Biomedical Engineering Reference
In-Depth Information
mixed with sodium bisulfi te solution. Next, 750
L of the sodium bisulfi te/
hydroquinone solution was transferred to each tube and overlaid with 750
μ
L of
heavy mineral oil. These aliquots were chilled on crushed ice for at least 30 min.
Restricted DNA samples were denatured by boiling for 10 min and incubated
with 0.33 M NaOH for 15 min at 50 °C. Two volumes of melted (50-55 °C) 2 %
low-melting-point agarose gel (NuSieve® GTG® Agarose; Cambrex Bio
Science Rockland Inc.) was mixed with DNA sample, and 25
μ
L of the sample
solution was immediately transferred to the mineral oil layer of the chilled
sodium bisulfi te solution to form agarose beads by chilling in the ice-cold min-
eral oil layer. The tubes were left on ice for 30 min, allowing the agarose beads
to sink into the bisulfi te solution. Then, the samples were incubated at 50 °C for
3.5 h for bisulfi te conversion. After briefl y chilling on ice, all solutions were
removed from the tubes, and agarose beads were washed subsequently with 1x
TE buffer (2 × 15 min), 0.3 M NaOH (2 × 15 min), 1x TE buffer (10 min), and
distilled water (10 min). Finally, the distilled water is completely removed, and
the samples were stored at 4 °C.
2 .
Bisulfi te treatment of genomic DNA in solution
: Four hundred nanogram of
genomic DNA, purifi ed from honeybee brains, was treated overnight with a
restriction enzyme that does not cut within the target region to be amplifi ed by
PCR. Sodium bisulfi te powder (3.8 g; mixture of NaHSO
3
and Na
2
S
2
O
5
; Sigma)
was dissolved in 2.5 mL distilled water and 750
μ
μ
L 2 M NaOH. Restricted DNA
samples are mixed well with 187
L of scav-
enger chemical [98.6 mg 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Sigma) in 2.5 mL dioxane (Sigma)] and incubated in a PCR cycler at 99 °C
for 15 min, at 50 °C for 30 min, at 99 °C for 5 min, at 50 °C for 1.5 h, at 99 °C
for 5 min, and at 50 °C for 1.5 h. After adding 150
μ
L of the bisulfi te solution and 73
μ
L distilled water to the DNA
samples, bisulfi te-treated genomic DNA is purifi ed and desulfonated using
MICROCON® Centrifugal Filter Devices (YM-30 membrane; Millipore) by
subsequent application of 500
μ
L 0.3 M
NaOH (15 min incubation followed by 15 min centrifugation), and 1x TE buffer
(15 min centrifugation). After elution using 50
μ
L 1x TE (2 × 15 min centrifugation), 50
μ
L of pre-warmed (50 °C) 1x TE,
bisulfi te-converted DNA is ready to use and can be stored at 4 °C.
μ
Recently, kits for bisulfi te treatment, which are designed to simplify and stream-
line the diffi cult procedures above, can be purchased from several companies, such
as TaKaRa, QIAGEN, Invitrogen, and Millipore.
10.6.2
Bisulfi te PCR and Cloning
PCR primer design for bisulfi te-converted DNA is challenging but crucial for effi -
cient amplifi cation. Oligo primers should be designed to be devoid of CpG posi-
tions. In order to amplify specifi cally from bisulfi te-converted DNA, all cytosines in
forward primers and all guanines in reverse primers for this PCR were replaced with
thymines and adenines, respectively. The length of the amplifi ed PCR products
