youPCR

 

Introducing Portable Genomics and Point of Grow Marker Assisted Selection

youPCR

Speed, Confidentiality and Precision

After consulting many legal firms in the cannabis industry, it has been made clear to us that shipping cotyledons (stamped onto FTA paper) across state lines is federally illegal under the current interpretation of the Controlled Substance Act (CSA). Three different reputable law firms were consulted with specific instructions to review the hemp stalk exemptions in Hemp Industries Association vs the DEA. There was consensus on DNA clean of cannabinoids being as legal as hemp stalk free from cannabinoids, but leaves and cotyledons were still off limits. Companies offering this type of PCR service become honey pot databases of customer addresses that have violated the CSA.

In addition to this, some of these companies are using this Male/Female DNA to sequence the genomes of their customers samples as a “massive data intake tool” to accelerate the release of their own strains.  These “database play” business models are well known in the human consumer genetics markets with companies like 23 & Me. In this more mature human sequencing market, customer consent forms aid in the transparency of these business models and provide disclosure about their intentions to resell or even patent findings from that human data. These business models are often a surprise to many growers in the cannabis space given the recent issuance of many cannabis patents. Pay close attention to the terms and conditions of such providers as the entire genome can be sequenced from the material required to perform an X/Y PCR.

To decentralize this risk and to honor the intent of the CSA law we have designed and assembled a genomics platform than can be installed for under $650 and can run on a USB port of a laptop or cell phone at the point of grow. This is the most congruent approach with the Cole memo and respects state law and interstate commerce.

To accomplish this, we have engineered a platform of cannabis genetic tests we term youPCR. This platform can detect various genetic signatures of the cannabis plant using a USB portable PCR device coupled with a Colorimetric read-out compatible with a mobile phone camera or visual interpretation.

The Colorimetric assay is designed for any Presence/Absence testing relevant for the cannabis industry. We have initially optimized this for Male/Female detection and the detection of the Bd allele known to produce CBD. The assay is sensitive for 10 fg to 100ng of DNA and can be run on a hole punch of leaf sample that is processed with a novel single step homogenous boil-prep method.

Females remain Pink, Positive targets (Y chromosome or the Bd allele) turn yellow.

The USB stick PCR device has 8 wells and 2 USB sticks can be run off any one laptop at a time. The colorimetric assay also works with any existing PCR cycler that has a heated lid and is 96 or 384 well compatible.

 

In addition to these two markers, we have a menu of over a dozen other assays under development that screen for microbes, pests and terpene genetics.

You can now perform marker assisted selection for very low costs at your grow without having to take unnecessary risks and delays shipping tissue to other labs that may intend to compete with you with your own data. This can all be done for costs lower than a single grow light and save tremendous growth space by growing only those genetics you are really after.

USB PCR stations and reagents can be purchased at our web-store. We have an agreement with Amplyus/miniPCR that affords a discount code for Medicinal Genomics customers. Please contact us with other +/- assays you would like to see on the youPCR platform.

Beta Trial at Colorado Seed Inc.

In march of 2017, we packed a few youPCRs in our bags and flew to Boulder CO and sex tested samples at Colorado Seeds Inc. We compared this to a method of attempting to visually identify the plants based on the shape of the seed.

As of April 2nd 2017, the plants used in the seed visualization technique are too young to confirm sex, but if the accuracy of the youPCR seen on known sexed plants holds up on those unknown samples, the results would imply a call rate no better than randomness with visual seed ID.

Genetics may help to optimize the ultimate goal of simple iPhone based sexing from seed shape alone. This is not an approach to give up on now that we have genetic tools that can train those algorithms with more rapid sex testing. Less soil, lights and space is required to iterate with genetics.


CBD Amp Reagents

Etienne de Meijer pioneered the work on the Bd:Bt allele governing cannabinoid expression. We used Pacific Biosciences single molecule sequencing to identify the variants that drive this genetic switch and turned those into a colorimetric assay known as youPCR CBD.

Our StrainSEEK data on 100s of CBD cultivars suggests a recent selection for these genetics and an AC/DC or Cannatonic bottleneck that has resulted from this selection. As a result if you encounter chemotyped CBD strains with more distant heritage and our test fails to detect your CBD gene, contact us and we will work together to sequence your strain and improve the test to capture your unique genetics. This test will turn yellow if 1 or 2 copies of active CBD allele exist. It cannot distinguish between Type II (50:50 CBD:THC) and Type III (all CBD) plants but can rule out a Type I plant and inform you if a male has potential CBD genetics worth breeding.

 

 

All of the plants on the X axis and at a 45 degree angle to the X axis would light up yellow with the SeeBD test. BD = Blue Dream and AK 47 = Ak47. The Negative Sign is a Negative control and should always be Pink. We also sell Positive controls for each assay which can be helpful for troubleshooting purposes.

To further validate this test we collected 25 HPLC confirmed samples from the Emerald Cup in 2015 with the help of Sonoma Labs. We collected 36 hemp samples from a colorado hemp farm for on site testing and 11 samples HPLC confirmed from  Massachusetts. We have 100% accuracy with the HPLC confirmed samples and are still awaiting 1 Hemp sample to mature to test for THC+ status.

 


Below is a subset of the diversity of CBD samples that this test was validated on.

Other markers under development-

1)E.coli/Salmonella

2)4 Aspergillus species (niger, flavus, fumigatus, terreus)

3)Vascularized Powdery Mildew (invisible to human eye but present)


 

Technique considerations

Prior to hole punching, it is helpful to wipe the surface of the leaf with an Isopropanol wipe to eliminate soils nutrients and other surface epiphytes. This same isopropanol wipe should be used to sterilize the hole punch by punching several holes in the wipe in between samplings. Fold the wipe over to make it thicker than a leaf and punch several holes in the wipe. Use the ejection syringe to clear the punch of isopropanol wipe hole punches.

The hole punch is sharp and can hole punch your finger if you are not careful . Use of a plastic plant identifier as a punch backstop is recommended as seen in this photo.

Hole punches can be placed in either a 96 well plate or 8 strip tubes. youPCR can be performed in 96 well plates in thermocyclers but heated lids are required to avoid condensation on the plate seals or lids. You do not need an qPCR instrument. A simple thermocycler will suffice.