Part:BBa_K4636025

RPA_0004771fw_2

RPA_0004771fw_2 (BBa_K4636025) is a reverse primer for recombinase polymerase amplification (RPA) reaction. This primer is designed to amplify Insert_0104771.

Main

Recombinase polymerase amplification (RPA) is a method for isothermal DNA amplification (Figure 1). Unlike polymerase chain reaction (PCR), it does not require temperature cycling to replicate DNA. RPA employs proteins such as single-stranded binding proteins and recombinases to facilitate reactions at room temperature[1]. Due to its distinctive reaction mechanism, the design of primers in RPA differs from that of PCR. The most notable distinction lies in the length of primers. In RPA reactions, the Tm value is not as critical; conversely, the length of primers must be sufficiently long to enable the binding of recombinase proteins and facilitate the RPA reaction.

Figure 1. This graph shows the process of RPA which needs recombinases and single-stranded binding proteins to carry out the reaction.

In our project, we aim to perform lateral flow tests using PCRD after amplifying the samples with RPA. Therefore, in terms of primer design, we are considering adding a biotin modification to RPA forward primer. Additionally, depending on the specific sample, the reverse primer will be linked to different modifications (DIG or FAM)(Figure 2). This approach allows the RPA product to be labeled at both ends, enabling subsequent PCRD detection[2]. The detailed principle is illustrated in the diagram below (Figure 3):

Figure 2. Schematic chart for modified RPA primer. Forward primer with biotin (blue) and reverse primer with DIG (orange) or FAM (green).

Figure 3. Schematic chart for lateral flow test, PCRD.

Design

RPA primers were designed according to TwistAmp® DNA Amplification Kits Assay Design Manual[3]. We should notice that primer size is important for RPA reaction. In addition, there are other tips for designing RPA primer. Here are the considerations:

(1) Primer size between 30 and 36 bps.
(2) Primer GC% between 20 % and 70 %.
(3) Primer Tm between 50°C and 100°C.
(4) Avoid continuously repeating sequences.
(5) Confirm that our product size is large enough (>200 bps).

Experiment

We used RPA_0101802fw_2 and RPA_0101802rv_2 primers to isothermally amplify IVT product of Insert_0101802 through RT-RPA. As demonstrated in the gel electrophoresis results (Figure 4), lane 2 confirms the accuracy of our primer design, for the band’s position conformed to our expectations.

Figure 4. Gel electrophoresis of diluted RNA sample from RT-RPA. Lane 1: 100 bp DNA Ladder, Lane 2: RT-RPA using IVT product of circ_0101802 (385 ng/μL), Lane 3: A 10^4-fold dilution of the IVT product from Insert_0101802 (385 ng/μL); Lane 4: A 10^6-fold dilution of the IVT product from Insert_0101802 (385 ng/μL), Lane 5: A 10^8-fold dilution of the IVT product from Insert_0101802 (385 ng/μL), Lane 6: A 10^10-fold dilution of the IVT product from Insert_0101802 (385 ng/μL), Lane 7: A 10^12-fold dilution of the IVT product from Insert_0101802 (385 ng/μL), Lane 8: No template control (NTC).

Reference

1. Lobato, I. M., & O'Sullivan, C. K. (2018). Recombinase polymerase amplification: Basics, applications and recent advances. *Trends in analytical chemistry : TRAC*, *98*, 19–35. https://doi.org/10.1016/j.trac.2017.10.015
2. El-Tholoth, M., Branavan, M., Naveenathayalan, A., & Balachandran, W. (2019). Recombinase polymerase amplification-nucleic acid lateral flow immunoassays for Newcastle disease virus and infectious bronchitis virus detection. *Molecular biology reports*, *46*(6), 6391–6397. https://doi.org/10.1007/s11033-019-05085-y
3. https://www.twistdx.co.uk/wp-content/uploads/2021/04/twistamp-assay-design-manual-v2-5.pdf