The mechanics of the polymerase chain reaction (PCR)...a primer -

The polymerase chain reaction (PCR) is a technique for copying a piece of DNA a billion-fold. As the name suggests, the process creates a chain of many pieces, in this case, the pieces are nucleotides and the chain is a new, short and specific strand of DNA.

*IMPORTED POST

PCR is an enzyme-mediated reaction, and as with any enzyme, the reaction must occur at the enzyme’s ideal operating temperature. The enzymes that are used for the PCR are DNA-dependent DNA polymerases (DDDP) derived from thermophilic (heat-loving) bacteria. As such, the enzymes function at higher temperatures than the enzymes we commonly use in the laboratory or have working in our bodies. These DNA polymerases operate at 60-75°C, and can even survive at temperatures above 90°C. This is important because a part of the PCR requires that the reaction reaches ~95°C as we shall see.

Apart from the DNA polymerase, PCR needs a DNA template to copy, and a pair of short DNA sequences called oligonucleotides or “primers” (described here) to get the DNA polymerase started.

Broadly speaking, there are three steps identified by incubating at different temperatures. The three steps make up a PCR “cycle” are…

Double-stranded DNA separation or denaturation (D in Figure 1)
Primer annealing to template DNA (A in Figure 1)
Primer extension (E in Figure 1)

In Figure 1 above, I’ve plotted the three temperatures which make up a single cycle and highlighted the section for the temperature which includes the “ramping time” it takes for the thermal cycler machine we use to get the contents of the PCR reaction mix, in a tube, to that temperature, hold it there for the desired time, and then ramp the contents to the next temperature.

The DNA denaturation section (D), oligonucleotide annealing section (A) and the primer extension (E) section are marked. The temperature range over which dsDNA duplexes can denature (T_D) or ‘melt’, and the range over which the oligonucleotide primers and probes can hybridize (T_M) are also marked.

Denaturation

At temperatures above 90°C, double-stranded DNA denatures or “melts”. That means the weak hydrogen bonds that usually hold the two complementary strands together at normal temperatures are disrupted resulting in two single-stranded DNA strands (shown below in an idealised form).

Primer Annealing

At the annealing temperature (T_A), primers that collide with their complementary sequence can hybridise or “bind” to it. The chance of such an encounter happening is increased because we use a vast excess of each primer in the reaction mixture compared to the number of template molecules present.

The test in the example below has been designed to amplify a region of the template spanned by and including, the primer sequences (highlighted by the grey box; could be 100 nucleotides, up to multiple hundreds of nucleotides long, depending on the type of PCR and the aim of the PCR).

Primer Extension

At the extension temperature (T_E), the DNA polymerase (the P in PCR) binds to the hybridized primer and begins to add complementary nucleotides (i.e. every time the polymerase reads a “G” on the template strand, its adds a “C”; an “A” for a “T”; a “G” for a “C” and a “T” for an”A”), chemically binding each new addition to the last to form a growing chain (the C in PCR).

The reaction (the R in PCR) process only occurs in one direction. In our example, the green primer is binding to its complementary template sequence and is facing toward the right. This is described as the 5′ (five-prime) to 3′ (three prime) direction if you want to sound sciency.

Extension proceeds in the direction that the primer faces. The result is a new double-stranded PCR product we usually call an “amplicon”. An amplicon can be defined as an amplified molecule of a single type, in this case, an exact (opposite strand though) replicate of the original template.

Exponential Template Duplication

The process is then repeated by cycling through the temperatures over and over again (35 to 55 times). Each cycle results in a new DNA duplex, each strand acting as a potential template for one or other primer.

The total number of cycles (what I’ve described above as “35-55 times”) is different from the cycle at which virus detection is clearly identifiable.
It gets a little confusing here. In the “old days”, before real-time PCR was in use in microbiology pathology labs (rPCR; see my specific post about what this is), all “PCR” involved running an agarose gel after opening the tube and pipetting out some of the completed PCR products. I call this “conventional PCR”.
These results indicated what had amplified at the end of 35-55 cycles. But this isn’t what we in the pathology lab today – not since about 2009.
Today we read a positive result as soon as it’s detectable – in a 55 cycle PCR run that might be at 17 cycles, or 28 or 35, whatever – I don’t think anyone calls a patient a true infection risk or even infected at C_T values above 40 cycles except when there are exceptional clinical and epidemiological reasons to do so, or when it’s being done in an academic/research/optimisation setting when you know beforehand that you have a known positive template present.

The whole process in one picture

Some interesting things stand out from the figure below.

Firstly, the original template strands (blue and red) continue to act as templates because the PCR process is not destructive. Secondly, each cycle produces a greater number of the shorter amplicon molecules. Notably, these are shorter in our example because the primers bind within the template sequence (which might, for example, be an entire very long gene) – the ends bits don’t get copied.

Example of an agarose gel after 5ul of 11 different PCR products (#18 – #28 shown) from a PCR looking for one rhinovirus (HRV-QIM; lab named) has been electrophoresed through it, alongside a molecular weight marker (aka “ladder, right-hand side) comprising known sized fragment used to estimate the size of the product. Five samples were positive given the correctly sized product of ~200 nucleotides.

Eventually, the majority of the amplicon in the reaction vessel will be the expected length, i.e. just the region spanned by, and including the primer sequences. We used to use that length (see the adjacent agaorse gel image) to say we’d amplified what we expected when we used gel-based conventional polymerase chain reaction methods.

It is possible to mathematically predict the pattern of amplicon accumulation. In our example, we have started with two strands. These strands, in a perfect PCR reaction (which rarely occurs!), would result in the production of two new strands making a total of four and after the second cycle we have eight strands, then 16, 32 and so on. The reaction is doubling the number of strands each cycle or to make that an equation, we have 2ⁿ

Note: To make the process easier to understand, I have drawn the DNA strands as straight lines – in reality, DNA does not exist in as simple a form as this.

*Imported Post

This post from 05MAY2015 was posted over on my old blog platform virologydownunder.blogspot.com.au. It has now been moved to here.

Virology Down Under

Facts, data, info, expert opinion and a reasonable, occasionally grumpy, voice on viruses: what they are, how they tick and the illnesses they may cause.

The mechanics of the polymerase chain reaction (PCR)…a primer

Denaturation

Primer Annealing

Primer Extension

Exponential Template Duplication

The whole process in one picture

Further reading…

*Imported Post

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Denaturation

Primer Annealing

Primer Extension

Exponential Template Duplication

The whole process in one picture

Further reading…

*Imported Post

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