Part:BBa_K4858004
F2RL3 LAMP NEB1 B3 Primer
The F2RL3 LAMP NEB1 B3 Primer is a carefully engineered oligonucleotide designed to amplify a specific region of the F2RL3 gene, notably encompassing the PAR4 mutation. When used in polymerase-based assays, this primer ensures targeted amplification of the desired segment, facilitating subsequent detection and analysis. It is suitable for both the LAMP reaction and asymmetric PCR as its design has been aided by the process described in Hyman et al. (2022) [1] and the Loop Primer Designer [2] from NEB.
Usage and Biology
General Design
Using the online NEB LAMP primer design calculator [2], we designed 5 primer sets for F2RL3 surrounding our SNP of interest. We tested each primer set on our ordered DNA, and saw significant products from 4 of the 5 sets (Figure 1a). We chose a single primer set from those that worked (the SP Set 1) and performed further confirmatory testing, including rigorous negative controls (Figure 1b) and qPCR measurements of dsDNA amplification over time using SYBR dye (Figure 1c). Note that the smear of DNA in each lane is caused by the stochastic extension of repeating LAMP amplicons, and is the expected distribution of sizes for a LAMP product.
Figure 1: LAMP primer set optimization. (a) shows agarose gel with the product of amplification with each primer set. Lanes from left: ladder, then LAMP products from reactions with the following – set SP1, set SP2, set NEB1, set NEB2, set NEB3. After selecting one of the working primer sets, (b) shows agarose gel with rigorous negative control testing the JP1 primer set. Lanes from left: ladder, standard LAMP conditions, control with nonspecific template DNA added (collected from purified E. Coli lysate), no polymerase control, and no template control. (c) shows measurement of dsDNA in solution as stained by fluorescent SYBR over time in LAMP reaction. We note one negative control replicate showed amplification, potentially due to contamination of the qPCR plate with the previous LAMP product.
Later on in our experimentation, we were faced with an unexplained failure to show differential fluorescence with our probes, and persistent amplification in no-template LAMP controls, both with dipstick samples from cultured cells and in various amplification reactions on ordered F2RL3 fragments. Thus, we began to question the identity of our product. As a preliminary experiment, we performed a diagnostic digest with a restriction enzyme targeted to break up the repeating structure of LAMP products into similar-sized fragments of around ~250 bp. We failed to observe digestion. Having evidence of an incorrect LAMP product, we returned to optimization of our LAMP conditions. We again tested all 5 primer sets, adding a diagnostic digest to our evaluation criteria of each condition, and found that there was only one primer set successful in producing amplification of the desired results - the NEB1 set. This was a different primer set than we had been using to that point (Figure 2).
Figure 2: Agarose gel showing LAMP primer set optimization with diagnostic digest as confirmation of product identity. Note NEB primer set 1 products showed a significant reduction in size to a consistent length of the expected digestion product following incubation with restriction enzyme
Design Steps
The first step to designing a suitable primer set is to find the sequence for your gene of interest and download it as a .txt file. Then, you must take 2000 bases from the gene sequence, being sure to include the SNP, and copy the sequence with the same strandedness. Paste this sequence in a new .txt file. (Naming convention: gene_upload_segment.txt). Upload this into the NEB LAMP Primer Calculator and click "Continue." This should yield you a set of LAMP Primers.
If no primer sequences are outputted, you can do one of a few things. First, you can set preferences to custom "Use values from Preferences tab." You may also increase the GC content to 85% and set the sorting method to "Easy." If the primers result in the SNP being in the improper location in the hairpin loop, change the start and end parameters around in order to achieve correct positioning. You must manipulate The F1/F2 and B1/B2 settings in order to put the SNP in the loop domain of the amplicon, as close to the tip of the loop as possible. Note: placing the SNP between B1 and B2 (closer to B2) or between F1 and F2 (closer to F2) must be done manually through trial and error. If done correctly, the primers should theoretically bind as in Figure 3.
Usage
We recommend testing the primers in strains that exhibit the F2RL3 gene carrying the mutation of interest.
After ordering our primers from IDT, we made a stock of 10x primers composed of 16 uM FIP, 16 uM BIP, 2 uM F3, and 2 uM B3 primer. Then, we created a LAMP master mix, added 10 copies of template DNA, and incubated for one hour at 65 C for one hour, as described in Hyman et al. (2022) [1] and shown in the two-step reaction below (Figure 4).
Figure 4: Two-Step Reaction to Amplify Target Sequence for 1 Hour at 65 C. Reproduced from Hyman et al. under Creative Commons license.
References
1. Hyman, L. B., Christopher, C. R., & Romero, P. A. (2022). Competitive SNP-lamp probes for rapid and robust single-nucleotide polymorphism detection. Cell Reports Methods, 2(7), 100242. https://doi.org/10.1016/j.crmeth.2022.100242
2. Biolabs, N. E. (n.d.). NEB LAMP Primer Design Tool. NEB LAMP. https://lamp.neb.com/#!/
Sequence and Features
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