Engineering

PCR troubleshoot

We planned to have several of the parts needed for the project, such as the GP1, FUS1, MAR1 to be amplified from C.reinhardtii genomic DNA, however that turned out to be more difficult than expected. While cloning genes through PCR is a rather straightforward method, the high CG content and highly repetitive genome regions provide a challenge when cloning C.reinhardtii genes.Our first approach was to adapt a cPCR protocol from Nouemssi et al. (2020)^[1] The cells were boiled for 10 min in EDTA buffer, and the lysates were then used in the PCR. The PCR was done using Taq DNA Polymerase (ThermoFisher Scientific) kit, following the manufacturers instructions. The PCR targeted the coding sequence for GP1 and used the primer pair GP1-F1/GP1-R1. The samples were then loaded on a 1% agarose gel, in order to visualize the results. We however did not see any product on the gel.

To rule-out the possibility, that the reagents we were using were the cause of the issue, we performed another PCR to test the PCR kits we had in stock: Taq DNA Polymerase (Thermo Scientific), Phusion (Thermo Scientific), DreamTaq DNA Polymerase (Thermo Scientific). We performed the PCR using primers and plasmid DNA, coding for the chromoprotein ScOrange, which were kindly provided by Prof. Anthony Forster. The results (Figure 1) showed that all of our kits were working as intended.

We then investigated if the issues with the PCR could be due to low quality cell lysates. We theorized that perhaps boiling the cells in EDTA was not enough to lyse cc-1690 strain cells, which have a thick, glycoprotein-rich cell wall, meaning that the DNA was not being released. We decided to try lysing both cc-1690 and cc-3403 (cell wall free strain) using both the previously mentioned protocol, as well as using a different protocol, which we adapted from an RNA prep protocol by Craig D. Amundsen (1999). ^[2] Here proteinase K is added to the reaction mixture to degrade proteins and inactivate nucleases, as well as SDS, which helps to disrupt cell membranes and denature proteins. However, we feared that SDS could potentially interfere with the DNA polymerase during PCR, so we decided to also try using a SDS-free lysis protocol, which we adapted from a genomic DNA purification with proteinase K protocol by Thermo Fisher Scientific.

For this PCR round we decided to use the same primer pair (PGK-F1/PGK-R1) as used in the cPCR protocol by Nouemssi et al. (2020)^[1], which is specific for the nuclear phosphoglycerate kinase (PGK) gene. We hoped that the PGK primers could serve as a positive control for future cloning attempts, as we could not be sure that the primers, which were designed by our team, were working as intended. The reaction was done using Taq DNA Polymerase (Thermo Scientific), following the manufacturers instructions. We once again did not see any product on the gel, meaning that PGK was not being amplified, regardless of which lysis method or strain was used.

We then tried to adapt the PCR for the high CG content of the C.reinhardtii. We first tried to follow the user manual for the Taq Polymerase, which suggested that in such cases up to 10% of DMSO could be added to the reaction mixture, as well as extending the denaturation time up to 3 min. We did another PCR, using the same primers (PGK-F/PGK-R) and the same cc-1690/cc-3403 lysates as in the previous try, but with these changes, however it again was unsuccessful (Figure 2).

At the same time, we also tried to perfectly recreate the cPCR protocol from Nouemssi et al. (2020) ^[1], as we were struggling to understand why we were not seeing PGK being amplified as described in the article, even when using the same primer pair. And for the first time, we were successful in amplifying a C.reinhardtii gene sequence. The main difference between this and previous attempts was that the extension temperature was lowered to 68 °C (we previously set the extension temperature at 72 °C, as recommended by the manufacturer). PGK was successfully amplified from both cc-1690 and cc-3403 lysates (figure 2, right side of the gel), although the band for cc-1690 was stronger. We decided to go on using the cc-1690 lysate.

Another factor that was considered was the DMSO amount. While DMSO is a great additive to increase PCR yield, it can also interfere with DNA polymerase activity. We decided to reduce the DMSO amount in the reaction mixture to 3%. We tried to run the PCR with the primer pairs for FUS1 (FUS1-F/FUS1-R), MAR1 (MAR1-F/MAR1-R), GP1 (GP1-F/GP1-R) and used PGK (PGK-F/PGK-R) as a positive control. We again were not able to amplify the target genes. However, based on PGK, we can clearly see that the addition of 3% DMSO did appear to greatly improve the PCR yield (Figure 3).

Finally, we decided to check if using a different polymerase could improve the results. We decided to try using Phusion (Thermo Scientific) high fidelity DNA polymerase, as the manufacturers claim that it is better suited for high GC templates. We used the GC buffer included in the kit, 3% DMSO was also added into the solution. All of these changes led to a much higher yield (Figure 4). Unfortunately, it also resulted in much lower specificity and with the amount of off-target products, it is impossible to say if we were successful in amplifying our actual targets. This might be caused by the low primer annealing temperature or improper primer design, which we hope to adjust in the future.

We conclud, that while cloning C.reinhardtii genes is without a doubt difficult, some changes can be implemented in order to greatly improve the results:

1. We did not see a noticeable difference between PCR efficiency when using cell wall free strain versus cell wall intact strain lysates, which shows that our lysis protocol can be used for both. But we did see a major difference between using lysates prepared from fresh, alive cell cultures, as opposed to from old cell samples.
2. Have a good positive control. This will make it much easier to adjust the PCR conditions and to troubleshoot any issues.
3. Try out different polymerases. Standard Taq polymerase can be used for simple applications, but it might struggle with more difficult amplicons. In our case, we found Phusion polymerase to be a good alternative.
4. Addition of DMSO to the reaction mixture can greatly increase the PCR yield for high GC DNA templates. It is best to start out with low amounts (2-3 %) and increase if needed. Keep in mind that this will decrease the primer melting temperature, and the annealing temperature will need to be adjusted accordingly. DMSO also reduces the effectiveness of the DNA polymerase, as well as changing the optimal MgCl2 concentration.