(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.
Subtitles by the Amara.org community