As part of a world wide effort from
Bernese Mountain Dog lovers, I participated in a fund raiser to
generate essential funds to be awarded to Dr Breen and his
research program. I was chosen at random from a considerable and
generous pool of donors to win a day in the lab!
It took me a bit to find the perfect
date to visit with Dr Breen and his staff at the NCSU Canine
Genomics lab, but on December 15th last year my husband Bill and
I learned how to isolate DNA from blood samples we had taken
from our dogs a few days before.
I brought Churchill and his Golden
Retriever siblings Riley and Mia to the lab to have blood drawn
ahead of our visit. It was quite a change of pace for the
dedicated research building to have three hairy beasts show up
and immediately hog all the attention from students and staff
alike. I couldn’t have asked for a better group of dogs and
we tried our best not to leave too many hair balls behind. I had
the feeling no one really minded since they don’t get the
chance to meet the dogs they are helping very often!
So with parking permits, agendas,
protocols and lab coats in hand I returned with Bill a few days
later to get started on our project. The work seemed daunting at
first. We had a full day ahead of us and at least half the terms
on our protocol were new to me. I had never held a pipette in my
life and I think the last time I was in any sort of lab had to
be high school. It didn’t matter though because we were
patiently guided through everything from lab safety to
vocabulary and instruments by the expert staff of researchers. I
was learning something new and exciting being around this
talented group of people.
To get started we became familiar with the lab. We had our
samples, blood from each of the dogs, that needed to be used at
room temperature and like a good chef we prepared for each stage
by making sure kits, pipettors, centrifuge tubes, buffers, and
QIAamp Midi columns were all prepared to go at each stage. The
task of isolating DNA from a blood sample is always seen as
magic on modern TV shows. It’s a squirt of this and a
scrape of that, and just like that you’ve caught the
criminal! Our protocol had at least 15 different steps and each
step had several components that would need to be repeated for
each of the Goldens and twice for Churchill so that we always
had a balanced and even distribution of four in the centrifuge.
Our original starting point was just 2
ml of blood. We were measuring things with the pipettors from
2.4 ml and smaller. The details included things like measuring
temperature, disposing of pipette tips for every single action,
vigorous shaking, (YES! By hand! The sample mixed with buffer
needed to yield a homogenous solution.)
Precise incubation times, more vigorous
shaking. We had to ,make sure all liquid was spun to the bottom
of the tubes so that no liquid remained at the cap. We then
divided the solution and buffer again and then taking that which
remained and placing it on to a QIAamp Midi column (a kind of
specialized DNA filtration device used for each sample) followed
by more incubation and more time spinning in the centrifuge.
It’s not a pretty color any more
at this stage, but before you know it the result will be a
crystal clear liquid in a small precious amount that will be
tested by the spectrophotometer for.concentration and quality.
This was the stage where we learned
whether our hard work during the day was precise enough to move
forward. We were lucky to have so much guidance and patience
from the research team. Our result was that we had beautiful
samples, all four samples showing a predictable curve,
indicating great yield and concentration.
We next had to determine if our
techniques had resulted in high molecular weight genomic DNA, or
if we had damaged it during isolation. For this we used agarose
gel electrophoresis. For this we used the tiniest of pipette
tips and the steadiest of hands we loaded our gel with the
samples – just 10 microlitres (1/100 of 1 ml – yes, a
fraction of one drop).
There are no fancy words needed to
describe the medium that we were using. “Gel” is just
right. Think, “JELLO”. And within that gel is a top
row of “wells”. (They’ll have to think of a
better word for that part though. When I think of a “well”
I think of a big hole in the ground you bring water up from.
These “wells” were the size of a baby fingernail
cutting.) So with our precious samples loaded into the smallest
of pipette tips we had to hold the pipette tip over the gel and
gently squeeze out the DNA into separate wells. Four times.
You can’t let yourself get too
excited or nervous. Extra shaky hands would not help in this
detailed work. Once all the wells are loaded a current is run
through the gel and that’s what produces that picture that
makes Churchill a Bernese Mt Dog and what makes Riley and Mia
Goldens.
It was an amazing day. And we did so
many more things than just isolate DNA from blood. Before we
even arrived Dr. Breen had taken a look at our three blood
samples at the overall chromosomal level. We were shown how the
chromosomes are viewed using a powerful and sophisticated
microscope. We also got a VIP tour of the new hospital and a
meeting with the Dean of the Vet School.
As you can see we are happy with our
DNA! I can not begin to imagine what the daily running of the
lab looks like. It must be a difficult and daunting task to
perform these essential tests day in and day out. The
professionals and students in this lab love their work. It’s
evident in the passion and patience they shared with us on our
day there. Beyond that, it is reflected with the information
they are arming us all in the public with so that we may one day
have the chance to prevent disease in our best friends and
families, whether they be canine or human.
The DNA samples we isolated are loaded
onto a gel in the order, Churchill, Mia, Riley. The last lane is
DNA ladder used to check the size of the DNA.
Dr. Breen's Biography
http://breenlab.org/
See also this recent article
http://www.newsobserver.com/2011/06/20/1285103/dogs-help-chase-rogue-genes.html
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