PCR in Real Time

I really like graphics, and I make a lot of them to represent my lab work.

Below is a graphic that represents my lab project. So far, I've gone through the greens, yellows, pinks & blues and am working in the orange to red zone. Well, I still ave some blue work to do, but I'm currently working on orange zone material

My project in a very small nutshell the size of your computer screen.

That is, I'm trying to amplify cDNA derived from frozen cell samples to make estimates of gene expression from my different lines of transformed diatoms.

 "Regular" or endpoint PCR can hit a duplication plateau that isn't limited by the amount of starting template in a reaction, and therefore usually only gives a positive or negative result (yes there is product or no there isn't). Real time-PCR on the other hand lets us see the amount of amplified product as PCR cycles progress and lets us accurately quantify the amount of DNA that is being amplified each round. By using specific primers that only amplify one region within a pool of DNA, we can determine how much particular DNA we had to start with.

Let's say the cartoon to the left here represents our real time-PCR experiment, and we have a pool of cDNA waiting to be amplified. We have primers ready to amplify the pink cDNA and the dark blue cDNA. Using these primers, we set up a reaction to amplify the pink cDNA and a separate reaction to amplify the dark blue cDNA. If we incorporate a special dye into each reaction, any double stranded DNA that is amplified by the primers with fluoresce, which can then be measure by the real time-PCR machine. This data is then put into graph form, and we can track the amplification of DNA in real time as the experiment progresses. In the cartoon, you can see the pink cDNA starts at 2x and the dark blue cDNA starts at 1x in terms of starting cDNA template. As such, the amount of cDNA that is made (and therefore the amount fluorescence given off) will vary.

In the graph on the right, we can see the difference in fluorescence between the different amplification reactions. Here the red, blue, and gray lines are almost identical, and are far ahead of the green line. This means that these three lines have much more starting cDNA template than the green line.

This is the approach I will take in measuring the gene expression in my experiments, if I can get my primers to work. But more on that in a later post.