Monday, July 18, 2011

Polymerase Chain Reactions

A.k.a. PCR!

The goal of PCR is to isolate a smaller segment within the entire DNA strand and amplify it billions and billions of times, so you have a lot of that segment with which to work. Primers are some of the key tools in PCR. They are even smaller segments that contain specific base pair sequences (usually only 5-15 bps). During PCR, they line up with the correct sequences within a DNA strand and isolate the desired segment. Then with the help from Taq polymerase, the strand is replicated millions of times.

There are actually three parts to PCR, namely denaturation, annealing, and extension. The thermal cycler, in principle, heats up the tubes, and the DNA strands denature (split apart). Then the primers attach to the correct segments (annealing), and Taq uses the dNTPs to construct new strands off the primers (in the 5' --> 3' direction of course... extension).

We PCRed (yes, it is a verb) the blood extractions from Holly's samples earlier, and we're doing another one today with extractions from Aaron, a graduate student from the University of Chicago. His work with the genus Tangara is being contributed to the Emerging Pathogens Project.

To start, we created a master mix of reagents to add to each of the samples. They usually include a 10x buffer, dNTPs (which comprises the new segments), di water, Taq, your primers, and give/take a few other reagents. The logic behind creating a master mix is to make one large solution of your reagents in the proper proportions and then dispense an equal amount into each tube. The alternative is to add each reagent into each individual tube... you do the math.

(this is our master mix "recipe")
Oh and it always helps to use an electronic pipette!

Then we transfered approximately 1 microliter of each DNA sample (or more depending on each sample's concentration) into its corresponding tube now containing master mix.

Once each tube contained master mix and DNA, we put them into a thermal cycler!

(they come in different colors!)

And when we pull them out a few hours later, voila! We have billions and billions of copies of one teeny little segment!


After PCR, we can run the samples through agarose using gel electrophoresis. Stained DNA is pipetted into mini wells in the gel and immersed in a buffer (essentially salt water). Then an electric current (negative to positive) is conducted through the solution, and the negatively-charged DNA will move through the gel towards the positive end. The shorter the DNA segment, the faster it will move.
 (a setting gel)

(a gel in action)

If DNA is present, it will glow under a UV light - so cool!
and then we look at it in a G:Box on the computer!

And here's my drawing of Tiktaalik -- the fish who can do push-ups!

- Kit -

No comments:

Post a Comment