During this year, we found an interesting method, linear array epitope (LAE), to produce a tandem-repeated sequence (TRS). Using the conventional method, it is hard to produce a repeat sequence on a commercial scale. We hope LAE can help future iGEMers when they need it!
Linear Array Epitope (LAE)
LAE is a technique that first produces the TRSs. Later in the procedure, those TRSs would be expressed as a long-chain peptide which can be cleaved by an enzyme, resulting in many identical peptides.
LAE involves three steps: the design of DNA oligo primers, template-repeated PCR (TR-PCR) and adaptor PCR (AD-PCR). The procedures are shown in Figures 1-3.
We design the primers with the desired sequence of peptides. In the primer design (Figure 1), TR-PCR primers would be partially complementary to each other and AD-PCR would have the restriction sites we needed.
Figure 1. The design DNA oligo primers.
In TR-PCR (Figure 2), those primers act as both primer and template and they are partially complementary shown in red. The TRSs would be produced during the PCR cycling.
Figure 2. The principle of TR-PCR.
In AD-PCR (Figure 3), the restriction sites would be introduced into the TR-products by adding the AD-PCR primers and PCR cycling.
Figure 3. The principle of AD-PCR.
- TR-PCR is sensitive to primer concentration. To optimize the results, you should try several concentrations to obtain the most repeating sequences.
- During AD-PCR, the gel will show many bands representing the products with different sizes. You can choose the specific product to meet the requirements of the experiment.
Originally, LAE was a method for preparation of an immunogen containing multiple copies of a defined peptide in a linear alignment. This method was designed to overcome the poor immune responses induced by traditional immunogenic peptide/protein antigens. The tandem-repeated epitopes are able to efficiently induce a strong immune response.
In summary, we hope that LAE can be useful to future iGEM teams.
Peng-Yeh Lai, Chia-Tse Hsu, Shao-Hung Wang, Jin-Ching Lee, Min-Jen Tseng, Jaulang Hwang, Wen-Tsai Ji, Hau-Ren Chen. Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique. Journal of General Virology (2014), 95, 2155–2165