Biomedical Engineering Reference
In-Depth Information
should not exceed 650 bp. If the bisulfi te treatment was performed with agarose
beads (Method 1), the beads were melted by heating at 70 °C just before mixing into
the PCR solution. PCR was performed using AccuPrime™
Ta q
DNA Polymerase
System (Invitrogen) or Ex Taq™ polymerase (TaKaRa) for 40 cycles of 95 °C for
30 s, 50 °C for 30 s, and 68 °C for 1 min. High-fi delity DNA polymerases are rec-
ommended for the reactions. Following agarose gel electrophoresis and purifi cation
of PCR products from gels with the QIAquick Gel Extraction Kit (QIAGEN), the
PCR products were inserted into a TA vector, such as pGEM®-T Easy vector
(Promega). Competent
Escherichia coli
cells were transformed with cloned vectors
and plated onto Luria-Bertani (LB) agar plates containing ampicillin and X-Gal/
IPTG, which allowed the easy screening of positive transformants by appearance of
white colonies. Positive white colonies were picked from the LB agar plates and
grown in liquid LB medium. Plasmids with successful insertions were purifi ed with
QIAprep Spin Miniprep Kit (QIAGEN) and sequenced using primers which are
located on the plasmid right upstream of the insertion (e.g., a T7 or SP6 primer in
case pGEM®-T Easy vector was used).
10.6.3
Results of Bisulfi te Sequencing
Twenty-one colonies were picked from the LB agarose plate for sequencing.
Figure
10.4b
represents the typical illustration of results of bisulfi te sequencing,
named lollipop scheme, to show the sites of 5-methylcytosine residues. We found
four highly methylated cytosines in the amplifi ed 475 bp of the promoter region
(Fig.
10.4b
). The amplifi ed region was partially methylated in more than half of the
sequences at the cytosines no. 142, 154, 188, and 214.
10.7
Single Nucleotide Primer Extension Assay
Although highly accurate, the described technique is still laborious and expensive,
so samples should be well chosen before bisulfi te sequencing is performed. Single
nucleotide primer extension (SNuPE) technique can be regarded as a fast and cost-
effective prescreening solution for quantitative analyses of methylation levels. This
method is able to assess DNA methylation at one or two specifi c single cytosine
residues representing the methylation state of the whole amplicon. Experimental
protocol can be divided into four parts: (1) generation of PCR products derived from
bisulfi te PCR as described above, (2) purifi cation of PCR amplicon, (3) SNuPE
reaction, and (4) separation of the SNuPE products by high-performance liquid
chromatography (HPLC) and quantifi cation of the peaks (El-Maarri
2004
). The
SNuPE reaction is similar to Sanger sequencing reactions using unlabeled dideoxy-
nucleotides which specifi cally detect the methylation status of the respective CpG
in bisulfi te PCR products (ddCTP and ddTTP when SNuPE primers are placed on
