Parts

Background

In this year's project, Team Michigan is experimenting with the production of primers for use in loop-mediated isothermal amplification (LAMP), and a probe/sink combination to measure the differential fluorescence between the wildtype and single nucleotide polymorphism (SNP)-containing sequence of the F2RL3 gene. LAMP requires 4-6 primers that recognize distinct regions in target DNA. This can include two forward primers, a forward internal primer, two backwards primers, and a backward internal primer. Once the LAMP reaction has been completed, fluorescent probes can be used to detect the presence of a SNP or other type of mutation. We adapted a probe/sink combination approach outlined by Hyman et. al in order to maximize differential fluorescence¹. The probe has a higher affinity for the SNP-containing sequence, while the sink has a higher affinity for the wildtype sequence. Therefore, the probe will fluoresce upon binding to the SNP-containing sequence if that sequence is present in the solution, or the sink will bind to the wildtype sequence if that sequence is present in the solution, blocking the fluorescent probe from binding and fluorescing. Note that our project was designed to show increased fluorescence in the presence of the Ala variant of F2RL3.

Our template sequences were synthesized by Twist Bioscience. The design process of the five different sets of primers used in our application was aided by the NEB Loop Primer Generation Tool² and the Primer Explorer Tool³, therefore allowing it to be suitable with both LAMP and PCR reactions. The final primer design was determined using an open source command line interface that runs a primer optimization simulation we created. Our probes were also generated from inputting the segment between the F2 and F1 primer binding sites into a thermodynamic modeling based probe design calculator outlined by Hyman et al.

Basic parts include our optimized primer set and our set of probe and sink sequences. Our HEK293T cell culture line was generously donated to us by the Liu lab at the University of Michigan, and was lysed and used as part of our LAMP and fluorescent experiments.

We created the following parts as part of our project this year. The following table describes the parts we used over the course of our project this year.

Basic Parts

F2RL3 LAMP NEB1 F3 Primer	BBa_K4858001
F2RL3 LAMP NEB1 B3 Primer	BBa_K4858004
F2RL3 LAMP NEB1 FIP Primer	BBa_K4858005
F2RL3 LAMP NEB1 BIP Primer	BBa_K4858006
F2RL3 Fluorescent Probe (ProbeF)	BBa_K4858000
F2RL3 Quencher Probe (ProbeQ)	BBa_K4858002
F2RL3 Sink1 Probe	BBa_K4858003
F2RL3 Sink2 Probe	BBa_K4858008