Loss of Heterozygosity

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This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

This method is used to detect genomic DNA deletions in tumor cells. For a more detailed discussion of applying this approach to microdissected samples, see Allelic Loss Studies in Prostate MP at NCI .

1. Reagents

1. DNA sample (see Processing of Microdissected Tissue - DNA-based Analysis )
2. Proteinase K (Sigma)
3. Proteinase K buffer (0.05 M tris-HCL, 0.001 M EDTA, 1% Tween 20, 0.1 mg/ml proteinase K, pH 8.0 )
4. Ampli Taq Gold Buffer (Perkin Elmer)
5. dNTP mixture (Perkin Elmer)
6. Primers
7. DEPC-treated H ₂ O
8. Ampli Taq Gold Polymerase (Perkin Elmer)
9. a - ³² P dCTP, 6000 Ci/mmol (NEN Dupont)
10. Formamide, 99% (Fluka)
11. Bromophenol blue-Xylene cyanole (Sigma), reconstituted as directed
12. Gel Mix-6 sequencing gel solution (Life Technologies)
13. Ammonium persulfate (Biorad)
14. 10X TBE buffer (0.89 M Tris Base, 0.89 M Boric Acid 0.02M Disodium EDTA ) (Advanced Biotechnologies)
15. Acrylease (Stratagene)
16. Glass cleaner (e.g., Windex, Glass Plus)
17. 95% ethanol

2. Equipment

1. Thermal cycler (MJ Research)
2. Sequencing gel electrophoresis apparatus (Gibco BRL)
3. High voltage power supply
4. Gel dryer (Life Technologies)
5. Glass plates, 31.0 x 38.5 cm (Life Technologies)
6. 0.4 mm spacers (Life Technologies)
7. Casting boot (Life Technologies)
8. Shark tooth comb (Life Technologies)
9. Small clamps
10. Whatman blotting paper, 3 mm thickness
11. Kodak Biomax MR or AR film
12. Film cassette (Amersham Life Science)
13. Film processor

3. Time Requirements

1. Gel preparation: 1.5 - 2 hours. Polymerization requires 1 hour, but may stand overnight.
2. LOH reactions: 2 .5 hours (approximately 1 hour for set-up, 1.5 for PCR)
3. High-resolution denaturing polyacrylamide gel electrophoresis: 1-3 hours. Twenty minutes for set-up. Electrophoresis time varies according to product size.
4. Gel drying: 1 hour
5. Autoradiography: 1 hour-2 days

4. Methods

TIP: Investigators must be especially careful when using this methodology 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. Therefore, when this technique is used to analyze archival samples, it is highly recommended that replicate experiments (multiple independent dissections, triplicate PCR reactions, etc.) be used to verify results.

A: LCM and Proteinase K Treatment

1. Obtain microdissected cells using the LCM procedure.
  TIP: 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 is recommended as a good starting point.
2. Suspend approximately 1000 microdissected cells in 20 µl proteinase K buffer.
3. Incubate overnight at 37°C.

B: Prepare the Glass Plates

TIP: Use Accuwipes for cleaning purposes, as they will not leave lint behind and are non-abrasive.

1. Clean glass plates twice with glass cleaner.
2. Repeat using 95% EtOH.
3. Spray small plate with Acrylease.
4. Spread Acrylease evenly using a circular motion.
5. Buff dry.
6. Quickly assemble the plates without touching the clean surface.
7. Place 0.4 mm spacers on the edges of the larger plate.
8. Place the smaller glass plate on top of the larger plate and spacers.
9. Secure the plates with a casting boot (tape or clamps may be substituted for the casting boot).

C: Polymerize the Gel

TIP: Acrylamide is a neurotoxin. Be sure to wear gloves and a labcoat when working with this substance.

Add 480 µl of 10% ammonium persulfate to 75 ml of Gel-mix-6.

Mix by inversion.

Hold the nozzle of the bottle at the corner of the gel cast.

Hold the gel cast at a 45 ^o angle to the bench and pour the gel between the plates. If bubbles get trapped between the plates, remove them by tapping the outside of the plates or by tipping the plates upright.

Insert the straight side of the comb 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 small bubbles.

Clamp the top of the plates together.

Allow the gel to polymerize for at least one hour.

TIP: 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 use.

D: PCR Reaction

TIP: Investigators must be especially careful when using this methodology 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, we highly recommend that replicate experiments (multiple independent dissections, triplicate PCR reactions, etc.) be used to verify results.

Remove reagents from the freezer before beginning the procedure.
- Thaw thoroughly before use.
- Prepare all reactions on ice.
- Prepare the reduced cytosine mixture prior to beginning the LOH reaction setup.
- Vortex all reagents, with the exception of Taq Gold Polymerase before beginning the PCR reaction setup.

Prepare 320 µl reduced nucleotide mixture:

<center> Reduced Nucleotide Mix <table> <caption> </caption> <tbody> <tr> <td> 10 µl </td> <td> dATP, 10 mM </td> </tr> <tr> <td> 10 µl </td> <td> dGTP, 10 mM </td> </tr> <tr> <td> 10 µl </td> <td> dTTP, 10 mM </td> </tr> <tr> <td> 2.0 µl </td> <td> dCTP, 10 mM </td> </tr> <tr> <td> 288 µl </td> <td> DEPC-treated H 2 O </td> </tr> </tbody> </table> </center>

References
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 6(13):2285-90, 1997.

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 Res 57:1855-8, 1997.

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 Res 55: 2959-62, 1995 .