Supporting Information Methods S1
MSAP relies on the differential efficiency of restriction enzymes to cut methylated and ummethylated
templates in the step prior to ligation and preamplification in conventional AFLP. The validity of
selecting MroI and BseAI as isoschizomers for MSAP was tested by assessing the relative
effectiveness of each enzyme to restrict template DNA differing only in the methylation status at the
restriction recognition site. This was tested using the following strategy:
1. Design of template for differential restriction
2. Selection of a plasmid into which the methylated/unmethylated fragment was cloned prior to
restriction and PCR amplification
3. Ligation of target fragment into plasmid
4. Quantification of ligation product
5. Restriction of ligation products using MroI or BseA1
6. Quantification of enyzme activity (as evidenced by PCR amplification) using RTqPCR
Each of the steps used in this experiment are described below.
1. Design of template for differential restriction
DNA fragments (Table 1 below) were designed to contain a MroI/BseAI recognition site (5´TCCGGA-3´) and also coadhesive ends for HindIII (5´-AGCT-3´)and EcoRI (5´-AATT-3´). Since the
restriction of the plasmid was conducted in the same reaction volume as the ligation of the
oligonucleotides, the bases adjacent to the coadhesive ends were selected in order to disrupt the
recognition sites of both enzymes to avoid restriction of the incorporated fragment (Figure 1 below).
Table 1 Oligonucleotide sequences: Methylated cytosines are shown as [5MedC]
Oligonucleotide name
Forward unmethylated
Reverse unmethylated
Forward methylated
Reverse methylated
Sequence (5´-3´)
AGCTACTCCGGACT
AATTAGTCCGGAGT
AGCTACTC[5MedC]GGACT
AATTAGTC[5MedC]GGAGT
2. Selection of plasmid
Plasmid pCR2.1-TOPO (invitrogen) was selected because it is provided in a linearized form. This,
together with the restriction using HindIII and EcoRI precludes illegitimate amplification from
plasmids lacking the target ligand.
3. Ligation of target fragment into plasmid
1ul of plasmid DNA was restricted using 1U of HindIII and 1U of EcoRI for two hours at 37⁰C in a
20uL reaction mix containing 1.5uL of the double stranded methylated or unmethylated fragment
(10uM) plus 1.1ul T4 Ligase buffer, 1.1ul NaCl (0.5mM), T4 Ligase 1U, BSA 1mg/mL.
Restriction/ligation products were diluted in 50uL of nanopure water
4. Quantification ligation product
The amount of ligation product obtained with each of the fragments was quantified using Real Time
Quantitative PCR (RT-qPCR). The ligation product obtained using the unmethylated fragment was
used to generated a serial dilution (1/10, 1/100, 1/1000, 1/10000). A PCR reaction mix containing
10uL of Sensimix, 1uL of Syto9, 0.8uL of each forward and reverse M13 primers (5uM) and 0.5 uL
of each of the ligation products (including the serial dilutions for the unmethylated fragment). Two
extra reactions were prepared using 1uL of the unmethylated ligation product and water instead of
DNA as a negative control. RT-qPCR was conducted using a Rotorgene 6000 (Corbett) under the
following conditions: 2minutes at 72⁰C followed by 40 cycles of 30 seconds at 94⁰C, 30seconds at
53⁰C and 30 seconds at 72⁰C with a final step of 2 minutes at 72⁰C. CT values were calculated using
the integrated software in the Rotorgene 6000.
5. Restriction of ligation products using MroI and BseAI
10uL from the ligation products obtained using plasmid pCR2.1 and both DNA fragments were
restricted in a 20uL reaction using 1U of MroI or BseAI, 2uL of the restriction buffer buffer supplied
by the supplier (Roche). Reaction mixes were incubated at 37⁰C (MroI) or 55⁰C (BseAI) for two
hours. Three replicates were obtained for each enyzme.
6. Quantification of enyzme activity (as evidenced by PCR amplification) using RT-qPCR
2uL of the restriction products obtained using MroI and BseAI were amplified using the reaction
conditions shown above. Since restriction/ligation products were diluted two times after the restriction
reaction using MroI/BseAI we used 2uL of the restriction products in order to allow the reaction to
leave the exponential phase of the amplification before 35 cycles. Three replicates were obtained from
each of the restriction products described above.