Pee shoots of heirloom varieties in the laboratory at Department of Agriculture and Ecology, Copenhagen University (Copyright Ingrid Nolde)
Last weekend I joined the Danish seed savers at the laboratory at the Department of Agriculture and Ecology (Copenhagen University). Gunter Backes and Jihad Orabi led us through a study of our heirloom pea collection. It was a fantastic and educational weekend!
In preparation, we had grown peas for 3-4 weeks, so they were ready for analysis. We had to use leaves that had not been given too much light. Chlorophyll can make it difficult to extract DNA material from the rest of the leaf tissue. For safety’s sake, we were two cultivating the same peas. It went well and we only used one set.
Test tubes on ice (Copyright Ingrid Nolde)
We were divided into four groups. That way we could manage to work with all the varieties we had brought. After a brief introduction to laboratory work, to use gloves and gowns – take them off when you leave the lab, we started slicing the pea leafes.
Once cut, the natural enzymes in the leaves activates. To inhibit them, we set all tubes in a tray of ice. Millimetre small pieces were placed in the tubes, a solution of caustic soda were added and the tubes were boiled for 1 minute to denature the proteins. Then we blended the boiled leaves with the tip of a plastic stick and added a resolution that brought the pH down to a desired level. To separate DNA from the rest of the leaf broth, test tubes were centrifuged.
Pea leaf broth bottom, DNA located at the top in the clear part of the liquid (Copyright Ingrid Nolde)
Now we could move the purified DNA into new tubes, still set in ice.
Then it was time to mix liquids with primer for the DNA fragments we searched for. We looked at 6 different micro satellites. Micro satellites are good to tell the difference between varieties of a species. A primer is the DNA sequence immediately before the micro satellite, leading the DNA strand to be cut at the desired location. We used a primer for each end of the micro satellite, because it must be “cut” at both ends. To the liquid is also added Taq DNA polymerase, an enzyme that replicates DNA at specific temperatures.
We used pipettes for several hours. Even with multi pipettes there was plenty to do (Copyright Ingrid Nolde)
Next the test tubes are undergoing repeated temperature changes between
94 °C, where DNA strand separates
64-55 °C, where the primer binds to the DNA strand, and
72 °C, where Taq DNA polymerase copies the DNA strand from the primer and forward
By the repeated changes in temperature, we multiplied the selected micro satellite exponentially. The first cycle gave very little DNA material, but for each cycle, there was more copies. Just like reproduction of yeast in a bread dough.
Finally, we let an electric field pull micro satellites through a viscous liquid in capillary thin tubes. A dye was coupled to micro satellites, for a photocell at the end of capillaries to detect how quickly they passed by. Small micro satellites migrate faster through capillary tubes than larger.
First presentation results
The photo shows the horizontal bands, each representing a pea variety. The small vertical bands are micro satellites. The red vertical band marks a scale, so we know how long the micro satellite strands are.
Jihad and Gunter did the analysis work Sunday morning, before we all met up – thank you!
It was best to group the peas in 7 groups
The results will soon arrive in my Inbox. I look forward to delve deeper into the results. I might have a more to write about then.
Big thanks to both Gunter and Jihad for a learning and rewarding weekend :-)
PS: I’m just a happy amateur. Feel free to comment if I’m wrong in some of the techicalities :-)