User:Torsten Waldminghaus/Methylation analysis with qPCR

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This protocoll describes methylation analyse by cutting bwith methylation sensitive REN followed by qPCR

Enzyme Site Notes Link
HphI GGTGA No star activity; reaction at 37°C; heatinactivation at 20 min 65°Chttp://www.fermentas.com/catalog/re/hphi.htm
  • HphI seems to be a good choice since it can be heatinactivated and has no star activity (does not cleave unspecific when DNA is overdigested).
  • In unsynchronized cultures the detection of hemimethylation will be difficult. If every GATC is hemimethylated for about 1 min and replication of the chromosome takes about 50 min, than about 2% of a specific GATC will be in a hemimethylated state giving only 1% cut with an overlapping enzyme. However, some GATCs stay hemimethylated much longer as for example in oriC, dnaA or the GATC-cluster (ref.)

Procedure

DNA digest

  • Dilute 625 ng DNA in 22.5 μL dH2O
  • add 2.5 μL of 10x restriction buffer (NEB Buffer 4)
  • take out 2 x 9 μL in new tubes
  • add 0.5 μL HphI (2.5 units) and 0.5 μL EcoRI to one tube and 0.5 μL 50% glycerol and 0.5 μL EcoRI to the other tube
  • Note: EcoRI fragments both chromosomal DNAs which is important because one would otherwise compare fragmented (HphI digested) with unfragmented chromosomal DNA (only glycerol) in the qPCR which will influence the result.
  • mix by pipetting and short spin in a centrifuge
  • incubate at 37°C for 1 h
  • incubate at 65°C for 20 min to denature enzymes
  • add 190 μL dH2O to each tube mix and spin down
  • use 10 μL per qPCR reaction (corresponding to 15 ng)

qPCR

  • to quantify the ammount of restriction and with that the ammount of methylation one can use qPCR with specific primers
  • primers need to border a region containing only the HphI site to be analyzed (note that HphI cuts 8 or 9 bp away from recognition site!)
  • as two controls one should include one set of primers for a region without any HphI site (for example uvrD primers) and a set of primers with a HphI site not overlapping a GATC (for example datA)
  • for primers refer to User:Torsten Waldminghaus/Primers
  • make primer mixes:
Primer Mix
for 250μL
22.5μL primer fw
22.5μL primer rv
6.25μL probe
198.75μL ddH2O
  • for each primer set 3 PCR reactions should be prepared for each DNA (triplicate)
  • calculate how many reactions for each primer set you need and mix a corresponding master mix containing the following (include 1-2 reactions extra in the calculation to be sure you have enough):
component for 1 reaction (μL)
TaqManMix 12.5
primer mix (see above) 2.5
  • Note: The probe is fluoreszens labeld and light sensitive so you should not let your stock lay arround. Pipetting without turning out all the light is however no problem.
  • Make a drawing of the 96 well PCR plate with the location of your primer sets and DNAs
  • Pipette in 15 μL of the master mix in the designated well (with one pipette tip per master mix)
  • Add 10 μL of the DNA to the corresponding well using a new tip for every 10 μL (wipe of the DNA at the corner of the well when pipetting the DNA into the well)
  • cover the 96 well plate with plastic tape cover
  • centrifuge short up to about 1000 rpm
  • put in qPCR machine and hope for nice results
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