The Master Mix contains High-Fidelity Polymerases, which comes from a Pyrococcus bacteria and is fused with an enhanced domain for improved fidelity and speed. This polimerase has a lower error rate than the Taq polymerase and is used for its superior performace and high-fidelity to the original sequence. Additionally, this polymerase has 3’ to 5’ exonuclase activity and delivers blunt-end products.
The Master Mix also has HF Buffer Pack and GC Buffer Pack. These buffer solution provide the conditions for high fidelity during amplification.
Another component the Master Mix has is the dNTP solution. This solution contains the nucleotide molecules necessary for the amplification of the sequence.
The annealing temperature is an important condition that helps the PCR be suficiently specific to be able to adequately amplify the DNA of choice. Usually, the annealing temperature is between 55°C and 72°C. The specific temperature depends on the length of the primer (which needs to bond to the template) and the GC content. The annealing temperature is calculated based on the melting temperature, which is the temperature at which half of the double stranded DNA disassociates from their pair. The DNA needs to be separated in order for the primers to bond to the template, therefore the temperature at which this happens needs to be considered. When a DNA molecule has a higher GC content, it needs a higher temperature in order to disassociate since Guanine and Cytocine are joined by 3 hydrogen bonds (stronger bond).
One protocol for linearizing fragments of DNA is the PCR. A PCR consists in 3 steps: denaturation, annealing, elongation. In denaturation, the DNA is exposed to a temperature that breaks the hydrogen bonds that keep both helixes together. During annealing the temperature is lowered enough for complementary nucleotides to bond together again. In this step, the primers bond to the template region. Finally, during elongation the polymerase joins the DNA chain and begins transcription on the OH end of the primer.
Another protocol for linearizing a fragment is a digestion using restriction enzymes. Restriction enzymes catalyze a double strand break in a specific sequence in the DNA. It recognizes the secuence, called the restriction site, and next to it breaks the DNA into blunt ends or cohesive ends. Within a plasmid or in a DNA molecule, there are several restriction sites that can be recognized by the enzymes. To linearize and isolate a specific sequence, you need to identify restriction sites that surround it. These restriction sites need to be outside the sequence of interest and do not appear in the within it. The source DNA is incubated with restriction enzymes and then purified to obtained the isolated segments.
There are some similarities between the PCR protocol and the restriction enzyme protocol. They both result in the linearized product of choice and have great applicability within synthetic biology. Additionally, for both of the protocols, the sequence needs to be known, at least partially. In the case of PCR, the sequence needs to be known in order to design the primers that surround the gene or region of interest. For the restriction enzyme protocol the sequence needs to be known in order to determine if there are restriction sites compatible with the enzymes available.
Despite the similarities, there are several differences between the PCR and RE protocols. First, the RE protocol needs less equipment than a PCR. To cut with RE, you only need an incubator for the part of the restriction. In the case of a PCR, you need a thermocycler for the reactions to work. Another difference is the end product. The RE protocol results in a lower amount of product than the PCR. In the case of the PCR, the fragment is isolated and amplified, which results in a bigger quantity of the product. The RE protocol just cuts up the existing DNA, but does not increase its quantity.
A digestion using PvuII as the restriction enzyme requires CutSmart buffer because it provides the specific conditions for the optimal functioning. PvuII is a high fidelity enzyme, which means it has been engineered to be very specific with the restriction sites, reducing off-target digestions. The buffer provides the conditions for the enzyme to fulfill its role, quickening the reaction and ensuring it stays on-target. PvuII also has the characteristic that it can finish a digestion in up to 15 minutes, but it can also stay overnight without damaging the DNA present.
Gibson cloning is a technique that joins fragments based in homology. In order for a sequence to be appropriate for Gibson cloning, it needs to have ends that are homologous to the fragment to which it will join. This homology can be induced if it is not already present. When designing the primers, homologus sequences can be added to the edges of the fragment so that when the digestion happens, the fragments have homologus ends.
In order to transform the E. coli cells, the protocol uses the heat shock method. In this method, competent cells are subjected to an abrupt temperature change, causing pores to open up in the cell wall. Once this happens, the plasmid solution is added to the bacterial culture and the genetic constructs enter through the open pores by diffusion. Fresh growth media is added to the bacteria so that the cells can recuperate and start to multiply with the plasmids inside. Finally, the transformed bacteria are inoculated to a selective media, in which only the cells with the plasmid can grow. This way, we can select for the transformed E. coli cells.
Golden Gate Assembly is a cloning method that was created in 2008 and is caracterized by being performed in one reaction and one tube. All the components (enzymes, DNA template, construct) are added to the same tube and the reaction takes place. The reaction consists of a digestion using the 2S restriction enzymes and a ligation of the final product with a T4 ligase. The 2S RE is a key characteristic of this type of assembly method; it is different from the regular RE enzyme since the cut is not done within the recognition site, it is shifted. The 2S restriction enzymes leave an overhang when cut, which can be used to assemble DNA fragments. The overhangs are in average 1 to 5 nucleotides, which can be coded with sequences that can be compatible with the overhang in the template DNA (where the fragment will be inserted). This feature allows the fragment to be inserted in the correct direction into the vector DNA. Additionally, when the fragment is correctly ligated, the restriction enzyme recognition site is eliminated, so the final construct is not cut by the enzymes still present in the solution. Once joined, the ligase creates the final bonds. Through this process, the resulting DNA does not have additional undesired nucleotides between the fragments.