2.3 MCA Coupled with RDA
2.3.1 Outline
For detection of differentially methylated sequences, you need to generate MCA amplicons from the tester samples (e.g., a cancer sample) and relatively large quantities of MCA amplicons from the driver samples (e.g., DNA from normal tissues) with the adaptors removed. The tester’s adaptors will then be changed and the DNA hybridized with driver DNA , followed by PCR amplification using the second set of adaptors. The subtraction is then repeated once, and the resulting amplicons are further cloned and characterized.
2.3.2 Removal of Adaptors from the Driver Amplicon
- Perform MCA on multiple aliquots of driver DNA . We typically run 10 reactions in parallel. Verify and pool the aliquots. Quantitate in a spectrophotometer.
- Digest the driver MCA amplicons using 2 Units/m g SmaI to remove the RMCA/RXMA adaptor. Inactivate SmaI by phenol/chloroform extraction.
- Remove the adaptors using a cDNA Spun column (Amersham).
- Electrophorese an aliquot of DNA before and after column filtration to check for complete elimination of adaptors.
- Add 1/30th volume of 3M Sodium acetate, two volumes of ethanol, chill at ?70℃, precipitate by centrifugation, resuspend in 200-400 m l TE and quantitate by spectrophotometry.
- 80 m g of processed driver DNA is required per tester sample (per condition).
2.3.3 Change of Adaptors on the Tester Amplicon
- Digest 5 m g of tester MCA amplicons using 20 Units XmaI to remove the RMCA/RXMA adaptor. Inactivate XmaI by phenol/chloroform extraction.
- Remove the adaptors using a cDNA Spun column (Amersham).
- Electrophorese an aliquot of DNA before and after column filtration to check for complete elimination of adaptors.
- Add 1/30th volume of 3 M Sodium acetate, two volumes of ethanol, chill at ?70℃, precipitate by centrifugation, resuspend in 10-20 m l TE and quantitate by spectrophotometry.
- Prepare JMCA and/or JXMA adaptors as described in section 3.1.2. Ligate the adaptors to 0.5 m g of the above DNA as described in section 3.1.2.
2.3.4 Competitive Hybridization
- Add 70 m l of TE to the ligation mix and purify DNA by phenol/chloroform extraction. Mix all of this DNA with 40 m g of MCA amplicons from driver DNA .
- Add 1/10th volume of 3 M NaOAc and 2 volumes of ethanol, chill at ?70℃ for 30 min, and centrifuge for 30 min.
- Dissolve DNA in 4 m l of 3 X EE (30 mM EPPS, 3 mM EDTA) solution and transfer to a 0.5 ml microcentrifuge tube.
- Denature DNA at 96℃ for 10 min, quick spin, add 1 m l of 5 M NaCl, cover with mineral oil and incubate at 67℃ for 20 hours (this is best done in a thermocycler).
2.3.5 Selective Amplification
- Heat 100 m l of 1 M NaCl at 67℃ for 5 min., and add 45 m l to the competitive hybridization solution.
- Prepare PCR mixture as follows: 10 m l of 10X PCR buffer, 5 m l (1/10th) of the hybridization mix, 1.2 m l of 25 mM dNTP mix, 100 pmol JXMA24 (or JMCA24) primers, 15 Units of Taq DNA polymerase, 0 m l (RXMA) or 5 m l (RMCA) DMSO, H2O to a total volume of 100 m l. Cover with mineral oil.
- Fill the ends at 72℃ for 5 min. PCR amplification is then done at 10 cycles of 95℃ for 1 min and 72℃ (JXMA24) or 77℃ (JMCA24) for 3 min. Final extension is at 72℃ for 10 min.
- Transfer the PCR products to a clean microcentrifuge tube.
- To digest the single stranded amplified MCA products, add 10 m l of 10X Mung bean nuclease buffer and 100 units of Mung bean nuclease, and incubate at 30℃ for 30 min.
- Purify DNA with phenol/chloroform extraction, add 2/3 volume of NH4 OAc, and two volumes of ethanol, chill at ?70℃ for 30 min. and precipitate by centrifugation.
- Resuspend DNA in 50 m l water. Add 10 m l of 10X PCR buffer, 1.2 m l of 25 mM dNTP mix, 100 pmol JXMA24 (or JMCA24) primers, 15 Units of Taq DNA polymerase, 0 m l (RXMA) or 5 m l (RMCA) DMSO, H2O to a total volume of 100 m l. Cover with mineral oil.
- PCR amplification is then done at 20 cycles of 95℃ for 1 min and 72℃ (JXMA24) or 77℃ (JMCA24) for 3 min. Final extension is at 72℃ for 10 min.
- After the reaction, electrophorese 10 m l of the PCR products in a 1.5% agarose gel to check the quality of the amplification. You should see a DNA smear, ranging from 200 bp to 1 kb.
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