Real-Time Polymerase Chain Reaction (PCR)

This short animation introduces the real-time polymerase chain reaction (PCR) procedure. Captions are available in multiple languages. This resource was developed by Yaw Adu-Sarkodie of the Kwame Nkrumah University of Science and Technology and Cary Engleberg of the University of Michigan. It is part of a larger learning module about laboratory methods for clinical microbiology. The full learning module, editable animation, and video transcript are available at http://open.umich.edu/education/med/oernetwork/med/microbiology/clinical-microbio-lab/2009). Copyright 2009-2010, Kwame Nkrumah University of Science and Technology and Cary Engleberg. This is licensed under a Creative Commons Attribution Noncommercial 3.0 License http://creativecommons.org/licenses/by-nc/3.0/.

(English captions by Andrea Matsumoto, University of Michigan.)

This program shows how a specific nucleic
acid in a clinical sample can be detected

and quantified using PCR.

This is accomplished by detecting the accumulation
of the amplified PCR products as they are

generated in the reaction.

And so the process is called real-time,
or RTPCR.

To understand how amplified PCR products,
also called amplicons, are detected in real-time,

let's first review the events that occur
during a normal cycle of the PCR reaction.

Recall that the first step in any PCR cycle
is to raise the reaction temperature and melt

double-stranded DNA.

Then, when the temperature is lowered, the
specific primers bind to the sequences at

each end of the target DNA.

The intervening DNA can then be synthesized
by polymerase reaction in opposite directions.

Other results, you produce two double-strand
copies of the target DNA, where you started

with only one.

If you have any confusion about this basic
process, it might be a good idea to review

the program on basic PCR once again.

To detect the generation of new amplicons
in real-time, the PCR reaction requires an

additional ingredient -a single-stranded DNA
probe, designed to hybridize to the part of

the DNA sequence synthesized between the two
primers.

However, unlike the primers, this probe is
more defined in a special way. One of its

nucleotides is labeled with a fluorescent
molecule and another nucleotide is labeled

with a fluorescence quencher molecule.

The quencher rapidly absorbs any light energy
emitted by the fluorescent molecule, as long

as it remains in close proximity.

Now, let's look at what happens when this
additional ingredient is present during a

single cycle of PCR.

Other primers bind to the separate strands
of DNA.

The probe also finds its complimentary sites
between them.

The enzyme synthesizes new DNA from the ends
of the primers also have a second activity:

an exonucleus activity.

So when it encounters double-stranded DNA
in its path, it will disassemble the strand

that is in its way, and replace all of the
nucleotides.

As polymerase pass through the probe, note
that the nucleotide bearing the fluorescent

marker and the one bearing the quencher are
separated from one another.

In the absence of a nearby quencher, the fluorescent
molecule can now emit detectable light when

stimulated.

Each time another amplicon is produced, another
fluorescent marker is released from its neighboring

quencher.

Therefore, just as the number of amplicons
doubles in each PCR cycle, the amount of emitted

fluorescent energy also doubles.

This light generation can be monitored during
the PCR reaction thermocycler that is equipped

with a fluorometer.

So, if you begin with a clinical sample that
had only one copy of the target DNA, it could

take 40 or more cycles before the amplicons
are detected by a fluorometer in a specialized

thermocycler.

However, if the original sample contained
32 times more copies of the target DNA, then

the fluorometric detection would occur after
5 fewer rounds of PCR.

And if there were 1,024 more target DNA sequences
in the original sample, then the fluorescent

signal would be detected 10 rounds earlier.

So, the amount of specific DNA in the clinical
sample is determined by a reference to the

round of PCR in which the amount of fluorescence
first crosses the threshold of detection.

RTPCR is most commonly used to quantify
the burden of viruses in the blood of patients

with HIV, Hepatitis B, and other viruses.

But HIV is an RNA virus; it has no DNA, and
the RNA that it possesses is single stranded.

So, how can this method work?

The answer is that RNA, from an RNA virus,
can be quantified after it has been copied

and converted to double-stranded DNA.

This animation shows how this is accomplished.
First, the viral RNA is released from the

virion.

Then, a complimentary DNA strand is synthesized
from the viral RNA using purified reverse

transcriptase, just as it does in natural
replication.

In some protocols, a specialized RNAse enzyme
is then added to make the RNA and allow it

to be degraded.

Whether or not this is part of the procedure,
the next key step occurs when a DNA polymerase

and a primer generate a complimentary DNA
strand, just as in the PCR reaction.

At the end of this reaction, a single strand
of viral RNA has been converted to a double-stranded

DNA that has the same sequence of nucleotide
bases.

The qualitative PCR reaction can proceed as
described previously.