend the pellet in 30 ^L 1X TE.

18. The DNA concentration is measured after the pellet is completely dissolved.

3.3.3. Generation of Probes by In Vitro DNA Amplification

1. In vitro DNA amplification using PCR is a very efficient method to synthesize probe DNA. It can be applied to amplify a particular DNA sequence, such as a part of the cloning vector (60), or with mixed-base primers to perform arbitrary amplification of virtually any sequence of interest (51,59,61). As illustrated in the following paragraphs, the former amplification can be applied to prepare DNA landmark probes, whereas the latter allows the preparation of probes to counterstain the fibers.

2. The generation of P1/PAC-, BAC-, and YAC-vector probe DNA takes advantage of the access to published vector sequences. PCR primers are typically designed to amplify fragments of 1100 to 1400 bp of vector sequence (51,52,60). Various oligonucleotide pairs have been designed in several laboratories including ours and are used in either single pairs or combinations (51,52,56,60). The PCR usually follows standard conditions, i.e., a Tris-HCl buffer containing 1.5 mM MgCl2 and

1 U Taq DNA polymerase per 50 ^L reaction is used, and annealing temperatures range from 50°C to 60°C.

3. The YAC cloning vectors pJs97 and pJs98, cloned in plasmid vectors (Invitrogen), can be used to prepare probes that are useful to determine the orientation of the YAC insert (56). For this purpose, plasmid DNA is extracted using the alkaline lysis protocol in Subheading 3.3.1. or a commercial kit and labeled by random priming as described below in Subheading 3.3.4.

4. The DNA probes for counterstaining the YAC DNA fibers are generated by mixed-base oligonucleotide-primed PCR (also referred to as degenerate oligonucleotide-primed PCR [DOP-PCR] (61,62). An aliquot of the high molecular weight DNA obtained by PFGE for fiber preparation is PCR amplified for a total of 42 cycles with oligonucleotide primers that anneal about every 200 to 800 nucleotides. In our preferred scheme, we use two different DNA amplification programs (62). Initially we perform a few manual PCR cycles using T7 DNA polymerase to extend the oligonucleotide primers at a relatively low temperature. Next, DNA copies prepared in these first cycles are amplified using the thermostable Taq DNA poly-merase and a rapid thermal cycling scheme.

5. In the first amplification stage, T7 DNA polymerase (Sequenase II, Amersham) is used in five to seven cycles to extend the mixed-base primer JUN1 (5'-CCAAGCT TGCATGCGAATTCNNNNCAGG-3'; N = ACGT) that is annealed at low temperature. Briefly, 2 to 3 ^L of high molecular weight DNA solution (after PFGE purification) are removed from the bottom of each tube and PCR amplified using the following conditions: denaturation at 92°C for 3 min, primer annealing at 20°C for

2 min, and extension at 37°C for 6 min. Sequenase II enzyme must be added after each denaturation.

6. In the second amplification stage, 10 ^L of the reaction products are resuspended in a 200-^L Taq DNA amplification buffer and amplified with primer JUN15 (5'-CCCAAGCTTGCATGCGAATTC-3') with the following PCR conditions: denaturation at 94°C for 1 min, primer annealing at 50°C for 1 min, and extension at 72° for 2 min, repeated for 30 cycles. After precipitation of the PCR products in 1.2 vol of isopropanol, the products are resuspended in 30 ^L of TE buffer. Subsequently, 1.5 ^L of this solution is labeled in a 25-^L random priming reaction incorporating digoxigenin-11-dUTP or FITC-12-dUTP.

7. DNA amplification is confirmed by electrophoresing a 5-^L aliquot on a 3% agarose gel in TBE buffer containing 0.5 ^g/mL ethidium bromide.

3.3.4. Probe Labeling via Random Priming and Hybridization

1. Labeling of DNA by random priming is a reliable method and, in our laboratory, is applied routinely to label DNA fragments from 100 bp to several hundred kb. The procedure involves an initial thermal denaturation of the DNA to allow the random oligonucleotides ("primers") to anneal. Thus, restriction or hydrolysis of large molecules is not necessary. Several companies now offer kits for random priming reactions. Slight differences exist with regard to enzyme activity, amount of random primers, and cost per reaction.

2. The concentration of PCR products can be estimated from the agarose gels run to confirm target amplification. If a sufficient amount of clonal or genomic DNA is available, 1 or 2 ^L can be used to accurately determine the concentration with Hoechst 33258 fluorometry using a TK100 fluorometer (Pharmacia).

3. Add 250 ng of DNA to water to a final volume of 7 ^L in a 0.5-mL microcentrifuge tube.

4. Boil DNA at 100°C for 5 min, and then quickly chill on ice.

5. For labeling with either digoxigenin-dUTP or FITC-dUTP, add:

2.5 ^L 10X modified nucleotide mixture 3.25 ^L 1 mM dTTP

1.75 ^L digoxigenin-11-dUTP or FITC-12-dUTP (1 mM each, Roche) 10 ^L 2.5X random primers (BioPrime kit, Invitrogen)

6. For labeling the DNA with biotin, add 2.5 ^L 10X dNTP mix provided with the BioPrime kit (containing biotin-14-dCTP), 5 ^L water, and 10 ^L 2.5X random primers).

7. Mix well, add 0.5 ^L DNA polymerase I Klenow fragment (40 U/^L, Invitrogen) and incubate in a water bath at 37°C for 120 min.

8. Add 2.5 ^L of 10X stop buffer (Invitrogen, part of the BioPrime kit).

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