iGEM 2023 — Team michigan

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{% block title %}Notebook{% endblock %}
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## Contents

* [May](#may)
* [June](#june)
* [July](#july)
* [August](#august)
* [September](#september)

## May

### Week 1

* [05/09/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-09-2023.pdf) - Make common stocks needed for wet lab experiments & dipsticks
* [05/11/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-11-2023.pdf) - Test primers and PCR for activity
* [05/12/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-12-2023.pdf) - Ran gels on primer activity and PCR

![Michigan member making dipsticks in a science lab.](https://static.igem.wiki/teams/4858/wiki/notebook/photos/photos-gdoc/making-dipsticks.jpg)

Making Dipsticks

### Week 2

* [05/15/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-15-2023.pdf) - Test gel electrophoresis ladder resolution
* [05/16/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-16-2023.pdf) - Test dipstick on E. coli and run PCR on gapA gene
* [05/18/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-18-2023.pdf) - Run first LAMP reaction and test/dilute our primers
* [05/19/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-19-2023.pdf) - Gel electrophoresis and analysis of first LAMP reaction

### Week 3

* [05/24/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-24-23.pdf), [05/25/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-25-2023.pdf), [05/26/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-26-23.pdf) - Test LAMP primers under different conditions
* Test stocks to make sure they are performing properly

### Week 4

* [05/30/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/05-30-23.pdf) - Identify our best set of primers under various conditions

## June

### Week 1

* [Cell Culture Week 1](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/cell-culture-week-1.pdf) - Start culturing human cells

### Week 2

* [06/21/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/06-21-23.pdf) - Try new SYBR Safe to resolve resolution problems with gel electrophoresis
* [06/22/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/06-22-23.pdf) - PCR on gapA gene
* [06/23/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/06-23-23.pdf) - Run gel electrophoresis on LAMP reactions with human cells

![Michigan team member preparing to run LAMP reaction](https://static.igem.wiki/teams/4858/wiki/notebook/photos/photos-gdoc/recording-data-next-to-thermocycler.jpg)

Preparing to run LAMP reaction

### Week 3

* [06/27/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/06-27-23.pdf), [06/28/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/06-28-23.pdf), [06/29/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/06-29-23.pdf) - Reproduce LAMP experiments to confirm that our JP1 primers and determined LAMP conditions are effective

## July

### Week 1

* [07/06/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-06-23.pdf) - Begin work on qPCR to obtain differential fluorescence between our two DNA fragment samples

### Week 2

* [07/11/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-11-23.pdf) - More LAMP on our DNA fragments with qPCR fluorescence readings
* [07/12/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-12-23.pdf) - Fluorescent testing of standard probe concentrations on LAMP product
* [07/13/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-13-23.pdf) - Testing how the concentration of probes will affect our fluorescence results
* [07/14/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-14-23.pdf) - LAMP on cultured human cells

### Week 3

* [07/17/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/cell-culture-07172023.pdf) - Testing dipstick method on human cells
* [07/18/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-18-2023.pdf) - Test how serial dilutions of amplified DNA affect fluorescence
* [07/19/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-19-23.pdf) - DNA cleanup and lysis on LAMP products
* [07/20/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/7-20-23.pdf) - Test how LAMP amplification is affected by the concentration of MgSO4 in reaction

### Week 4

* [07/27/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-27-2023.pdf) - Testing HEK cells with and without our SNP on qPCR
* [07/28/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-28-23.pdf) - Testing water bath method for LAMP optimization

![Michigan team member pipetting samples into a gel](https://static.igem.wiki/teams/4858/wiki/notebook/photos/photos-gdoc/pipetting-into-wells-of-gel.jpg)

Pipetting samples into a gel

## August

### Week 1

* [07/31/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/07-31-2023.pdf) - Testing cell dilutions and seeing if our negative control is contaminated
* [08/01/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/08-01-23.pdf) - Probe optimization
* [08/02/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/08-02-23.pdf) - Probe testing with increaced replicates to test technical variation
* [08/04/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/08-04-2023.pdf) - Primer optimization

### Week 2

* [08/08/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/08-08-23.pdf) - dNTP optimization and testing for negative control contamination
* [08/11/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/08-11-23.pdf) - Testing fluorescence with 5x more quencher

### Week 3

* DNA sequencing
* [08/16-08/17 & 08/23-08/24](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/08-16-23-08-17-23-08-23-23-08-24-23.pdf) - Serial dilutions with DNA to test differential fluorescence

### Week 4

* [08/29/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/08-29-23.pdf) - Restriction enzyme test on AleI
* [08/30/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/8-30-23.pdf) - Gel electrophoresis on restriction enzyme test

![Michigan team member pouring gel to run electrophoresis](https://static.igem.wiki/teams/4858/wiki/notebook/photos/photos-gdoc/making-a-gel.jpg)

Pouring gel to run electrophoresis

## September

### Week 1

* [09/05/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/9-05-23.pdf) - Diagnostic digest
* [09/07/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/09-07-23.pdf) - Run qPCR on sink titration experiment
* [09/07/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/09-07-23.pdf) - PCR of oligos using 5 LAMP primer sets
* [09/08/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/09-08-23.pdf) - Rerun restriction digest test to validate product identity

### Week 2

* [09/12/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/09-12-23.pdf) - Run asymmetric PCR
* [09/13/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/9-13-23.pdf) - Run probes on asymmetric PCR, clean up LAMP product, run overnight digest on LAMP product
* [09/14/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/9-14-23.pdf) - Run probes on asymmetric PCR, clean up LAMP product, run overnight digest on LAMP product

### Week 3

* [09/18/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/09-18-2023.pdf) - Replication of LAMP reaction from Hyman paper again
* [09/19/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/9-19-2023.pdf) - Asymmetric PCR of our templates
* [09/20/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/9-20-2023.pdf) - Clean up the LAMP product and submit for sequencing

### Week 4

* [09/25/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/9-25-23.pdf) - Asymmetric PCR of template DNA
* [09/26/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/9-26-23.pdf) - Restriction enzyme digests of LAMP product
* [09/29/2023](https://static.igem.wiki/teams/4858/wiki/notebook/notebook/9-29-23.pdf) - Run probes on asymmetric PCR

![Michigan team member preparing qPCR plate for probes testing](https://static.igem.wiki/teams/4858/wiki/notebook/photos/photos-gdoc/pipetting-qpcr-well-plate.jpg)

Preparing qPCR plate for probes testing

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## Contents

* [Engineering Process](#engineeringProcess)
* [Cycle 1: LAMP Primers](#cycle1)
* [Cycle 2: Fluorescent Probe Assay](#cycle2)
* [References](#engineeringSources)

## Engineering Process

The engineering cycle — design, build, test, learn — is imperative when aiming to engineer biology and to characterize any biological component of interest. This year, our team’s project aimed to develop a complete end-to-end single nucleotide polymorphism (SNP) detection platform for use in point-of-care settings. With our goal use case being low resource, we aimed to use primitive and inexpensive reagents throughout the development of our test. Though this strategy is ultimately optimal for the end product, using lower fidelity reagents tends to introduce more variability and a greater need for troubleshooting. Accordingly, our team employed the engineering process extensively throughout our project in order to achieve an optimally functional test.

## Cycle 1: LAMP Primers

Our first iteration of the engineering cycle was performed in order to test our custom LAMP primers for F2RL3, the gene containing our SNP of interest.

### 1 — Design & Build

To design LAMP primer sets for the F2RL3 gene, our team first employed the [PrimerExplorer V5](https://primerexplorer.jp/e/) tool. We uploaded the first segment of the gene as a target sequence, then allowed the software to generate primer options. We edited default design parameters to allow sequences with 40-100% GC content, as our SNP is located in a GC rich region of the gene. We manually searched through the remaining results until obtaining a primer set which would yield a LAMP primer where the SNP was located in the hair pin loop structure. It is required that the SNP be placed in this position so that there are weakened interactions between complementary amplicon strands to allow fluorophore access. We selected two primer sets of varying melting points from these results, which we named JP1 and JP2.

In order to diversify our design approach to maximize the chances of obtaining functional primers, our team also employed the [NEB LAMP Primer Design Tool](https://lamp.neb.com/#!/) to create potential primer sequences. We inputted the first portion of the F2RL3 sequence, set the GC content to a maximum of 85%, then selected three resulting primer sets — denoted NEB1, NEB2, and NEB3. The NEB1 primer sequences are uploaded to the Part Registry with the following part numbers: [BBa\_K4858001](http://parts.igem.org/Part%3ABBa_K4858001), [BBa\_K4858004](http://parts.igem.org/Part%3ABBa_K4858004), [BBa\_K4858005](http://parts.igem.org/Part%3ABBa_K4858005), [BBa\_K4858006](http://parts.igem.org/Part%3ABBa_K4858006).

Each primer set contained 4 individual DNA oligos: F3, B3, FIP, and BIP. These sequences were ordered from IDT.

### 2 — Test

To test whether our LAMP primer designs were successful in amplifying our gene of interest, we ran a LAMP reaction for each primer set. More details regarding how LAMP was run can be found on the [Experiments](https://2023.igem.wiki/michigan/experiments) and [Protocols](https://2023.igem.wiki/michigan/protocols) pages. Following amplification, we ran the LAMP product from each reaction on an agarose gel to visualize our product. The gel produced from this experiment can be seen below in Figure 1.

![Agarose gel displaying LAMP products from all five custom primer sets.](https://static.igem.wiki/teams/4858/wiki/engineering/figure1.png)

Figure 1. Agarose gel displaying LAMP products from all five custom primer sets.

### 3 — Learn

From the gel shown in Figure 1, we concluded that all primer sets yielded substantive product, with a possible exception of the NEB3 primer set. LAMP products form concatemers, so they form a large smear when run on a gel, as is shown in our data. This result gave our team confidence that we were successful in designing LAMP primers capable of amplifying the F2RL3 gene.

### 2 — Test

We next sought to perform more extensive testing on a specific primer set of interest. From all the sets which showed definitive amplification, we arbitrarily chose the JP1 set for continued use. For this additional testing, we included several negative controls in order to verify our product was real and not an artifact. These negative controls included a LAMP reaction with DNA not containing the F2RL3 gene, a LAMP reaction with no DNA template, and a LAMP reaction with no polymerase. The results from this experiment are shown in Figure 2.

![Agarose gel displaying LAMP product using JP1 primer set.](https://static.igem.wiki/teams/4858/wiki/engineering/figure2.png)

Figure 2. Agarose gel displaying LAMP product using JP1 primer set.

### 3 — Learn

The gel shown in Figure 2 provided more convincing evidence that the JP1 primer set was producing real LAMP product, as all negative controls showed no amplification on the gel. This prompted our team to continue using the JP1 primer set.

## Cycle 2: Fluorescent Probe Assay

Our team next needed to apply the engineering cycle to designing and troubleshooting the use of our fluorescent probes for detection of our SNP of interest.

### 1 — Design & Build

To design fluorescent probes that would yield a maximal difference in signal between the cases of SNP presence and absence, our team utilized a thermodynamic modeling based probe sequence calculator provided by Hyman et al1. We copied the sequence of the LAMP product that fell between the F2 and F1 binding sites and inputted the wildtype and SNP-containing versions of this sequence into the probe calculator. The tool provided us with sequences for the fluorescent probe, quencher, and both sinks. The information and sequences for each of these parts can be found on the Part Registry with the following part numbers: [BBa\_K4858000](http://parts.igem.org/Part%3ABBa_K4858000) (fluorescent probe), [BBa\_K4858002](http://parts.igem.org/Part%3ABBa_K4858002) (quencher), [BBa\_K4858003](http://parts.igem.org/Part%3ABBa_K4858003) (sink 1), and [BBa\_K4858008](http://parts.igem.org/Part%3ABBa_K4858008) (sink 2).

To build these biological parts, we submitted their sequences for synthesis by IDT.

### 2 — Test

The goal of our fluorescent probe assay is to achieve differential fluorescence levels in samples with and without the SNP. To test whether this was achieved, we executed the fluorescent probe assay using JP1 LAMP product. More information on how this assay is performed can be found on the [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) and [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) pages. The fluorescence readings from this initial test of the assay are shown in Figure 3.

![Fluorescence readings from fluorescent probe assay.](https://static.igem.wiki/teams/4858/wiki/engineering/figure3.png)

Figure 3. Fluorescence readings from fluorescent probe assay.

### 3 — Learn

As seen in Figure 3, we did not observe any difference in fluorescence between samples with and without the SNP. Because the fluorescence of the sample without an SNP present was well above our negative control, we hypothesized that the fluorescent probe may be nonspecifically binding in this sample. We proposed that this may be due to a large amount of LAMP product present, creating an excess of opportunity for the probe to nonspecifically bind.

### 2 — Test

We next sought to test our hypothesis that an excess of sample DNA was causing nonspecific fluorescent probe binding and a lack of fluorescent signal difference between samples. To do so, we performed a titration of our LAMP product when performing our fluorescent probe assay, using maximal dilutions which produced equivalent results to the 1:25 condition in Figure 4 and decreasing concentration to a 1:200 dilution, which showed little difference with samples containing no DNA.

![Fluorescence readings from fluorescent probe assay with DNA titration.](https://static.igem.wiki/teams/4858/wiki/engineering/figure4.png)

Figure 4. Fluorescence readings from fluorescent probe assay with DNA titration.

### 3 — Learn

The continued lack of differential fluorescence seen in Figure 4 suggested to our team excess amplicon DNA was not the cause of poor probe performance. In identifying potential other rationales for not observing differences in fluorescence, we supposed that the fluorescence reading from a reaction is ultimately an indication of the equilibrium point at which the reaction falls. We hypothesized that our reaction parameters produced an equilibrium with a nonoptimal difference in the amount of fluorescent probe bound, thus removing any potential difference in the fluorescence reading. We reasoned that by altering the relative concentrations of the four interacting components of the probe reaction, we might be able to skew the equilibrium point such that we obtain a larger separation between the fluorescence values of samples positive and negative for the SNP.

### 2 — Test

In order to evaluate the possibility that adjusting reaction parameters may enable an alternative, more optimal equilibrium point, we performed our fluorescence assay with a variety of conditions that perturbed the concentration of one or both sinks. The results of this experiment are shown in Figure 5.

![Fluorescence readings from fluorescent probe assay with sink titration.](https://static.igem.wiki/teams/4858/wiki/engineering/figure5.jpeg)

Figure 5. Fluorescence readings from fluorescent probe assay with sink titration.

### 3 — Learn

As seen in Figure 5, altering the concentration of one
or both sinks in solution did not improve the differential fluorescence observed between samples with and without the SNP. Our next hypothesis proposed that we may be able to induce greater difference in signal by increasing the concentration of quencher in solution. This may make it more difficult for the fluorescent probe to bind to the sample, effectively enhancing differences in binding probability and increasing the relative fluorescence of the SNP-containing sample.

### 2 — Test

To assess whether increasing the concentration of quenchers in our fluorescent probe assay would increase the difference in fluorescence between samples, we performed a titration of our quencher. Results from this experiment are shown in Figure 6.

![Fluorescence readings from fluorescent probe assay with quencher titration.](https://static.igem.wiki/teams/4858/wiki/engineering/figure6.png)

Figure 6. Fluorescence readings from fluorescent probe assay with quencher titration.

### 3 — Learn

We did not observe any difference in fluorescence between the SNP and wildtype samples, despite increased quencher concentrations. We next began to question whether our LAMP product was in fact the sequence we were expecting. A different sequence would explain the high fluorescent signal in the wildtype sample and the lack of differential fluorescence. Pursuit of this research avenue can be read about further on our [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

## 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>

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{% block title %}Experiments{% endblock %}
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## Contents

* [Overview](#experimentsOverview)
* [DNA Extraction](#experimentsDipsticks)
* [LAMP](#experimentsInVitro)
* [Fluorescent Probes](#experimentsInVitro)
* [Asymmetric PCR](#experimentsAsymmetric)

## Overview

Our work in lab this year can be chronologically separated into four main categories — DNA extraction, LAMP, our fluorescent probe assay, and asymmetric PCR.

## DNA Extraction

We proposed cellulose dipsticks as a low cost approach to genomic DNA purification. To determine whether dipsticks were capable of transferring sufficient DNA from cell lysate for amplification, we used this approach to purify DNA from E. Coli, then used the extracted sample to amplify the gapA gene via PCR. We grew up E. Coli, lysed them, then dipped the unwaxed end of the dipstick into the solution to make the DNA stick to the paper. After washing the contaminants off with a wash buffer, we ran PCR amplification of the gapA gene, and we tested the success of the dipstick method by running gel electrophoresis to see if the corresponding gapA gene size was visible. We noticed that the dipsticks began to crumble during dipping and washing, so we switched to cellulose squares used with mulitple clean tweasers. This method proved successful, and was thus used in future DNA extraction from HEK293T cells for cellular LAMP.

## LAMP

Before moving forward with the amplification from cellular DNA, we had to confirm that we could amplify the F2RL3 fragments via LAMP, serving as our positive and negative controls for further experiments. We designed five primer sets with standard LAMP design software and performed *in vitro* LAMP on ordered Twist DNA to confirm amplification. Following this success, we opted to perform further confirmatory and optimization testing. This included using SYBR Green to measure dsDNA amplification over time in a qPCR machine, as well as SYBR Safe in gel electrophoresis to confirm the presence of our product (a long band revealing the presence of multiple different-sized concatemer products).

## Fluorescent Probes

Now that we could amplify the target gene using LAMP, we proceeded to combine dipstick extraction of F2RL3 from HEK293T cells with the amplification portion of LAMP

Since we confirmed we could amplify the wild-type and mutant F2RL3 fragments *in vitro*, we began testing our fluorescent probes. We used an online calculator to create a set of probes which bound differential amounts of fluorophore to the SNP region depending on the presence or absence of the SNP. Thus, after amplifying template and cellular DNA, we added a 1 uM mixture of probes and ran a fluorescent binding experiment (as described in our protocols). After initially not seeing promising results, we opted to dilute the amount of DNA mixed with probes and changed the ratios of our probes and sinks.

## Asymmetric PCR

We ran a restriction enzyme digest (the Ale1 enzyme) to reconfirm the identity of our product. This experiment, designed to cut the concatemer in the flat region of the DNA, suggested that we needed to switch to a different primer set than the one we had been using for LAMP so far. Repeating probe-based fluorescent experiments did not yield promising results again, which is when we switched to asymmetric PCR, which produces a simple, single-stranded product that might bind to our single-stranded probe and sink sequences more easily.

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{% block title %}Results{% endblock %}
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## Contents

* [Overview](resultsOverview)
* [Dipstick Extraction of Prokaryotic Genome](resultsDipstick)
* [LAMP Amplification of F2RL3](resultsLAMP)
* [Initial Testing of Fluorescent Probes on Ordered DNA](resultsInitial)
* [Discovery of Incorrect Amplicon and Reoptimization of LAMP](resultsDiscovery)
* [Asymmetric PCR As Alternative Amplification Strategy](resultsAsymmetric)
* [Development of Improved Probe Design Algorithms](resultsDevelopment)
* [Future Directions](resultsFuture)
* [References](resultsSources)

## Overview

To explore the use of SNP-LAMP detection of F2RL3, we divided our project into 3 specific aims. First, we adapted a published method for genomic extraction via simple cellulose dipstick1. Second, we developed and optimized options for amplification of extracted F2RL3 compatible with our extraction methodology 2, 3. Finally, we adapted a published design methodology for competitive fluorescent probes to our chosen SNP 2, 3.

## Dipstick Extraction of Prokaryotic Genome

We began work on our first aim by making cellulose dipsticks and testing their compatibility with downstream amplification methods. As a model, we used PCR amplification of gapA from cultured E. coli. While there are differences between mammalian and prokaryotic DNA, we felt this simple model would allow us to address any unforeseen challenges before moving to a more complex system.

Our dipsticks were made by cutting a Whatman paper filter into standardized thin rectangles and dipping in candle wax to make a handle (Figure 1a). Then, we lysed bacterial cells with a simple, SDS-based chemical lysis buffer1 and extracted genomic material by dipping the exposed end of our stick into the lysate. We found the wax crumbled a bit upon vigorous dipping and switched to a simple square of cellulose and fine-point tweezers. Besides this, however, the experiment was successful, producing a clean band of amplified gapA (Figure 1b). Total time for lysis and dipstick extraction was around 6 minutes using prepared materials.

![Simple cellulose dipstick made from a Whatman filter and the successful results of extraction testing.](https://static.igem.wiki/teams/4858/wiki/results/slide1.png)

Figure 1: Simple cellulose dipstick made from a Whatman filter next to 0.2 mL PCR tube for size reference (a), and the successful results of extraction testing (b). Lanes from left: ladder, no template PCR control, gapA PCR from dipstick extracted genome.

## LAMP Amplification of F2RL3

Our team hypothesized that the most difficult part of our project would be fluorescent probe optimization. To expedite this process, we ordered two fragments of F2RL3 from Twist Bioscience, one for the A allele and one for the G rs773902 allele. Then, we tested our amplification reaction on these fragments to produce material for probes testing. The rest of this results page will consider only samples which contain just one of the two alleles. We recognize that many patients in the clinic will be heterozygous, and that they will likely show an intermediate test result between the fluorescence levels from homozygous patients.

We chose LAMP as our primary method of amplification because of its speed and isothermal simplicity. However, LAMP is also known to be a finicky reaction. To develop an optimized amplification, we ordered 5 primer sets designed with standard LAMP design software 4, 5. We tested each primer set on our ordered DNA, and saw significant products from 4 of the 5 sets (Figure 2a). We chose a single primer set from those that worked and performed further confirmatory testing, including rigorous negative controls (Figure 2b) and qPCR measurements of dsDNA amplification over time using SYBR dye (Figure 2c). 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.

![LAMP primer set optimization.](https://static.igem.wiki/teams/4858/wiki/results/slide2.png)

Figure 2: 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 JP1, set JP2, 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.

With a seemingly well-suited reaction in vitro, we proceeded to combine amplification with dipstick extraction of F2RL3 from human cell line HEK293T. After harvesting and counting cells, we performed chemical lysis with a Tween20/TritonX-100 based buffer1 and dipstick extraction of genomic DNA into the LAMP reaction mix. We demonstrated amplification of our gene from the cells, but noted an inability to produce a clean no-template negative control over several tests (Figure 3). This was reminiscent of our earlier negative control issue (Figure 1c), but we hypothesized that it was again the product of contaminating F2RL3 gene fragments in our workspace. This is especially a concern with LAMP, as its hypersensitive, continuous amplification mechanism makes it prone to producing a disproportionately large amount of product from as little as 10 copies of contaminating template6.

![LAMP amplification of genomic material extracted from HEK293T via dipstick.](https://static.igem.wiki/teams/4858/wiki/results/slide3.png)

Figure 3: LAMP amplification of genomic material extracted from HEK293T via dipstick. (a) shows LAMP product on agarose gel. Lanes from left: ladder, sample from dipsticked DNA from cell lysate, no template control, and positive control with ordered F2RL3 template. We note some unexpected products present in negative control. (b) shows measurement of dsDNA in solution over course of LAMP reaction via SYBR visualization. Samples include starting with lysate from 10,000 cells, 8,000 cells, and a no template control. Again, we note an unexpected product in the negative control.

## Initial Testing of Fluorescent Probes on Ordered DNA

With amplified F2RL3 from our ordered fragments containing either the G or A variant of rs773902, we began testing our fluorescent probes. Our chosen system relies on a competition for binding the SNP region of F2RL3 between a DNA sequence attached to a fluorophore (F) and a similar sequence with no fluorophore, sink 1 (S1) (Figure 4). When F is bound to the SNP region, it fluoresces. However, if it is not bound to the DNA, it will bind a complementary sequence attached to a quencher (Q), decreasing its fluorescence. Likewise, when S1 is not bound to the SNP region, it has a complement sink sequence (S2) with which to bind. To produce differential fluorescence between alleles, we used an online calculator to create a set of probes which produce an increased level of fluorescence in the presence of the Ala variant vs Thr variant2.

![Competitive probe-quencher-sink mechanism.](https://static.igem.wiki/teams/4858/wiki/results/slide4.png)

Figure 4: competitive probe-quencher-sink mechanism, see above for detailed description. Reproduced from Hyman et al. under Creative Commons license2. The diagram at left shows an interaction probe, quencher, sinks, and F2RL3 amplicon in the presence of the Thr SNP.

To facilitate this process, we combined raw amplification products with a mixture of each of the probes at 1 µM, according to the procedure described in Hyman et al. 2 After mixing, the temperature was increased to 95 °C and then allowed to slowly cool back to room temperature. We expected to observe differential fluorescence between samples with different F2RL3 alleles. However, as seen below, we did not observe any meaningful differences between samples over numerous trials (Figure 5).

![Fluorescence of probe-quencher-sinks-LAMP product mixture by temperature, as read by qPCR machine.](https://static.igem.wiki/teams/4858/wiki/results/slide5.png)

Figure 5: Fluorescence of probe-quencher-sinks-LAMP product mixture by temperature, as read by qPCR machine. This trial was representative of the several we conducted but do not show here. As the annealing samples approach room temperature, we expect them to show differential fluorescence between alleles with higher fluorescence for F2RL3 Thr in a successful test. However, this was not observed.

This led us to suspect an issue with the conditions of the probe reaction. To address this, we tried to optimize our probe conditions by testing various combinations of probe and DNA concentrations. This included testing the following hypotheses:

1. Our LAMP reaction was producing SNP sites in excess of our fluorophore, such that 100% of F was bound to template DNA in the presence of both variants (Figure 6a). We were able to titrate DNA between maximum possible concentration (no DNA dilution, results of which were not significantly different than the 1:25 dilution pictured here) and a 1:200 dilution of our LAMP product, which did not produce a curve with a significant difference from conditions with no amplicon added. None of the tested conditions produced significant differential fluorescence.
2. Concentrations of S1, S2, & Q were not optimal, and did not produce differentially fluorescent equilibria under the present conditions. We tested various increased and decreased concentrations of each sink component, together and individually. No combination convincingly produced improved results (Figure 6b, c, d). While thermodynamic modeling may be able to predict correct concentrations for each component, we did not have a working model and relied on a simple trial and error approach of conditions relatively close to those successfully used by Hyman et al. 2

![Condition optimization testing of fluorescent probes reaction.](https://static.igem.wiki/teams/4858/wiki/results/slide6.png)

Figure 6: Condition optimization testing of fluorescent probes reaction. Each LAMP product was produced using the same reaction conditions and ran for an excess amount of time to ensure consistent DNA concentration between samples and trials. (a) shows titration of final DNA concentration relative to the maximum produced by our LAMP reaction. (b) shows titration of S1 and S2 sequences in concert. (c) shows titration of S1 and S2 alleles individually. (d) shows titration of Q.

## Discovery of Incorrect Amplicon and Reoptimization of LAMP

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, we began to question the identity of our product (Figure 2c, 3). We were concerned that we may have observed amplification of an incorrect amplicon, especially in light of the contamination concerns explained above.

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 one primer set that produced a digestible result. This was a different primer set than we had been using to that point (Figure 7a).

We proceeded to again evaluate the efficacy of our probes on the products of each primer set, including the digestible product, but again failed to observe convincing differential fluorescence in any condition (Figure 7b). We recognize that our probe reaction optimization experiments should be repeated on this improved product, but did not have time in the short iGEM season to conduct such testing.

![Agarose gel showing LAMP primer set optimization with diagnostic digest as confirmation of product identity.](https://static.igem.wiki/teams/4858/wiki/results/slide7.png)

Figure 7: (a) 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. Up to this point we had used set JP1, which did not show confirmatory digestion. (b) shows fluorescence data of probes reactions with LAMP product produced with each primer set. No condition showed convincing differential fluorescence.

## Asymmetric PCR As Alternative Amplification Strategy

While our initial literature review revealed LAMP as a promising candidate for F2RL3 amplification due to its speed and sensitivity, we were concerned that our probe experiments may have been affected by the primary or secondary structure of the LAMP amplicon. To address this, we adapted a method from another research paper that used a similar competitive fluorescence method to detect an SNP in a gene amplified by asymmetric PCR3. Asymmetric PCR is standard PCR amplification with an excess of one primer, producing a large number of single stranded products complementary to our probe sequence. Although asymmetric PCR requires a thermocycler and is not as user-friendly as LAMP, it addresses several of LAMP's drawbacks. In addition to a more simple amplicon structure, the quantized nature of PCR amplification means a small amount of amplified contamination present at the beginning of the reaction was less likely to compose a disproportionately large amount of the product.

We designed and tested PCR primers for an asymmetric PCR reaction, producing a correctly sized, diagnostically digestible product (Figure 8a). However, testing this product with our probes again failed to produce a convincingly differential result between F2RL3 alleles (Figure 8b). As above, we recognize that our probe optimization experiments should be repeated on this improved product, but did not have time in the short iGEM season to conduct such testing.

![Alternative amplification and probe testing with asymmetric PCR.](https://static.igem.wiki/teams/4858/wiki/results/slide8.png)

Figure 8: Alternative amplification and probe testing with asymmetric PCR. (a) shows agarose gel of PCR product along with identity confirmation via diagnostic digest as with LAMP above. (b) shows fluorescence readings of probes reaction on the asymmetric PCR product, tested at several dilutions of the raw product.

## Development of Improved Probe Design Algorithms

When we began our project, we realized that the single probe and sink combination, even if produced by the online calculator from Hyman et al. may not be optimal or effective for our test. To address this, we developed our own algorithm to design and evaluate alternative probe and sink options. We have produced a command-line tool to implement our algorithm, which is explained in detail on our software page.

## Future Directions

While we did not produce convincing differential fluorescence in the short iGEM experimental season, we learned a great deal about our methodology that is helpful for our own future work and other iGEM teams that may pursue similar projects.

Towards the simple, point-of-care extraction of patient genomic information, our future work will include optimization of extraction conditions from human cells by testing the compatibility and specificity of our improved amplification reactions with a dipstick extraction model. Given the resources, we would hope to conduct this testing on more realistic patient samples, such as human blood or saliva.

Towards rapid, point-of-care amplification of F2RL3, we would like to improve our methodology of confirming product identity through the use of a Sanger sequencing experiment on our PCR product. LAMP amplicon sequencing is not as straightforward due the complexity of its primary structure; however, we think properly analyzed next-gen sequencing could produce reliable results. Additionally, we have considered the idea that the total fluorescence signal of a sample may be dependent on the amount of DNA in solution. This means a valid test must standardize the amount of DNA produced in an amplification reaction. Our plan to address this is to ensure reagents, such as primers or dNTPs, for each reaction are limiting, such that each amplification produces the maximum amount of product. The sigmoidal curve of our SYBR experiments suggests that this is feasible, although we recognize more rigorous testing must be conducted with a finalized amplification reaction.

Additionally, to convert our methodology into a usable point-of-care test, further work must be done to show that the amplification procedure can be performed with simple equipment. This would include testing a LAMP reaction in a simple, temperature controlled heat block, and developing strategies to robustly minimize contamination of samples with non-patient-derived F2RL3. We hypothesize that this could be done by including a restriction enzyme like the one we used for diagnostic digestion in a commercial LAMP master mix. This enzyme could destroy contaminating DNA, and can be denatured at temperatures which do not affect LAMP polymerase, such that a restriction enzyme could keep the mix contamination free up until deactivation immediately before addition of a patient sample.

Towards developing reliable differential fluorescent probes and sinks, we will perform concentration and buffer optimization with our confirmed amplification product. Additionally, we recognize that our system relies on a manageable number of well-characterized chemical interactions, making it a good candidate for mathematical modeling. We realized this late in the season, and while our team is currently developing this capability through DNA interaction chemistry and thermodynamic modeling, we do not yet have a working model.

Once probe conditions are optimized, development of a usable point-of-care test further work must include demonstration of efficacy via simple heat block melting and annealing. Further, since fluorescence is dependent on temperature, a temperature range must be selected to be the end-point at which fluorescence is measured.

Finally, extensive trials must test the statistical properties of this test to determine whether controls for each F2RL3 allele must be included in each test. Further, heterozygous individuals will likely show intermediate fluorescence, which may be labeled with less confidence than a homozygous sample. Testing must be conducted to demonstrate test efficacy in this scenario.

## References

1. Mason, M.G., Botella, J.R. (2020). Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks. *Nat Protoc* 15, 3663-3677. <https://doi.org/10.1038/s41596-020-0392-7>
2. 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>
3. Wang, J., Zhang, D. (2015) Simulation-guided DNA probe design for consistently ultraspecific hybridization. *Nature Chem* 7, 545-553. <https://doi.org/10.1038/nchem.2266>
4. NEB LAMP Primer Design v1.4.1, New England BioLabs. <https://lamp.neb.com/#!/>
5. Primer Explorer V5, Elkien Chemical Co, <https://primerexplorer.jp/e/>
6. Loop-Mediated Isothermal Amplification (2023), New England BioLabs <https://www.neb.com/en-us/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp>

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