Team:EPF-Lausanne/2-step PCR

From 2008.igem.org

2-step PCR

The 2-step PCR is a technique used to add small DNA sequences to a PCR product. In the context of iGEM, this can be used as an alternative to classical cloning: instead of digesting and ligating each biobrick, small parts can be directly added by PCR, saving a lot of time.

The 2-step PCR requires three pairs of primers and three PCR runs:

A first pair is used to amplify a sequence of interest (for example an ORF) and provides annealing sites for the second pair of primers:


2step1.png

The second pair anneals with the first and contains the sequence we want to add to the PCR product. The third pair of primers finally amplifies the initial PCR product along with the added sequence.

2step2.png

Results:

2step3.png


During our project, we used this technique to clone the ORF of the gene luxI (the corresponding Biobrick, F1610, seemed to be inconsistent). The first PCR was performed on a plasmid to amplify the ORF coding for LuxI. During the second step, an RBS and the prefix were added upstream, whereas a terminator and the suffix were added downstream. The result of the this 2-step PCR was digested and inserted in pSB1A2, resulting in the Biobrick BBa_K092400. We are awaiting results on the generated sequence quality of this procedure.

Time saved

The major advantage of this technique is that the time needed to create clones can be reduced significantly. A promoter, an RBS and a terminator can be added to an ORF in a few hours. The result of this 2-step PCR can be digested and ligated into a vector. In total, this operation takes approximately 30 hours. In comparison, generating the same clone using classical cloning methods would take six days.

A primer library?

Several cloning steps can be considered as being standard in an iGEM project. Adding RBS, promoter or terminator to an ORF is a common feature of almost all projects. For this reason, we suggest that a library containing primers designed for 2-step PCR should be created. This would give an easier and quicker way to assemble components. The new parts created with this technique keep all the Biobricks properties, meaning that classical cloning can also be used afterwards to assemble big parts together.

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