Multiplex Combined Bisulfite Restriction Assay (COBRA) for BAGE

1)        Nested PCR

• First round PCR:
  for each PCR use
  • 5 µl 10x buffer
  • 5 µl dNTPs (2mM each)
  • 3 µl MgCl₂
  • 0.5 µl primer BS.BAGE32f+ (TttagaggaTTaggagaagggggagT) 100µM
  • 0.5 µl primer BS.BAGE1540r+ (AcctAccaAttAAcattAttActAacattA) 100µM
  • 1 µl Taq Polymerase
  • 32.5 µl sterile distilled water
  • 2.5 µl bisulfite treated genomic DNA as template
• PCR cycle conditions:

• Label tubes with "sample name I" and store PCR product at -80°C for later use
• Second round PCR:
  As for first round but use

  • 0.5 µl primer BS.BAGE99f+ (gatggtggtggTaaTagagatggT) 100µM
  • 0.5 µl primer BS.BAGE1371r+ (ccttaAAcaAtAtaAaccctAataA) 100µM
  • 2.5 µl of the first round PCR as template
• PCR cycle conditions:

• Check size and quantity of PCR product and absence of amplification in the negative control by gel electrophoresis.
• If everything went all right, do again 2 more second round PCRs

• Combine all 3 second round PCR products in one tube and precipitate with PEG (see protocol "PEG purification of PCR products")
• Dissolve pellet in 60 µl 10 mM TRIS/Cl pH 7.5 - 8.0
• Label tubes with "sample name II"

• Dilute PCR products 10fold in 60 µl (6 µl PCR product + 54 µl water)
• Measure optical density (OD) at 260 nm (OD1 corresponds to 50 ng/µl DNA) and calculate concentration of PCR products. Note ration OD_{280 nm}/OD_{260 nm}. If ratio is below 1.8 there is probably an impurity in your preparation (and quantification is incorrect). If you have more than one sample, adjust concentration of samples by dilution. Do not dilute to less than 200 ng/µl.

• 700 ng PCR product are digested with MboI (tube M) and HphI (tube H) in a total volume of 20 µl:
• tube M:         
  • 2 µl NEB buffer 3
  • 0.2 µl MboI (1 unit)
  • x µl PCR product (700 ng)
  • (17.8 - x) µl sterile distilled water
• tube H:         
  • 2 µl NEB buffer 4
  • 0.2 µl HphI (1 unit)
  • x µl PCR product (700 ng)
  • (17.8 - x) µl sterile distilled water
• incubate at 37°C 1 - 2 hours

5)        Gel electrophoresis

• Prepare 200 ml 2% agarose in 0.5x TBE, weight the solution before heating, heat to dissolve agarose completely and replace evaporated water. Add 1 µl ethidiumbromid to 100 ml agarose solution, let cool to approximately 60°C and pour a gel of 50 g agarose solution using combs of 1 mm thickness.
• Add 2 µl loading buffer to digest and load the entire volume on the gel.
• Migrate at 80 - 100 V until upper light-blue band (Xylencyanol) reaches the upper part of the handle. Check under UV if bands are well separated.

• Take a picture under UV in 46 cm distance, aperture 2.8. Use expose time that gives a maximum of intensity without saturation (usually around 1 sec).
• Save image to diskette.
• Load image into quantification software (ImageQuant 2.1) and create a profile along the lane covering all visible bands (electropherogram). Calculate baseline and perform quantification for each peak. Since absolute values can vary from gel to gel use relative values in % band intensities for conversion into % methylation. Here, 100% corresponds to the sum of band intensities for all bands observed in a single lane.

• If the original genomic DNA was methylated, the MboI digest delivers characteristic bands at 287 and 241 bp. Theoretical simulation and experimental tests with M.SssI methylated DNA show that if these bands are occupied by 62.6% of the digested PCR product, the original genomic DNA was fully methylated at the investigated site.
• For the HphI digest the diagnostic bands lie at 266 and 246 bp. These bands cannot be separated on the agarose gel. If 16.3% of the digested PCR product are located in these bands the original genomic DNA was fully unmethylated.
  • MboI: % methylation = [% band intensity(287 bp) + % band intensity(241 bp)] * 100 / 62.6
  • HphI: % methylation = 100 - [(% band intensity(266,246 bp)) * 100 / 16.3]