You’ve seen the pictures of laddered DNA spots, with a scientist deciphering what this spot means, and what that spot means.  Well….how in the heck do you get those spots, and how does the Seed Industry use them?

We want spots!

Well it’s kind of complicated, and is born from many years of basic work from many scientists, but…here’s the simple version of how it works with plants.

Get some plant material, usually a leaf but sometimes a root or even a seed.  Then crush it in a chemical that rips the DNA free from the other parts of the plant.  Clean away the non-DNA plant parts using chemistry techniques and then dilute into a liquid that stabilizes the pH.  The DNA is now ready.

Ready for what!

Ready for a process called Polymerase Chain Reaction (PCR), which was developed by an eccentric scientist named Kary Mullis, who won the Nobel Prize in Chemistry for his discovery.

DNA is much, much too small to observe, so we have to do something to it, in order to observe it.  PCR is what we do.  Add several ingredients into the DNA sample and place it into a very precise oven of sorts called a Thermocycler or PCR machine.  The Thermocycler goes through heating and cooling cycles that loosen the DNA particle enough to enable the added ingredients and the DNA to combine and duplicate the original DNA particles.  Each cycle produces a copy of the DNA, so one piece of DNA becomes two, which become four, which become eight, and so on, until there is enough DNA to test.  The DNA is now in larger quantities, but it’s all mixed up in a tiny mass, so you have to sort it out.   

Sorting DNA by size through electrophoresis

Place the duplicated DNA inside, and at one end of a flat sheet of gel that looks something like Jell-O, and is submerged into a special liquid. DNA has a negative charge, so if you place a small electrical current through the gel , the negatively charged DNA at the top of the gel will move through the gel toward the positive charge at the bottom of the gel.  The gel acts like a filter of sorts.  The bigger DNA particles have a harder time getting through the gel and move slower than the smaller particles.  DNA is separated as it moves down the gel.  The smaller DNA particles that move faster end up closer to the bottom of the gel sheet, and the larger, slower moving DNA particles stay nearer the top of the gel.  DNA particles that are intermediate in size end up somewhere in the middle, depending on their size.  What you end up with are clusters of DNA that are similar in size i.e. the ladder.  At this point though, you still can’t see the DNA in the gel, but it’s there.  To see it, you have to soak the sheet of gel in a solution that stains the DNA so the spots appear.  You can then take a picture of the stained gel to analyze later.  The following website has a nice pictorial on how electrophoesis works.  http://learn.genetics.utah.edu/units/biotech/gel/

A Couple of Ways the Seed Industry uses the ladder

OK…now for the good part.  How do we use this ladder?  Go back to the PCR.  One of those ingredients that you put into the DNA before you duplicate it in the PCR machine is called a “primer” (small piece of starter DNA). 

DNA will only duplicate itself, if there is a primer or “starter DNA” in the mix, which is compatible with the DNA.  The difficulty is finding a “primer” that duplicates a piece of the DNA that corresponds to a plant characteristic that you’re interested in.  This process is mostly trial and error, but once you find a primer, you can start to use the technique to test for the characteristic of interest.  With the correct primer, a spot will appear at a particular point on the ladder, only if the plant has the characteristic of interest.  Several biotech companies now produce and sell different DNA primers for seed companies to test on their varieties. 

The vegetable seed industry uses this type of testing technique mostly to determine if a plant is actually a hybrid or not (genetic purity), and sometimes to identity a particular variety.  Plant breeders can use the process in a conventional breeding program to quickly identify crosses with desired characteristics such as disease resistance.  The speed and efficiency of this technique has been improved over recent years, so labs with the latest equipment sometimes don’t have to go through all of these steps, but the basic principles are still the same.