Biomedical Engineering Reference
In-Depth Information
added at the 3
-end of the primer depending on the methylation status of the target
cytosine residue. The ddCTP and ddTTP were incorporated at the cytosine site
which remains cytosines when methylated or show up as thymines when unmethyl-
ated, respectively, after bisulfi te PCR. The ratio of the SNuPE product signals
(C-extended primer and T-extended primer) is measured by HPLC.
′
10.7.1
SNuPE Reaction and HPLC
The procedure for SNuPE analysis has been modifi ed from that previously
described (El-Maarri
2004
; Tierling et al.
2010
). Bisulfi te treatment of genomic
DNA and subsequent PCR was performed using the same procedure described
above for bisulfi te sequencing. PCR products were purifi ed with the QIAquick
PCR Purifi cation Kit (QIAGEN) or by Exonuclease I/Shrimp Alkaline Phosphatase
(1 U ExoSAP) treatment. When larger PCR products are used, treatment with a
restriction enzyme for 2 h, which digests the internal site of the PCR products, can
be helpful to prevent forming of secondary structures and thereby inhibiting the
SNuPE reaction. SNuPE primers were placed immediately adjacent to the target
cytosine residues. The reaction was carried out in a total volume of 20
l contain-
ing 100-130 ng purifi ed PCR product, 1x buffer C (Solis BioDyne), 1.5 mM
MgCl
2
, 50
μ
M each ddNTP, 3.675 pmol of each primer, and 2.5 U TermiPol (Solis
BioDyne) DNA polymerase, which shows high performance for incorporating
ddNTPs. The thermal cycling was as follows: initial denaturing step of 96 °C for
2 min, followed by 50 cycles of 96 °C for 30 s, 50 °C for 30 s, and 60 °C for 60 s.
Extension products were separated on an HPLC system (WAVE® DNA Fragment
Analysis System; Transgenomic) at 50 °C and a fl ow rate of 0.9 mL/min using
acetonitrile gradients, which were generated by increasingly mixing buffer B
(0.1 M TEAA, 25 % acetonitrile) to buffer A [0.1 M triethylammonium acetate
(TEAA)]: 25-37 % buffer B for 15 min for cytosine 142, 27-39% buffer B for
15 min for cytosine 154, and 24-32 % buffer B for 10 min for cytosine 188 and
214. After estimation of peak areas or heights, the methylation index (MI) was
calculated as the ratio of the methylated signal divided by the sum of methylated
and unmethylated signals.
μ
10.7.2
Results of SNuPE Assay
We performed SNuPE analyses to exhaustively investigate the methylation level at
the four sites of 5-methylcytosines identifi ed by bisulfi te sequencing (Fig.
10.5b
).
At cytosines 142 (C142), 188 (C188), and 214 (C214), large peaks indicating meth-
ylation were clearly observed together with small peaks representing unmethylated
cytosines (Fig.
10.5b
). These data clearly corresponded with the data obtained by
bisulfi te sequencing shown in Fig.
10.4b
. We detected a peak of methylation at
