1 00:00:00,000 --> 00:00:02,000 (English captions by Andrea Matsumoto, University of Michigan.) 2 00:00:02,000 --> 00:00:09,000 This program shows how a specific nucleic acid in a clinical sample can be detected 3 00:00:09,000 --> 00:00:12,000 and quantified using PCR. 4 00:00:12,000 --> 00:00:19,000 This is accomplished by detecting the accumulation of the amplified PCR products as they are 5 00:00:19,000 --> 00:00:23,000 generated in the reaction. 6 00:00:23,000 --> 00:00:29,000 And so the process is called real-time, or RTPCR. 7 00:00:29,000 --> 00:00:38,000 To understand how amplified PCR products, also called amplicons, are detected in real-time, 8 00:00:38,000 --> 00:00:46,000 let's first review the events that occur during a normal cycle of the PCR reaction. 9 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 10 00:00:53,000 --> 00:00:55,000 double-stranded DNA. 11 00:00:55,000 --> 00:01:03,000 Then, when the temperature is lowered, the specific primers bind to the sequences at 12 00:01:03,000 --> 00:01:06,000 each end of the target DNA. 13 00:01:06,000 --> 00:01:15,000 The intervening DNA can then be synthesized by polymerase reaction in opposite directions. 14 00:01:15,000 --> 00:01:21,000 Other results, you produce two double-strand copies of the target DNA, where you started 15 00:01:21,000 --> 00:01:23,000 with only one. 16 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 17 00:01:30,000 --> 00:01:35,000 the program on basic PCR once again. 18 00:01:35,000 --> 00:01:41,000 To detect the generation of new amplicons in real-time, the PCR reaction requires an 19 00:01:41,000 --> 00:01:50,000 additional ingredient -a single-stranded DNA probe, designed to hybridize to the part of 20 00:01:50,000 --> 00:01:54,000 the DNA sequence synthesized between the two primers. 21 00:01:54,000 --> 00:02:02,000 However, unlike the primers, this probe is more defined in a special way. One of its 22 00:02:02,000 --> 00:02:11,000 nucleotides is labeled with a fluorescent molecule and another nucleotide is labeled 23 00:02:11,000 --> 00:02:16,000 with a fluorescence quencher molecule. 24 00:02:16,000 --> 00:02:23,000 The quencher rapidly absorbs any light energy emitted by the fluorescent molecule, as long 25 00:02:23,000 --> 00:02:27,000 as it remains in close proximity. 26 00:02:27,000 --> 00:02:36,000 Now, let's look at what happens when this additional ingredient is present during a 27 00:02:36,000 --> 00:02:39,000 single cycle of PCR. 28 00:02:39,000 --> 00:02:44,000 Other primers bind to the separate strands of DNA. 29 00:02:44,000 --> 00:02:49,000 The probe also finds its complimentary sites between them. 30 00:02:49,000 --> 00:02:56,000 The enzyme synthesizes new DNA from the ends of the primers also have a second activity: 31 00:02:56,000 --> 00:03:00,000 an exonucleus activity. 32 00:03:00,000 --> 00:03:07,000 So when it encounters double-stranded DNA in its path, it will disassemble the strand 33 00:03:07,000 --> 00:03:12,000 that is in its way, and replace all of the nucleotides. 34 00:03:12,000 --> 00:03:18,000 As polymerase pass through the probe, note that the nucleotide bearing the fluorescent 35 00:03:18,000 --> 00:03:24,000 marker and the one bearing the quencher are separated from one another. 36 00:03:24,000 --> 00:03:32,000 In the absence of a nearby quencher, the fluorescent molecule can now emit detectable light when 37 00:03:32,000 --> 00:03:34,000 stimulated. 38 00:03:34,000 --> 00:03:41,000 Each time another amplicon is produced, another fluorescent marker is released from its neighboring 39 00:03:41,000 --> 00:03:43,000 quencher. 40 00:03:43,000 --> 00:03:50,000 Therefore, just as the number of amplicons doubles in each PCR cycle, the amount of emitted 41 00:03:50,000 --> 00:03:52,000 fluorescent energy also doubles. 42 00:03:52,000 --> 00:04:00,000 This light generation can be monitored during the PCR reaction thermocycler that is equipped 43 00:04:00,000 --> 00:04:02,000 with a fluorometer. 44 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 45 00:04:08,000 --> 00:04:15,000 take 40 or more cycles before the amplicons are detected by a fluorometer in a specialized 46 00:04:15,000 --> 00:04:16,000 thermocycler. 47 00:04:16,000 --> 00:04:24,000 However, if the original sample contained 32 times more copies of the target DNA, then 48 00:04:24,000 --> 00:04:30,000 the fluorometric detection would occur after 5 fewer rounds of PCR. 49 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 50 00:04:38,000 --> 00:04:42,000 signal would be detected 10 rounds earlier. 51 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 52 00:04:48,000 --> 00:04:56,000 round of PCR in which the amount of fluorescence first crosses the threshold of detection. 53 00:04:56,000 --> 00:05:04,000 RTPCR is most commonly used to quantify the burden of viruses in the blood of patients 54 00:05:04,000 --> 00:05:08,000 with HIV, Hepatitis B, and other viruses. 55 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. 56 00:05:19,000 --> 00:05:23,000 So, how can this method work? 57 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 58 00:05:30,000 --> 00:05:34,000 and converted to double-stranded DNA. 59 00:05:34,000 --> 00:05:42,000 This animation shows how this is accomplished. First, the viral RNA is released from the 60 00:05:42,000 --> 00:05:43,000 virion. 61 00:05:43,000 --> 00:05:51,000 Then, a complimentary DNA strand is synthesized from the viral RNA using purified reverse 62 00:05:51,000 --> 00:05:56,000 transcriptase, just as it does in natural replication. 63 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 64 00:06:06,000 --> 00:06:08,000 to be degraded. 65 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 66 00:06:15,000 --> 00:06:22,000 and a primer generate a complimentary DNA strand, just as in the PCR reaction. 67 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 68 00:06:31,000 --> 00:06:37,000 DNA that has the same sequence of nucleotide bases. 69 00:06:37,000 --> 00:06:41,000 The qualitative PCR reaction can proceed as described previously.