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Dialogue: 0,0:00:00.00,0:00:02.00,Default,,0000,0000,0000,,(English captions by Andrea Matsumoto, University of Michigan.)
Dialogue: 0,0:00:02.00,0:00:09.00,Default,,0000,0000,0000,,This program shows how a specific nucleic\Nacid in a clinical sample can be detected
Dialogue: 0,0:00:09.00,0:00:12.00,Default,,0000,0000,0000,,and quantified using PCR.
Dialogue: 0,0:00:12.00,0:00:19.00,Default,,0000,0000,0000,,This is accomplished by detecting the accumulation\Nof the amplified PCR products as they are
Dialogue: 0,0:00:19.00,0:00:23.00,Default,,0000,0000,0000,,generated in the reaction.
Dialogue: 0,0:00:23.00,0:00:29.00,Default,,0000,0000,0000,,And so the process is called real-time,\Nor RTPCR.
Dialogue: 0,0:00:29.00,0:00:38.00,Default,,0000,0000,0000,,To understand how amplified PCR products,\Nalso called amplicons, are detected in real-time,
Dialogue: 0,0:00:38.00,0:00:46.00,Default,,0000,0000,0000,,let's first review the events that occur\Nduring a normal cycle of the PCR reaction.
Dialogue: 0,0:00:46.00,0:00:53.00,Default,,0000,0000,0000,,Recall that the first step in any PCR cycle\Nis to raise the reaction temperature and melt
Dialogue: 0,0:00:53.00,0:00:55.00,Default,,0000,0000,0000,,double-stranded DNA.
Dialogue: 0,0:00:55.00,0:01:03.00,Default,,0000,0000,0000,,Then, when the temperature is lowered, the\Nspecific primers bind to the sequences at
Dialogue: 0,0:01:03.00,0:01:06.00,Default,,0000,0000,0000,,each end of the target DNA.
Dialogue: 0,0:01:06.00,0:01:15.00,Default,,0000,0000,0000,,The intervening DNA can then be synthesized\Nby polymerase reaction in opposite directions.
Dialogue: 0,0:01:15.00,0:01:21.00,Default,,0000,0000,0000,,Other results, you produce two double-strand\Ncopies of the target DNA, where you started
Dialogue: 0,0:01:21.00,0:01:23.00,Default,,0000,0000,0000,,with only one.
Dialogue: 0,0:01:23.00,0:01:30.00,Default,,0000,0000,0000,,If you have any confusion about this basic\Nprocess, it might be a good idea to review
Dialogue: 0,0:01:30.00,0:01:35.00,Default,,0000,0000,0000,,the program on basic PCR once again.
Dialogue: 0,0:01:35.00,0:01:41.00,Default,,0000,0000,0000,,To detect the generation of new amplicons\Nin real-time, the PCR reaction requires an
Dialogue: 0,0:01:41.00,0:01:50.00,Default,,0000,0000,0000,,additional ingredient -a single-stranded DNA\Nprobe, designed to hybridize to the part of
Dialogue: 0,0:01:50.00,0:01:54.00,Default,,0000,0000,0000,,the DNA sequence synthesized between the two\Nprimers.
Dialogue: 0,0:01:54.00,0:02:02.00,Default,,0000,0000,0000,,However, unlike the primers, this probe is\Nmore defined in a special way. One of its
Dialogue: 0,0:02:02.00,0:02:11.00,Default,,0000,0000,0000,,nucleotides is labeled with a fluorescent\Nmolecule and another nucleotide is labeled
Dialogue: 0,0:02:11.00,0:02:16.00,Default,,0000,0000,0000,,with a fluorescence quencher molecule.
Dialogue: 0,0:02:16.00,0:02:23.00,Default,,0000,0000,0000,,The quencher rapidly absorbs any light energy\Nemitted by the fluorescent molecule, as long
Dialogue: 0,0:02:23.00,0:02:27.00,Default,,0000,0000,0000,,as it remains in close proximity.
Dialogue: 0,0:02:27.00,0:02:36.00,Default,,0000,0000,0000,,Now, let's look at what happens when this\Nadditional ingredient is present during a
Dialogue: 0,0:02:36.00,0:02:39.00,Default,,0000,0000,0000,,single cycle of PCR.
Dialogue: 0,0:02:39.00,0:02:44.00,Default,,0000,0000,0000,,Other primers bind to the separate strands\Nof DNA.
Dialogue: 0,0:02:44.00,0:02:49.00,Default,,0000,0000,0000,,The probe also finds its complimentary sites\Nbetween them.
Dialogue: 0,0:02:49.00,0:02:56.00,Default,,0000,0000,0000,,The enzyme synthesizes new DNA from the ends\Nof the primers also have a second activity:
Dialogue: 0,0:02:56.00,0:03:00.00,Default,,0000,0000,0000,,an exonucleus activity.
Dialogue: 0,0:03:00.00,0:03:07.00,Default,,0000,0000,0000,,So when it encounters double-stranded DNA\Nin its path, it will disassemble the strand
Dialogue: 0,0:03:07.00,0:03:12.00,Default,,0000,0000,0000,,that is in its way, and replace all of the\Nnucleotides.
Dialogue: 0,0:03:12.00,0:03:18.00,Default,,0000,0000,0000,,As polymerase pass through the probe, note\Nthat the nucleotide bearing the fluorescent
Dialogue: 0,0:03:18.00,0:03:24.00,Default,,0000,0000,0000,,marker and the one bearing the quencher are\Nseparated from one another.
Dialogue: 0,0:03:24.00,0:03:32.00,Default,,0000,0000,0000,,In the absence of a nearby quencher, the fluorescent\Nmolecule can now emit detectable light when
Dialogue: 0,0:03:32.00,0:03:34.00,Default,,0000,0000,0000,,stimulated.
Dialogue: 0,0:03:34.00,0:03:41.00,Default,,0000,0000,0000,,Each time another amplicon is produced, another\Nfluorescent marker is released from its neighboring
Dialogue: 0,0:03:41.00,0:03:43.00,Default,,0000,0000,0000,,quencher.
Dialogue: 0,0:03:43.00,0:03:50.00,Default,,0000,0000,0000,,Therefore, just as the number of amplicons\Ndoubles in each PCR cycle, the amount of emitted
Dialogue: 0,0:03:50.00,0:03:52.00,Default,,0000,0000,0000,,fluorescent energy also doubles.
Dialogue: 0,0:03:52.00,0:04:00.00,Default,,0000,0000,0000,,This light generation can be monitored during\Nthe PCR reaction thermocycler that is equipped
Dialogue: 0,0:04:00.00,0:04:02.00,Default,,0000,0000,0000,,with a fluorometer.
Dialogue: 0,0:04:02.00,0:04:08.00,Default,,0000,0000,0000,,So, if you begin with a clinical sample that\Nhad only one copy of the target DNA, it could
Dialogue: 0,0:04:08.00,0:04:15.00,Default,,0000,0000,0000,,take 40 or more cycles before the amplicons\Nare detected by a fluorometer in a specialized
Dialogue: 0,0:04:15.00,0:04:16.00,Default,,0000,0000,0000,,thermocycler.
Dialogue: 0,0:04:16.00,0:04:24.00,Default,,0000,0000,0000,,However, if the original sample contained\N32 times more copies of the target DNA, then
Dialogue: 0,0:04:24.00,0:04:30.00,Default,,0000,0000,0000,,the fluorometric detection would occur after\N5 fewer rounds of PCR.
Dialogue: 0,0:04:30.00,0:04:38.00,Default,,0000,0000,0000,,And if there were 1,024 more target DNA sequences\Nin the original sample, then the fluorescent
Dialogue: 0,0:04:38.00,0:04:42.00,Default,,0000,0000,0000,,signal would be detected 10 rounds earlier.
Dialogue: 0,0:04:42.00,0:04:48.00,Default,,0000,0000,0000,,So, the amount of specific DNA in the clinical\Nsample is determined by a reference to the
Dialogue: 0,0:04:48.00,0:04:56.00,Default,,0000,0000,0000,,round of PCR in which the amount of fluorescence\Nfirst crosses the threshold of detection.
Dialogue: 0,0:04:56.00,0:05:04.00,Default,,0000,0000,0000,,RTPCR is most commonly used to quantify\Nthe burden of viruses in the blood of patients
Dialogue: 0,0:05:04.00,0:05:08.00,Default,,0000,0000,0000,,with HIV, Hepatitis B, and other viruses.
Dialogue: 0,0:05:08.00,0:05:19.00,Default,,0000,0000,0000,,But HIV is an RNA virus; it has no DNA, and\Nthe RNA that it possesses is single stranded.
Dialogue: 0,0:05:19.00,0:05:23.00,Default,,0000,0000,0000,,So, how can this method work?
Dialogue: 0,0:05:23.00,0:05:30.00,Default,,0000,0000,0000,,The answer is that RNA, from an RNA virus,\Ncan be quantified after it has been copied
Dialogue: 0,0:05:30.00,0:05:34.00,Default,,0000,0000,0000,,and converted to double-stranded DNA.
Dialogue: 0,0:05:34.00,0:05:42.00,Default,,0000,0000,0000,,This animation shows how this is accomplished.\NFirst, the viral RNA is released from the
Dialogue: 0,0:05:42.00,0:05:43.00,Default,,0000,0000,0000,,virion.
Dialogue: 0,0:05:43.00,0:05:51.00,Default,,0000,0000,0000,,Then, a complimentary DNA strand is synthesized\Nfrom the viral RNA using purified reverse
Dialogue: 0,0:05:51.00,0:05:56.00,Default,,0000,0000,0000,,transcriptase, just as it does in natural\Nreplication.
Dialogue: 0,0:05:56.00,0:06:06.00,Default,,0000,0000,0000,,In some protocols, a specialized RNAse enzyme\Nis then added to make the RNA and allow it
Dialogue: 0,0:06:06.00,0:06:08.00,Default,,0000,0000,0000,,to be degraded.
Dialogue: 0,0:06:08.00,0:06:15.00,Default,,0000,0000,0000,,Whether or not this is part of the procedure,\Nthe next key step occurs when a DNA polymerase
Dialogue: 0,0:06:15.00,0:06:22.00,Default,,0000,0000,0000,,and a primer generate a complimentary DNA\Nstrand, just as in the PCR reaction.
Dialogue: 0,0:06:22.00,0:06:31.00,Default,,0000,0000,0000,,At the end of this reaction, a single strand\Nof viral RNA has been converted to a double-stranded
Dialogue: 0,0:06:31.00,0:06:37.00,Default,,0000,0000,0000,,DNA that has the same sequence of nucleotide\Nbases.
Dialogue: 0,0:06:37.00,0:06:41.00,Default,,0000,0000,0000,,The qualitative PCR reaction can proceed as\Ndescribed previously.