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

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

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and quantified using PCR.

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

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generated in the reaction.

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

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

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

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

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double-stranded DNA.

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

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each end of the target DNA.

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

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

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with only one.

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

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the program on basic PCR once again.

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

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

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the DNA sequence synthesized between the two[br]primers.

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

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nucleotides is labeled with a fluorescent[br]molecule and another nucleotide is labeled

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with a fluorescence quencher molecule.

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

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as it remains in close proximity.

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

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single cycle of PCR.

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Other primers bind to the separate strands[br]of DNA.

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The probe also finds its complimentary sites[br]between them.

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

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an exonucleus activity.

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

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

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

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marker and the one bearing the quencher are[br]separated from one another.

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

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stimulated.

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

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quencher.

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

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fluorescent energy also doubles.

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

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with a fluorometer.

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

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

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thermocycler.

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

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

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

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signal would be detected 10 rounds earlier.

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

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

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

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with HIV, Hepatitis B, and other viruses.

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

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So, how can this method work?

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

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and converted to double-stranded DNA.

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

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virion.

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

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transcriptase, just as it does in natural[br]replication.

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

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to be degraded.

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

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

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

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DNA that has the same sequence of nucleotide[br]bases.

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The qualitative PCR reaction can proceed as[br]described previously.