PCR reaction

Because the antisense sequences overlap with the sense sequences for 20 bp in the designed primers, they will base pair with each other. Therefore, the whole CEA sequence can be constructed in the polymerase chain reaction (PCR) by DNA polymerase.

5‘ 3‘ 5‘ 3‘ 5‘ 3‘

3‘ 5‘

3‘ 5‘ 3‘ 5‘

DNA polymerase Figure 1. The scheme of CEA and SARS synthesis.

The CEA P1~P6 primers and the CEA P1~P5, P7 were added into a 0.2 ml tube, respectively, each taken 1 μl. The concentration of the first and the last primers in the sequence was 10 μM (P1, P6, P7) while the rest was 1 μM. 5 μl Taq buffer, 4 μl 10 nmol/ml dNTP, 0.5 μl Pro Taq, and 34.5 μl dd H2O were added. The reaction cycle is: 94 for 30 ℃ sec, 50 for 30 sec, and 72 for 20 sec for 2 cycles, 94 for 30 sec, 60 for 30 sec, and ℃ ℃ ℃ ℃ 72 for 20 sec for 34 cycles, 72 for 5 min to ℃ ℃ complete the reaction.

2.2.2.2 SARS synthesis

The SARS P1~P8 primers were added into a 0.2 ml tube, each taken 1 μl. The concentration of the first and the last primers in the sequence was 10 μM (P1, P8) while the rest was 1 μM. 5 μl Taq buffer, 4 μl 10 nmol/ml dNTP, 0.5 μl Pro Taq, and 34.5 μl dd H2O were added. The reaction cycle is: 94 for 30 sec, 55 for 30 sec, and 72 for 20 sec for ℃ ℃ ℃ 2 cycles, 94 for 30 sec, 65 for 30 sec, and 72 for 20 sec for 33 cycles, 72 for 5 min ℃ ℃ ℃ ℃

to complete the reaction.

2.2.2.3 SARS mutation

The m1 plasmid was mutated by Gene Tailor^TM Site-Direted Mutagenesis System (Invitrogen, USA). The site-directed mutation has been designed on the primer beforehand.

Original plasmid DNA is methylated before PCR so that it can be distinguished from the PCR product. After transformation into DH5α^TM-T1 competent cells, McrBC endonuclease in the host cell digests the methylated template DNA, leaving only unmethylated, mutated product.

100 ng plasmid DNA, 1.6 μl methylation buffer, 1.6 μl 10X SAM, 1.0 μl DNA methylase (4 U/μl) were added into a 0.2 ml tube, sterile, distilled water was added to make the total volume 16 μl. These reagents were incubated at 37 for 1 hr. Then, 2℃ μl methylated DNA, 5 μl 10X PCR buffer, 1.5 μl 10 mM dNTP, 1.5 μl SARS m1 5’ and 3’ primers (10 μM each), and 0.5 μl TaqXL (Protech, Taipei, Taiwan) were added into a 0.2 ml tube. The PCR condition is as the following: 94 for 2 min, 94 for 30 sec, 53 for 30 sec, 68 for 10 ℃ ℃ ℃ ℃ min (the last three steps were run for 24 cycles), and 68 for 10 min to finish the unfinished ℃ reaction.

The m2 and m3 plasmids were constructed by the following methods. The original CEA-SARS sequence was run twice to create two segments in which mutation sequence was designed in the primers. Then, these two segments served as the template for a second run of PCR, in which the very beginning and the end of the original CEA-SARS primers were added.

After the construct of m2, m3 was created by the m2 template by the same process.

CEA+front SARS CEA 5’

2nd muta 3’

Rear SARS 2nd muta 5’

SARS 3’

CEA+front SARS

Rear SARS CEA 5’

SARS 3’

CCEA-m2

Figure 2. The scheme of m2, m3 plasmid construction.

The m1 plasmid serves as the template for m2 PCR. 5 μl template (~4.0 μg/ml), 5 μl 10X buffer, 4 μl 10 mM dNTP, 0.5 μl Pfu were added into a 0.2 ml tube. 1 μl CEA P1 and SARS m2 3’ primers were added for the CEA+front SARS PCR while 1 μl SARS P8 and SARS m2 5’ primers were added for the rear SARS PCR. To make the total volume be 50 μl, an appropriate amount of dd H2O was added. The PCR condition for the CEA+front SARS sequence is as the following: 94 for 2 min, 94 for 30 sec, 55 for 30 sec (the l℃ ℃ ℃ ast three steps were run for 34 cycles), 72 for 1 min, and 72 for 5 min. The PCR condition for the ℃ ℃ rear SARS sequence is: 94 for 2 min, 94 for 30 sec, 53 for 30 sec (the last three steps ℃ ℃ ℃ were run for 34 cycles), 72 for 1 min, and 72 for 5 min. ℃ ℃

To combine the CEA+front SARS and the rear SARS sequences, 1 μl each PCR product was added, along with 5 μl 10X buffer, 4 μl dNTP, 0.5 μl Pfu, and 1 μl CEA P1 and SARS P8.

The PCR condition is: 94 for 30 sec, 57 for 30 sec, 72 for 1 min. This step was run f℃ ℃ ℃ or 3 cycles. The annealing temperature was set at 57 for the annealing of the PCR products. ℃ Then, the next round of PCR condition is: 94 for 30 sec, 50 for 30 sec, 72 for 1 min. ℃ ℃ ℃ This step was run for 34 cycles. The annealing temperature was set at 50 ℃for the annealing of the primers onto the templates.

The m2 plasmid serves as the template for m3 PCR. 3 μl template (~4.5μg/ml), 5 μl 10X buffer, 4 μl 10 mM dNTP, 0.5 μl Pfu were added into a 0.2 ml tube. 1 μl CEA P1 and SARS m3 3’ primers were added for the CEA+front SARS PCR while 1 μl SARS P8 and SARS m3 5’ primers were added for the rear SARS PCR. To make the total volume be 50 μl, an appropriate amount of dd H2O was added. The PCR condition for the CEA+front SARS sequence is as the following: 94 for ℃ 2 min, 94 for 30 sec, 52 for 30 sec (the last three ℃ ℃ steps were run for 34 cycles), 72 for 1 min, and 72 for 5 min. The PCR condition for the ℃ ℃ rear SARS sequence is: 94 for 2 min, 94 for 30 sec, 48 for 30 sec (the last three steps ℃ ℃ ℃ were run for 34 cycles), 72 for 1 min, and 72 for 5 min.℃ ℃

To combine the CEA+front SARS and the rear SARS sequences, 1 μl each PCR product

was added, along with 5 μl 10X buffer, 4 μl dNTP, 0.5 μl Pfu, and 1 μl CEA P1 and SARS P8.

The PCR condition is: 94 for 30 sec, 58 for℃ ℃ 30 sec, 72 for 1 min. This step was run for ℃ 3 cycles. The annealing temperature was set at 58 for the annealing of the PCR products. ℃ Then, the next round of PCR condition is: 94 for 30 sec, 50 for 30 sec, 72 for 1 min. ℃ ℃ ℃ This step was run for 34 cycles. The annealing temperature was set at 50 for the annealing ℃ of the primers onto the templates.