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.
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
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.