Testing the sensitivity of each component of our method – how low can we go in detecting snow leopard DNA?

Dear friends of the project,

Sincere apologies for the lateness of this update. Despite the simplicity of our results, the lab work to get to this stage has many different facets and we’ve been spending time planning the best strategy forward…..and….I really wanted to complete the first milestone before sending this update out!

After the first 6 months in the lab, we were successfully able to create many copies of DNA from a short sequence of snow leopard DNA and link that amplification to a simple colour response using a paper template…..we had proof of concept!

However, we were unable to detect very low concentrations of DNA, just like what may be found in fecal or bone samples. So for the next 6 months our goal is to break down the components of the reaction and test their sensitivity, in other words, what concentrations of DNA are we able to detect?

Once we have this basic information, we can then work on ways to increase the sensitivity to the levels required to detect DNA in the field…..and we have some clever plans in how we might be able to do that!

The most basic component of the reaction, where we create lots of copies of snow leopard DNA, is the rolling circle amplification reaction or RCA. RCA in its simplest form consists of a circular DNA template and a short DNA sequence which is designed to specifically bind to the DNA sequence of snow leopards (primer). When those components bind together in the presence of an enzyme which builds DNA molecules (DNA polymerase), many copies of the circular DNA template are produced to make one big long strand……..with that long strand we can then initiate other reactions which then produce the colour change as in the diagram below.

So in the last few months we’ve been working to determine the lowest concentration of DNA circle + primer that can produce RCA sequence we can see using a special type of gel.

We knew that the RCA method can be really sensitive to detecting low quantities of DNA but we never knew it could be this sensitive!

This gel shows that RCA sequence can be produced from about 0.1fmol of primer + circle……that basically means we can detect about 1 million cells…..which is about 1000 times less sensitive than we need it. This is an awesome start because by simply adding a colour response instead of using the gel we could make it 10X more sensitive. And by using more than one primer, we could make the method many times more sensitive again!!!

I hope this hasn’t made your mind explode…..we see this as a very positive and encouraging result, and I once again thank you all for helping us get to this stage.

Without the help of people we can never achieve effective, sustainable conservation.

Blessings to you all.

Natx