Molecular Photocopying – PCR.

Now PCR or Polymerase Chain Reaction is a vital part of many laboratory procedures involving DNA, it is a process for producing exponential numbers of copies of a given DNA molecule, that is, if you start off with one molecule of your template DNA, and run 20 cycles of amplification, you will get twenty doublings’ worth.

Before I delve into the depths of the technique, I think it is worth introducing the concept to you with the help of an amazingly funny (and appropriate) video from BioRad.

😀

Right, getting down to business now, PCR basically involves the replication of a given DNA segment in-vitro or ex-vivo(outside cells). We need a template (the DNA segment that we want to amplify) , free nucleotides needed to build new strands, a buffer containing ions to stabilize the mixture, PCR primers to help start off replication, a DNA polymerase enzyme & a method to heat and cool the reaction mixture.

The polymerase that is routinely used these days is called Taq Polymerase, this comes from a bacterium called Thermus aquaticus which lives in hot springs, this is used because it can withstand the high temperatures that are a part of the PCR process. All of the aforementioned ingredients have to be put into reaction tubes before we start cycling them through different temperatures (this is one reason that PCR machines are also called Thermocyclers). In some cases, if the Taq polymerase that is being used needs to be activated by heat, the reaction tube is heated to 96’C for about 10 minutes.

I think it is now time to move on to the process itself, which can be divided into three phases.

[1]Denaturation

In this step, the reaction tube is heated to 94-98′ C for around half a minute, this separates the double stranded DNA template into its constituent single strands.

[2] Annealing

In this step, temperature is reduced to 50′ C – 60′ C for 1 minute, this enables primers (short single stranded sequences of DNA) to bind to complementary sequences of the single stranded DNA derived from step [1] , the diagram should show this clearly. This is required because DNA polymerase requires a primer to be present in the 5′ -> 3′ direction to produce a new strand successfully.

[3]Extension

In this step, the temperature is raised to 72’C or so for 2 minutes or so, this is the optimum operating temperature for Taq Polymerase and the primers are extended such that a new double stranded copy is synthesized using the single stranded copy generated in step [1].

To sum up, we start with two strands of DNA in one molecule, which is split into two single strands which are bound to primers, followed by replication to produce two new double stranded molecules, in the next cycle we’ll start of with two double stranded molecules and end up with four, and then four and sixteen, and sixtenn and two hundred fifty six and so on, you get the idea. 😛

A video summary that explains it all is available here

Finally, there is a technique called quantitative PCR, or qPCR, which can be used to estimate how much of your given fragment is present in a sample. This works on the principle that you can have dyes that fluoresce when they bind DNA, and you can see how many cycles of amplification it takes for a certain level of fluorescence to be reached in order to figure out how much of your DNA is present in the sample. This becomes possible because you can have DNA of known concentration that permits you to work out how many cycles a particular amount of DNA will take to produce a certain amount of fluorescence, alternatively you can just compare two samples – the more template DNA you have, the quicker it’ll reach that threshold…

 

I’ll leave you with another funny video pertaining to PCR from BioRad.

That is it until the next post, I must say this has been one of my shorter posts of late.

– Ankur

Advertisements

4 responses to “Molecular Photocopying – PCR.

  1. This is the optimum operating temperature for produce two new double stranded molecules its has been allows many temperatures with in model level separate a models.

    • I’m not sure what exactly you were trying to say there; however, generally speaking, yes, these temperatures, although they’re part of a range, are optimum operating temperatures, a cursory glance at enzyme kinetics will enable you to figure out that there is a characteristic optimum for every enzyme in terms of temperature or pH or whatever else might catch your fancy. It is quite possible, also, to produce variants that have different operating temperatures.

  2. it’s always was kind of interesting to me to look at this kind of stuff, like pictures of dead people with bones that are going in to the lab to get researched on. its not my hobby, but it’s interesting though.

  3. This was a great post. I didn’t understand the technicalities of it but now I can explain PCR to a layman. Cool enough. Thank you.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s