WEBVTT 00:00:00.000 --> 00:00:02.000 (English captions by Andrea Matsumoto, University of Michigan.) 00:00:02.000 --> 00:00:09.000 This program shows how a specific nucleic acid in a clinical sample can be detected 00:00:09.000 --> 00:00:12.000 and quantified using PCR. 00:00:12.000 --> 00:00:19.000 This is accomplished by detecting the accumulation of the amplified PCR products as they are 00:00:19.000 --> 00:00:23.000 generated in the reaction. 00:00:23.000 --> 00:00:29.000 And so the process is called real-time, or RTPCR. 00:00:29.000 --> 00:00:38.000 To understand how amplified PCR products, also called amplicons, are detected in real-time, 00:00:38.000 --> 00:00:46.000 let's first review the events that occur during a normal cycle of the PCR reaction. 00:00:46.000 --> 00:00:53.000 Recall that the first step in any PCR cycle is to raise the reaction temperature and melt 00:00:53.000 --> 00:00:55.000 double-stranded DNA. 00:00:55.000 --> 00:01:03.000 Then, when the temperature is lowered, the specific primers bind to the sequences at 00:01:03.000 --> 00:01:06.000 each end of the target DNA. 00:01:06.000 --> 00:01:15.000 The intervening DNA can then be synthesized by polymerase reaction in opposite directions. 00:01:15.000 --> 00:01:21.000 Other results, you produce two double-strand copies of the target DNA, where you started 00:01:21.000 --> 00:01:23.000 with only one. 00:01:23.000 --> 00:01:30.000 If you have any confusion about this basic process, it might be a good idea to review 00:01:30.000 --> 00:01:35.000 the program on basic PCR once again. 00:01:35.000 --> 00:01:41.000 To detect the generation of new amplicons in real-time, the PCR reaction requires an 00:01:41.000 --> 00:01:50.000 additional ingredient -a single-stranded DNA probe, designed to hybridize to the part of 00:01:50.000 --> 00:01:54.000 the DNA sequence synthesized between the two primers. 00:01:54.000 --> 00:02:02.000 However, unlike the primers, this probe is more defined in a special way. One of its 00:02:02.000 --> 00:02:11.000 nucleotides is labeled with a fluorescent molecule and another nucleotide is labeled 00:02:11.000 --> 00:02:16.000 with a fluorescence quencher molecule. 00:02:16.000 --> 00:02:23.000 The quencher rapidly absorbs any light energy emitted by the fluorescent molecule, as long 00:02:23.000 --> 00:02:27.000 as it remains in close proximity. 00:02:27.000 --> 00:02:36.000 Now, let's look at what happens when this additional ingredient is present during a 00:02:36.000 --> 00:02:39.000 single cycle of PCR. 00:02:39.000 --> 00:02:44.000 Other primers bind to the separate strands of DNA. 00:02:44.000 --> 00:02:49.000 The probe also finds its complimentary sites between them. 00:02:49.000 --> 00:02:56.000 The enzyme synthesizes new DNA from the ends of the primers also have a second activity: 00:02:56.000 --> 00:03:00.000 an exonucleus activity. 00:03:00.000 --> 00:03:07.000 So when it encounters double-stranded DNA in its path, it will disassemble the strand 00:03:07.000 --> 00:03:12.000 that is in its way, and replace all of the nucleotides. 00:03:12.000 --> 00:03:18.000 As polymerase pass through the probe, note that the nucleotide bearing the fluorescent 00:03:18.000 --> 00:03:24.000 marker and the one bearing the quencher are separated from one another. 00:03:24.000 --> 00:03:32.000 In the absence of a nearby quencher, the fluorescent molecule can now emit detectable light when 00:03:32.000 --> 00:03:34.000 stimulated. 00:03:34.000 --> 00:03:41.000 Each time another amplicon is produced, another fluorescent marker is released from its neighboring 00:03:41.000 --> 00:03:43.000 quencher. 00:03:43.000 --> 00:03:50.000 Therefore, just as the number of amplicons doubles in each PCR cycle, the amount of emitted 00:03:50.000 --> 00:03:52.000 fluorescent energy also doubles. 00:03:52.000 --> 00:04:00.000 This light generation can be monitored during the PCR reaction thermocycler that is equipped 00:04:00.000 --> 00:04:02.000 with a fluorometer. 00:04:02.000 --> 00:04:08.000 So, if you begin with a clinical sample that had only one copy of the target DNA, it could 00:04:08.000 --> 00:04:15.000 take 40 or more cycles before the amplicons are detected by a fluorometer in a specialized 00:04:15.000 --> 00:04:16.000 thermocycler. 00:04:16.000 --> 00:04:24.000 However, if the original sample contained 32 times more copies of the target DNA, then 00:04:24.000 --> 00:04:30.000 the fluorometric detection would occur after 5 fewer rounds of PCR. 00:04:30.000 --> 00:04:38.000 And if there were 1,024 more target DNA sequences in the original sample, then the fluorescent 00:04:38.000 --> 00:04:42.000 signal would be detected 10 rounds earlier. 00:04:42.000 --> 00:04:48.000 So, the amount of specific DNA in the clinical sample is determined by a reference to the 00:04:48.000 --> 00:04:56.000 round of PCR in which the amount of fluorescence first crosses the threshold of detection. 00:04:56.000 --> 00:05:04.000 RTPCR is most commonly used to quantify the burden of viruses in the blood of patients 00:05:04.000 --> 00:05:08.000 with HIV, Hepatitis B, and other viruses. 00:05:08.000 --> 00:05:19.000 But HIV is an RNA virus; it has no DNA, and the RNA that it possesses is single stranded. 00:05:19.000 --> 00:05:23.000 So, how can this method work? 00:05:23.000 --> 00:05:30.000 The answer is that RNA, from an RNA virus, can be quantified after it has been copied 00:05:30.000 --> 00:05:34.000 and converted to double-stranded DNA. 00:05:34.000 --> 00:05:42.000 This animation shows how this is accomplished. First, the viral RNA is released from the 00:05:42.000 --> 00:05:43.000 virion. 00:05:43.000 --> 00:05:51.000 Then, a complimentary DNA strand is synthesized from the viral RNA using purified reverse 00:05:51.000 --> 00:05:56.000 transcriptase, just as it does in natural replication. 00:05:56.000 --> 00:06:06.000 In some protocols, a specialized RNAse enzyme is then added to make the RNA and allow it 00:06:06.000 --> 00:06:08.000 to be degraded. 00:06:08.000 --> 00:06:15.000 Whether or not this is part of the procedure, the next key step occurs when a DNA polymerase 00:06:15.000 --> 00:06:22.000 and a primer generate a complimentary DNA strand, just as in the PCR reaction. 00:06:22.000 --> 00:06:31.000 At the end of this reaction, a single strand of viral RNA has been converted to a double-stranded 00:06:31.000 --> 00:06:37.000 DNA that has the same sequence of nucleotide bases. 00:06:37.000 --> 00:06:41.000 The qualitative PCR reaction can proceed as described previously.