Protocol for assembly
The program was experimentally verified by using the
oligodeoxynucleotides determined by the program for the two-step
assembly PCR construction of a DNA molecule that is to be used to
produce an RNA molecule. The desired RNA product is a 191-nucleotide
moleculeconsisting of 5’ and 3’ cis hammerhead ribzoymes
and a core 20 nucleotide region that forms a hairpin structure and
is having its structure studied in our lab by nuclear magnetic resonance
(NMR) methods. The program broke this 191-nucleotide DNA molecule
into four segments for the first PCR reaction and produced the two
oligodeoxynucleotide molecules for the second PCR reaction (Figure
2.a, b, c, d).
Upon receipt, the oligodeoxynucleotides for the first
step of assembly PCR were diluted to 0.125 µg/µL with
double distilled water, while the oligodeoxynucleotides for the
second PCR step were diluted to 0.25 µg/µL. For the
first PCR reaction, 4 µL of each oligo, 4 µL of 5 mM
dNTPs, 10 µL of 10x thermopol buffer (NEB), 1.5 µL of
Vent DNA polymerase (2000 U/mL), and 68.5 µL of double distilled
water were combined. This mixture was then subjected to 8 cycles
of amplification at 94 °C (1.5 min), 54 °C (2 min), and
72 °C (3 min). During the first cycle, the 94 °C step was
performed for 7 min. After the last cycle completed, an additional
5 min 72 °C elongation step was performed.
For the second PCR reaction, 1 µL of the crude
mixture from the first PCR reaction was mixed with 4 µL of
each primer, 4 µL of 5 mM dNTPs, 10 µL of 10x thermopol
buffer (NEB), 1.5 µL of Vent DNA polymerase, and 75.5 µL
of double distilled water. This mixture was then subjected to 25
cycles of amplification. Each cycle consisted of a 30 second 94°C
step, a 2 min 54 °C step, and a 1.5 min 72 °C step. Prior
to the first cycle, a 5 min 94 °C step was used. A 5 min 72
°C elongation step was included following the final cycle.
The PCR mixtures were analyzed by agarose gel electrophoresis.
For each reaction a 6 µL sample was mixed with 2 µL
of blue-green dye. The gel was stained with ethidium bromide for
20 minutes, and observed under UV light. As shown by gel analyses
(Figure 2.e), the first PCR reaction produces a diffuse band or
smear, while the desired full length product results from the second
PCR reaction. This behavior is consistent with previous reports
of assembly PCR gene construction. The product of the second PCR
reaction was cloned in to the pUC18 plasmid, and the correctness
of its sequence was verified by DNA sequencing.
Figure 2. The construction of a 191-nucleotide
DNA molecule using the oligodeoxynucleotides determined by the Assembly
PCR Oligo Maker program. (a) The sequence of the 191-nucleotide
DNA target to be produced. (b) The DNA sequences reported by Assembly
PCR Oligo Maker program. Sequences for both step of the assembly
PCR reaction are reported. (c) Diagram showing how the four oligodeoxynucleotides
anneal to produce the full-length dsDNA product (d) The final dsDNA
product produced by the assembly PCR reaction (e) Agarose gel showing
the results of the first (lane 2) and second (lane 3) PCR steps.
The desired 191-nucleotide molecule is visible after the second
step PCR step. DNA length markers are shown in lane 1.