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MOLECULAR BIOLOGY: WORKING WITH DNA
POLYMERASE CHAIN REACTION
MOLECULAR PROFILING: LOSS OF HETEROZYGOSITY
Molecular Profiling: Loss of Heterozygosity
Contributor: The Molecular Profiling Initiative of the National Cancer Institute
URL: Molecular Profiling Initiative
Loss of Heterozygosity (LOH) is a method used to detect genomic DNA deletions in tumor cells (see Citations #1 to #4). Unlike other methods such as Comparative Genome Hybridization, LOH is capable of identifying small interstitial deletions (see Hints #1 and #2). Procedure
A. LCM and Proteinase K Treatment (see Hint #1 and #2)
1. Obtain microdissected cells by the LCM procedure (see Protocol ID#2236 and Protocol ID#2237).
2. Suspend approximately 1000 microdissected cells in 20 μl of Proteinase K Buffer (see Hint #3).
3. Incubate the samples overnight at 37°C.
B. PCR Reaction
1. Remove the reagents from the freezer before beginning the procedure.
2. Thaw the reagents thoroughly before use.
3. Prepare all the reactions on ice.
4. Prepare the reduced cytosine mixture prior to beginning the LOH reaction setup.
5. Vortex all the reagents, except the Taq Gold Polymerase, before beginning the PCR reaction setup (see Hint #4).
6. Prepare 320 μl of Reduced Nucleotide Mixture as follows:
10 μl of 10 mM dATP
10 μl of 10 mM dTTP
10 μl of 10 mM dGTP
2 μl of 10 mM dCTP
288 μl of DEPC-treated ddH2O
7. Aliquot 1 μl of each DNA sample into a separate PCR tube and set aside (see Hint #5 and Hint #6).
8. Prepare a sufficient volume of the reaction mixture in each of the reaction tubes as follows (reaction mixture/reaction tube):
1.0 μl of 10 X Taq Buffer
0.8 μl of Reduced Nucleotide Mixture
0.2 μl of Forward Primer
0.2 μl of Reverse Primer
0.1 μl of α[P32] dCTP
0.1 μl of Taq Gold Polymerase
The total volume should be 9 μl per tube
9. Thoroughly mix the reaction mixture with a pipette and dispense 19 μl of this mixture into each tube containing DNA sample (see Hint #7).
10. Cap the reaction tubes and place them in a thermal cycler.
11. Cycle the reactions according to the Tm of the specific primer set. Calculate the initial melting temperature (Tm) of each primer according to the following rules:
Each T or A is equivalent to 2°C
Each G or C is equivalent to 4°C
12. After PCR, remove the samples from the thermal cycler and dispense 20 μl of Loading Dye into each reaction tube.
13. Store the reactions at 4°C until the gel is ready for loading.
Prepare the glass plates
1. Clean the glass plates twice with glass cleaner. Repeat with 95% Ethanol (see Hint #8).
2. Spray the small plate with Acrylease™.
3. Spread Acrylease™ evenly in a circular motion.
5. Quickly assemble the plates without touching the clean surface.
6. Place 0.4 mm spacers on the edges of the larger plate.
7. Place the smaller glass plate on top of spacers and the larger plate.
8. Secure the plates with a casting boot (tape or clamps may be substituted for the casting boot).
Polymerization of the Gel
9. Prepare gel consisting of 6% acrylamide by adding 480 μl of 10% Ammonium Persulfate to 75 ml of Gel-Mix-6 (see Hint #9).
10. Mix by inversion.
11. Hold the nozzle of the bottle at the corner of the gel cast.
12. Hold the gel cast at a 45° angle to the bench and pour the gel between the plates. If bubbles are trapped between the plates, remove them by tapping the outside of the plates or by tipping the plates upright.
13. Insert the sharkstooth comb upside down with the straight edge inserted approximately 1 cm into the gel. If bubbles are introduced at this point, remove the comb and use the teeth of the comb to sweep out any small bubbles.
14. Clamp the top of the plates together.
15. Allow the gel to polymerize for at least one hour (see Hint #10).
Final Gel Preparation
16. Remove the gel from the casting boot.
17. Push the spacers into the gel until they are flush with the smaller glass plate to prevent the buffer from leaking during electrophoresis (spacers tend to get pushed out of the gel during polymerization).
18. Place the gel in the sequencing apparatus and close the buffer release valve.
19. Pour 500 ml of 0.5X TBE buffer in the upper chamber and 500 ml of 1X TBE buffer in the lower chamber.
20. Clear bubbles from the loading area with a pipette.
21. Insert the comb right side up with the points inserted approximately 1 mm into the gel.
22. Pre-heat the gel at 1700 volts for 15 to 20 min.
23. Remove the samples from the freezer.
24. Add 20 μl of Loading Dye to the PCR reaction.
25. Denature the samples in a thermal cycler at 95°C for 5 min.
26. Remove samples from the thermal cycler and immediately place on ice, with an ice pack on top of the samples, for 1 min.
27. Turn off the power supply.
28. Adjust the comb if it has been pushed out of the gel during the pre-heating run.
29. Load 3 μl of each sample per well (see Hint #11 and #12).
30. Run the gel at 1800 volts for 1 to 2 hr (running time based on PCR product size).
31. Turn off the power supply.
32. Drain buffer chambers (buffer must be disposed of in a liquid radioactive waste carboy).
33. Remove the gel from the sequencing apparatus.
34. Separate the plates by removing the spacers and inserting the tips of two thin spatulas in their place.
35. Gently lift the spatulas until the top plate separates from the lower plate and gel.
36. Place Whatman paper on the gel.
37. Slowly peel the Whatman paper and gel off the glass plate.
38. Cover the gel with plastic wrap and dry on a gel dryer for 1 hr.
39. Remove the plastic wrap from the gel.
40. Place the gel in an autoradiography cassette.
41. Expose the gel to film for 1 hr to 2 days with Kodak BioMax MR or AR film (see Hint #13).
1. A sample is considered to be composed of a monoclonal cell population when two alleles are recognized in the undigested DNA sample and complete absence of one allele is seen in the digested DNA sample.
2. DNA that is recovered from microdissected samples and "semi-purified" with a one-step proteinase K buffer will sometimes produce "non-specific" PCR products in addition to the true alleles. Normal-cell DNA from archival specimens from control subjects serves as a good comparator and can assist in interpretation of allele patterns.
Proteinase K Buffer 0.001 M EDTA
1% Tween 20
0.1 mg/ml Proteinase K, pH 8.0
0.05 M Tris-HCl
Proteinase K Sigma Gel Mix-6 Sequencing Gel Solution CAUTION! see Hint #14
Loading Dye 0.05% Xylene Cyanol
0.05% Bromophenol Blue
95% (v/v) Formamide
20 mM EDTA
99% (v/v) Formamide CAUTION! see Hint #14
α [P32] dCTP 6000 Ci/mmol (NEN Dupont) Ampli Taq Gold Polymerase Perkin Elmer DEPC-treated ddH2O See Biotools -Working with RNA for handling of RNA Reverse primer Also see Protocol ID# 2238
3' gene specific primer
Forward Primer Also see Protocol ID# 2238
5' Gene Specific Primer
dNTP Mixture 10 mM dATP
10 mM dTTP
10 mM dCTP
10 mM dGTP
95% Ethanol Ampli Taq Gold Buffer Perkin Elmer Glass Cleaner Windex, Glass Plus TBE (10X) Acrylease
0.01 M EDTA
0.9 M Boric Acid
1 M Tris
Ammonium Persulfate BioRad BioReagents and Chemicals
Iń [P32] dCTP
Gel Mix-6 Sequencing Gel Solution
Polymerase, Ampli Taq Gold
1. The Molecular Profiling Group used this method successfully for prostate tissue and for their specific objectives. Investigators will need to tailor the following protocol for their own research objectives and particular tissue under study.
2. Investigators must be especially careful when using this method to analyze archival tissue specimens. Formalin-fixation in particular results in DNA that is difficult to amplify and often produces inconsistent PCR results, including artifactual allelic loss and poor amplification of large products. If this technique is to be utilized for analysis of archival samples, replicate experiments (multiple independent dissections, triplicate PCR reactions, etc.) should be used to verify results.
3. The number of cells needed to successfully perform the assay varies depending on the quality and processing conditions of the tissue samples. One thousand cells are recommended as a good starting point.
4. Investigators may want to consider the "touchdown" procedure (see Citation #5) for PCR Advantages and include much cleaner bands, since by starting with a high annealing temperature of 66 degrees and lowering 1 degree every cycle, the first PCR products are the most specific ones. The exact same protocol can be used for all primers.
5. DNA that is recovered from microdissected samples and "semi-purified" with a one-step proteinase K buffer will sometimes produce "non-specific" PCR products in addition to the true alleles. Moreover, larger alleles will sometimes amplify less effectively than smaller alleles. Thus, normal cell DNA recovered from the same tissue section as the tumor DNA serves as the best control for determining the presence or absence of allelic loss.
6. Parallel analysis of control DNA that is known to be from a monoclonal population is recommended to verify the efficiency of the restriction digest. DNA recovered from a tissue specimen that was processed similar to the tissue sample under study is ideal.
7. Use a pipette to mix the LOH reaction mixture with the DNA sample. This is especially critical for DNA from microdissected samples that have been processed through a one-step proteinase K-based "purification."
8. Use Accuwipes for cleaning purposes.
9. Acrylamide is a neurotoxin. It is important to wear gloves and a lab coat when working with this substance.
10. The gel can be left to polymerize overnight. However, if bubbles appear, the gel has begun to separate from the plates. To minimize separation, wrap the gel in plastic film and store at 4°C until needed.
11. To avoid contamination caused by leaking, it is best to only load every second lane with sample.
12. The PCR products are approximately 200 bp. Therefore, do not overload the gel with sample and run the gel for a longer period of time to sharpen the bands and increase separation.
13. Use MR film for maximum resolution of bands. An intensifying screen is useful when analyzing PCR products from small numbers of microdissected cells. Try a short exposure time (1 hour) first and only re-expose depending on the results.
14. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
Citation and/or Web Resources
2. Debelenko LV, Brambilla E, Agarwal SK, Swalwell JI, Kester MB, Lubensky IA, Zhuang Z, Guru SC, Manickam P, Olufemi SE, Chandrasekharappa SC, Crabtree JS, Kim YS, Heppner C, Burns AL, Spiegel AM, Marx SJ, Liotta LA, Collins FS, Travis WD, Emmert-Buck MR. Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung. Hum. Mol. Genet 1997;6:2285-5590.
1. Molecular Profiling Initiative.
4. Emmert-Buck M R, Vocke C D, Pozzatti R O, Duray P H, Jennings S B, Florence C D, Zhengping Z, Bostwick D G, Liotta L, and Linehan WM. Allelic loss on chromosome 8p12-21 in microdissected prostatic intraepithelial neoplasia. Cancer Research 1995;552959-2962.
3. Emmert-Buck, MR, Lubensky, IA, Dong, Q, Chandrasekharappa, C, Guru, SC, Manickam, P, Keseter, M, Olufemi, S-E, Agarwal, S, Burns, AL, Spiegel, AM, Collins, FS, Marx, SJ, Zhuang, Z, Liotta, LA, Debelenko, LV. Localization of the multiple endocrine neoplasia Type I (MEN1) gene based on tumor deletion mapping. Cancer Research 1997;57:1855-1858.
5. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. "Touchdown" PCR to circumvent spurious priming during gene amplification. Nucl Acids Res 1991;19:4008.