Executive Summary
| Proposed End User | Proposed Word Flow | |
|---|---|---|
| Vision | Small-scale/ family farm operations farmers and farm workers | 1. Obtain gDNA samples from cattle 2. Extract gDNA for samples 3. Amplify target genes using isothermal amplification 4.Detect genetic polymorphisms with Flappase assay |
| Implementation Criteria | User-friendly Cost-effective Effective at detecting multiple genetic polymorphisms Requires minimal specialized equipment | 1- 4: easy to obtain, minimal distress to user and animal 2 - 4: cost effective, easy to perform, no expensive equipment required, ability to create "kits" 3 and 4: sufficient sensitivity, creating of accessory equipment |
| Progress | Collaborated with local farmer for preliminary work | 1 -3: Proof of concept - see below for details 3 and 4: Development - see below for details |
| Safety Concerns | System must be usable with no lab experience Clear instructions provided System must be self-contained and requires no complex set up | Minimal hazardous materials and wastes Requires minimal PPE Minimal danger to user or animal when obtaining DNA samples Safe, reliable equipment |
| Potential and Current Challenges | User buy-in Meeting implementation criteria (especially cost) | Affordability of RPA as amplification method Strength of signal - requires testing Difficulties purifying Flappase in suffieicient amounts and concentrations Adapting system to new polymorphism targets Sensitivity of devices |
Background
Last year, our team’s project was centered around creating a field deployable genetic testing system that could be used to detect alleles of the beta casein gene (A1 or A2). We envisioned a system that would permit farmers to conduct their own genetic testing to determine the A status of their cows, allowing them to breed A2 herds. A2 milk is an emerging niche market, that in North America is estimated to exceed 4,759 600 million dollars by 2025 (1). Entry into this expanding niche market will help farmers command a high price for their product (milk), helping to improve the prospects of farmers that struggle with fluctuating commodity prices, high overhead costs, and competition with factory farms.
For this year’s project, we expanded the scope of our genetic testing system. Our goal is to make our Flappase-based detection system more useful to farmers, and we decided to focus our efforts on adapting our system to expand its use. Instead of developing a method to only detect SNPs in the beta casein gene, we designed a system that can detect different types of molecular phenotypes in a variety of different advantageous or deleterious traits. This ability to conduct a panel of at-home genetic tests would allow farmers to make informed breeding decisions based on many desired traits, including milk production/composition, fertility, and genes associated with causing disease in cattle. For a small-scale farm, current genetic testing requires significant financial investment and takes a long time to receive results. Because of this, we have been designing our system to allow genetic testing with an eye to affordability.
Detecting polymorphisms in target genes
Figure 1: Proposed use of devices to facilitate the proposed workflow and information to end user.
Once DNA has been extracted, target genes must be amplified to increase any signal used to detect genetic polymorphisms. To do this, our team selected RPA as an appropriate amplification method. This method was selected due to the isothermal nature of the reaction. This eliminates the need for an expensive thermocycler, but still requires a heat source. Our team has been working to develop a reliable, affordable heat block that a farmer could use for the RPA amplification. This heating device would also be used to maintain a constant temperature for conducting the Flappase-based detection of polymorphisms within the amplified DNA targets. More information about the heat block device can be found here.
All these reactions can be done by simply mixing pre-labeled reaction components in a single tube using a step-by-step guide. We have developed a simple protocol for conducting the Flappase assay, which once optimized, could be easily adapted into a one-pot reaction. Detection of the fluorescent signal emitted from the assay will require a simple fluorimeter device. Our team has conducted some preliminary research into some simple designs that could be adapted for our use, and even possibly integrated into our heat block.
References
- A2 Milk Market Size, Share, Industry Demand, Global Analysis, 2025: MRFR. (n.d.). Retrieved October 24, 2020, from https://www.marketresearchfuture.com/reports/a2-milk-market-6495