Understanding the Polymerase Chain Reaction (PCR)

A "must" understand

J. Enrique Salcedo-Sora

February 2014

Scope

In this tutorial you will be presented with the fundamental processes that make the in vitro amplification of DNA possible. For the PCR is exactly that: the logarithmic amplification of DNA doing the equivalent (in a test tube) to what cells naturally do. Thus, understanding this in vitro reaction helps to visualise the natural DNA replication in its most simple terms. The opposite also applies, revising DNA replication will help to understand PCR:

• DNA directionality
• DNA replication
• Role of temperature oscillation
• DNA polymerases
• deoxynucleotides (dNTP)
• Role of magnesium in DNA polymerisation

The importance of PCR .... is HUGE

The Polymerase Chain Reaction (PCR) technique, invented in 1985 by Kary B. Mullis, has allowed scientists to do the impossible otherwise: make millions or more! of copies of a sample of DNA. A sample of DNA that can be scarce of many situations.

The technique has revolutionised every aspect of molecular biology and its applications:

• PCR has made cumbersome gene-based research and diagnostics much easier and sometimes even trivial
• Paleontobiology started to "see" what was in ancient samples
• Forensics became a much stronger and assertive branch of the justice system
• Can you think of other examples where amplifying DNA is important? ...

... Well, lots of them !

The template

Any sample of DNA or presummed to contain DNA. The practical size limits of DNA to amplify with PCR are in the thousands of nucleotides, or more accurately "base pairs" abbreviated bp (because DNA is a two-strand molecule). More probably just over 10 kb (kilobases).

DNA is a vectorial entity: magnitude and direction

The magnitude is given by the number of consecutive base pairs.

The direction is given by the carbon of the sugar (ribose) to which phosphate is conjugated (attached). The carbon 5' (read five prime) carries the phosphate while the OH (hydroxyl) group of carbon 3' receives the phosphate. The end of a strand of DNA with a terminal phosphate is the 5' end, the other end (without a terminal phosphate) is the 3' end.

Positions of the ribose are numbered as 1' to 5', with a ' (prime) symbol tagged to the numbers to differentiate them from the numbers of the different atoms of the bases (Adenine, Thymidine, Guanosine, Cytosine).

The consequences of the vectorial nature of DNA are several

• The complementary strands of DNA are anti-parallel
• DNA polymerases evolved to amplify in the 5'->3' direction
• The small DNA fragments needed to 'prime' the DNA polymerase are designed as 5'->3' sequences
• The nucleotides that can be added (polymerised) into the new DNA strand have to be deoxynucleotides
• Any new nucleotide to be added is loaded onto the 3' end of the new growing strand of DNA

Is it dNTPs or NTPs

The nucleotides that can be added (polymerised) into the new DNA strand have to be deoxynucleotides or dNTP.

The nucleotides added to the growing DNA strands are tri-phosphorylated. When linked they "lose" two phosphates and use the remaining phosphate to link the hydroxyl (OH) group of 3' carbon of the previous nucleotide.

Because they have only one OH group (other types of nucleotides have two) in the ribose ring they are called deoxynucleotides. Without that OH group DNA polymerisation won't occur.

High temperature replaces the job of the helicases

A crucial step to initiate the replication of DNA is to separate the two strands normally entwisted. At the temperature under which cells live (37 degrees in humans) DNA is happily a double helix. Helicases are in charge of opening this helix (denaturation). In PCR the sample is heated to temperatures that guarantee denaturation of the helix.

Important note: you might have realised that those high temperatures will harm enzymes such as the one enzyme we use in PCR, a DNA polymerase. A landmark for this technique was the isolation and, nowadays industrial production, of DNA polymerases from bacteria that live is very hot environments (thermophilic bacteria). We use routinely a DNA polymerase from Thermococcus aquaticus (Taq). This Taq polymerase is thermostable.

Let's put the mix together:

1) Template (DNA)

2) Primers (usually two and of 15 to 25 bp long)

3) DNA polymerase (comes with a buffer solution)

4) dNTPs

5) Magnesium

6) Water to complete a given volume

Now the cycle (temperature cycling)

Denaturation at usually 94 degrees (separating the double helix)

Annealing at 50 to 60 degrees (allowing primers to align to their complementary template). The length of this step is approximately 0.5 to 1 min per kilobase (kb) of template to amplify.

Elongation at 68 to 72 degrees (DNA polymerisation at a temperature that does not allow the helix to reform)

These three steps are repeated usually 30 to 40 times. An initial denaturation for 5 min and a final and longer elongation step for about 10 minutes are added before and after the cycles, respectively. Samples can be cool down before collecting.

No Magnesium, no can do. But why?

This is a very overlooked part of the PCR. It is usually just mentioned that Magnesium is esssential. Magnesium (Mg2+) is needed to "shelter" highly energetic negative groups such as phosphates to reduce the activation energy of the reaction. Without it, enzymes (here DNA polymerases) using phosphorylated substrates (e.g. dNTPs) could not catalyse the reaction. However, very seldom this is clarified when explaining PCR. Mg2+ is essential for PCR to work, but we need to bring about what the biochemistry books mention to understand the reason.

Given your knowledge on PCR:

Clickable answers.

1) Taq polymerase starts copying at:

A the end of free single-stranded RNA

B any open point

C RNA primers attached to the end of the desired gene

D DNA primers attached to the end of the desired gene

2) When DNA is heated, primers anneal to DNA strands.

True

False

3) Which of the following is an application of PCR technology?

A gene mapping

B epidemiology

C in vitro mutagenesis

D forensics

E all of the above

4) Which of the following is NOT required for a PCR reaction?

A dNTPs

B Taq polymerase

C a target sequence

D a primer

E RNA transcriptase

5) Taq polymerase is the commonly used enzyme in PCR because this enzyme is:

A a primer replicase

B not prone to errors

C a faster polymerase

D thermolabile

E thermostable

6) At the beginning of each cycle the temperature of the PCR reaction is raised in order to:

A attach the primer

B elongate the primer

C denature the double DNA strands

D polymerize the DNA

E renature the double DNA strands

7) In a PCR reaction, which of the following statements is NOT true?

A Polymerization occurs from 5' to 3'

B A thermostable enzyme is necessary.

C Primers are necessary.

D Nucleotides (NTPs) are necessary.

Before you go ... something for the road

Do you know how DNA replication is primed in vivo? If we use short fragments of DNA for PCR, what do cells use to prime polymerisation of DNA?