This is a protocol for the rapid, non-destructive purification of=20
oligonucleotides, which may help someone. This is currently being=20
published in Anal. Biochem., and should appear on this year. Please,=20
if someone uses this method, let me know how it works on his/her hands.
Best wishes,
Dr. E. Hardy
CIGB
La Habana-CUBA
E-mail: protchem@cigb.edu.cu
NOTES and TIPS
Title: Elution of Unmodified Oligodeoxynucleotides from Zinc-Imidazole
Negatively Stained Polyacrylamide Gels. Authors: Eugenio Hardy (*),
Elder Pupo (*), Jos=E9 A. Silva, Ricardo Silva, Edelgis Coizeau, and
Lila Castellanos-Serra. Address: Center for Genetic Engineering and
Biotechnology, P. O. Box 6162, Havana 10600 Cuba. FAX: 0537 21 80 70;
E-mail: protchem@cigb.edu.cu (*) To whom correspondence may be sent
Running title: Elution of unmodified oligodeoxynucleotides=20
UV-shadowing is the most commonly used method for the micropreparative
detection of polyacrylamide gel electrophoresis (PAGE)-separated
oligodeoxynucleotides; it is simple, rapid and appropriate for a
variety of uses (1). Unfortunately, the short wavelength UV-light may
damage the integrity of nucleic acids (2-5) and has the added
disadvantage of being hazardous to eyes and skin (1). To avoid these
problems, we have recently developed a new zinc-imidazole-based method
for the visualization of polyacrylamide-separated DNA under UV
light-free conditions (2). As zinc-imidazole is a reversible fixative
method, double-stranded DNA can be eluted with a high recovery, 85% or
higher (6); it is supposed that oligodeoxynucleotides can also be
recovered from the gel if required. To verify this, we used our
recently developed purification method (6) for the rapid elution of a
45-mer oligodeoxynucleotide from gel microparticles (Fig. 1, =9E).
However, we found that an appreciable (approx. 20%) amount of DNA
diffuses to the solution during the originally established
mobilization step for zinc-imidazole stained dsDNA, which includes
zinc-chelation with EDTA followed by water washes to remove excess
EDTA. Recently, it has been demonstrated that diffusion of ethidium
bromide-stained DNA on agarose gels can be prevented by inclusion of
70% ethanol in the storage buffer (7). Taking advantage of this
knowledge, we used 70% ethanol in water to wash the gel slice after
zinc chelation; looses of oligodeoxynucleotides were significantly
lowered to 1.4% and the overall elution yields increased to 89% (Fig.
1, n). We have eluted a variety of oligodeoxynucleotides from
zinc-imidazole stained gels by using this modified procedure, ranging
from 8 to 50 mer, with recoveries similar to those described in Fig.
1. The recovered oligodeoxynucleotides migrated as single, highly
purified bands upon re-electrophoresis, without discernible
degradation products. The quality of our oligodeoxynucleotide
preparations was further evaluated by standard sensitive techniques.
5'-End radioactive labeling. The synthetic oligodeoxynucleotide
5=B4-ATCCCGATCCAAAACAGC-3=B4 was labeled with [ g-32P ] ATP using T4
Polynucleotide Kinase (1). The percent of radioactivity incorporated,
as measured by trichloroacetic acid precipitation (1), averaged 88% in
three independent experiments with an specific activity of 109 cpm/mg
by using primers prepared by the zinc-imidazole-passive diffusion
technique or 78% after the conventional method [detection by
UV-shadowing after PAGE followed by elution with the crush and soak
method (1)]. PCR. The subtype P1.7,16 from the porA gen-containing
chromosomal DNA was prepared from cell cultures of N. meningitidis
H44/76 strain as described in (8). The PCR was performed in a DNA
thermal cycler (MiniCyclerTM, USA). Approximately 1 mg of chromosomal
DNA was added to the PCR mixture in a volume of 100 mL. The mixture
comprised 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.5% (v/v)
glycerol, 0.01 mM dithiothreitol, 0.2 mM each dNTP, 50 pmoles each
primer (oligodeoxynucleotides 5=B4-ATCCCGATCCAAAACAGC-3=B4and
5=B4-ATCCGATCCTGGCTTGC-3=B4), and 3U Taq DNA polymerase. After
denaturation at 94 =BAC for 4 min, we carried out 2 amplification
cycles, comprising denaturation at 94 =BAC for 1 min, annealing at 40
=BAC, and extension at 72 =BAC for 30 sec were carried out. This PCR was
completed after other 30 amplification cycles, comprising denaturation
at 94 =BAC for 1 min, annealing at 55=BAC for 1 min and extension at 72 =BA=
C
for 30 sec. The PCR-amplified products were analyzed by 2% (w/v)
agarose gel electrophoresis followed by ethidium bromide staining as
usual. The DNA amplification value by using primers prepared by our
zinc-imidazole-passive diffusion technique versus the conventional
crush and soak method after UV-shadowing (1) was 38 x 103 vs. 28 x
103. Values represent the average of two independent experiments. In
addition to the above experiments, good readable sequencing ladders
with appropriate signal-to-noise ratio have been obtained (not shown)
by using primers prepared by this technique. Together, these data
suggest that PAGE-zinc-imidazole followed by enhanced passive elution
from gel microparticles can be used as a reliable oligodeoxynucleotide
micropurification method. Although not tested, this method may be
useful for oligodeoxynucleotide structural analysis (e. g., by mass
spectrometry), as it does not introduce any alteration in the
structure of nucleic acids (2, 6), something that can not be assured
with the traditional transillumination-based methods.
REFERENCES
1. Sambrook J., E. F. Fritsch, T. Maniatis. (1989). Molecular cloning,
a laboratory manual. 2nd edition. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York, USA. 2. Hardy, E., Pupo, E., Casalvilla,
R., Sosa, A.E., Trujillo, L.E., L=F3pez, E., and Castellanos-Serra, L.
(1996) Electrophoresis 17, 1537-1541. 3. Hartman, P.S. (1991)
Biotechniques 11, 747-748. 4. Carielo, N.F., Keohavong, P., Sanderson,
B.J.S., Thilly, W.G. (1988) Nucleic Acids Res. 16, 4157. 5. Gr=FCndeman,
D., Sch=F6ming. (1996) BioTechniques 21, 898-903. 6. Castellanos-Serra,
L., Hardy, E., S=E1nchez, J.C. (1998) Anal. Biochem. 257, 227-228 7. =20
Jacobs, D., Neilan, B.A. (1995) BioTechniques. 19, 892-894. 8.
Guill=E9n, G., Alvarez , A., Lemos, G., Paredes, T., Silva, R., Mart=EDn,
A. (1993) Biotecnolog=EDa Aplicada 10, 108-113. LEGEND TO FIGURE Figure
1. Recovery of oligodeoxynucleotides from zinc-imidazole stained gels
by rapid passive elution. Identical samples of a 32P-labeled
oligodeoxynucleotide (45 mer, 522 ng, about 20,000 cpm) with the
sequence 5=B4-AGTCGTCCGGCAGCGAGGGGGGAGGAGGTGGTGGTGGTGGTGGTC-3=B4 were
electrophoresed in parallel lanes of a 15% polyacrylamide gel (1).
Triplicate lanes were stained with zinc-imidazole (2). The visualized
bands were excised and processed for oligodeoxynucleotide mobilization
as described in (6) (=9E), which includes an incubation (2 x 5 min) in
100 mM EDTA to chelate zinc ions (destaining) and three changes in
distilled water for gel washing (wash). Alternatively, distilled water
was replaced with ethanol 70% (v/v) in water for gel washing (n).
After oligodeoxynucleotide mobilization, bands were crushed through a
1 mL-polypropylene syringe containing two metal sieves at its bottom
to yield gel microparticles of 32-mm. The resulting gel slurry was
incubated in elution buffer (20 mM Tris-acetate, 0.5 M NH4Ac, 1 mM
EDTA, pH 7.4) under vortexing (2 x 10 min) and the eluted
oligodeoxynucleotides collected in the supernatant after
centrifugation (elution). On-gel corresponds to total radioactive
counts remaining in the gel after elution. Results are the average of
three independent experiments, followed by the standard deviation in
parenthesis.
<protchem@.cigb.edu.cu>
Prot. Chem. Dept.
Div. Phys. Chem. CIGB
FAX: 537-218070
Havana Cuba