captions.json

{"1": {"seconds": 2, "content": "This program shows how a specific nucleic acid in a clinical sample can be detected", "num": "1"}, "2": {"seconds": 9, "content": "and quantified using <a target='_blank' href='https://www.google.com/search?q=PCR'>PCR</a>.", "num": "2"}, "3": {"seconds": 12, "content": "This is accomplished by detecting the accumulation of the amplified PCR products as they are", "num": "3"}, "4": {"seconds": 19, "content": "generated in the reaction.", "num": "4"}, "5": {"seconds": 23, "content": "And so the process is called real-time, or RTPCR.", "num": "5"}, "6": {"seconds": 29, "content": "To understand how amplified PCR products, also called amplicons, are detected in real-time,", "num": "6"}, "7": {"seconds": 38, "content": "let's first review the events that occur during a normal cycle of the PCR reaction.", "num": "7"}, "8": {"seconds": 46, "content": "Recall that the first step in any PCR cycle is to raise the reaction temperature and melt", "num": "8"}, "9": {"seconds": 53, "content": "double-stranded DNA.", "num": "9"}, "10": {"seconds": 55, "content": "Then, when the temperature is lowered, the specific primers bind to the sequences at", "num": "10"}, "11": {"seconds": 63, "content": "each end of the target DNA.", "num": "11"}, "12": {"seconds": 66, "content": "The intervening DNA can then be synthesized by polymerase reaction in opposite directions.", "num": "12"}, "13": {"seconds": 75, "content": "Other results, you produce two double-strand copies of the target DNA, where you started", "num": "13"}, "14": {"seconds": 81, "content": "with only one.", "num": "14"}, "15": {"seconds": 83, "content": "If you have any confusion about this basic process, it might be a good idea to review", "num": "15"}, "16": {"seconds": 90, "content": "the program on basic PCR once again.", "num": "16"}, "17": {"seconds": 95, "content": "To detect the generation of new amplicons in real-time, the PCR reaction requires an", "num": "17"}, "18": {"seconds": 101, "content": "additional ingredient -a single-stranded DNA probe, designed to hybridize to the part of", "num": "18"}, "19": {"seconds": 110, "content": "the DNA sequence synthesized between the two primers.", "num": "19"}, "20": {"seconds": 114, "content": "However, unlike the primers, this probe is more defined in a special way. One of its", "num": "20"}, "21": {"seconds": 122, "content": "nucleotides is labeled with a fluorescent molecule and another nucleotide is labeled", "num": "21"}, "22": {"seconds": 131, "content": "with a fluorescence quencher molecule.", "num": "22"}, "23": {"seconds": 136, "content": "The quencher rapidly absorbs any light energy emitted by the fluorescent molecule, as long", "num": "23"}, "24": {"seconds": 143, "content": "as it remains in close proximity.", "num": "24"}, "25": {"seconds": 147, "content": "Now, let's look at what happens when this additional ingredient is present during a", "num": "25"}, "26": {"seconds": 156, "content": "single cycle of PCR.", "num": "26"}, "27": {"seconds": 159, "content": "Other primers bind to the separate strands of DNA.", "num": "27"}, "28": {"seconds": 164, "content": "The probe also finds its complimentary sites between them.", "num": "28"}, "29": {"seconds": 169, "content": "The enzyme synthesizes new DNA from the ends of the primers also have a second activity:", "num": "29"}, "30": {"seconds": 176, "content": "an exonucleus activity.", "num": "30"}, "31": {"seconds": 180, "content": "So when it encounters double-stranded DNA in its path, it will disassemble the strand", "num": "31"}, "32": {"seconds": 187, "content": "that is in its way, and replace all of the nucleotides.", "num": "32"}, "33": {"seconds": 192, "content": "As polymerase pass through the probe, note that the nucleotide bearing the fluorescent", "num": "33"}, "34": {"seconds": 198, "content": "marker and the one bearing the quencher are separated from one another.", "num": "34"}, "35": {"seconds": 204, "content": "In the absence of a nearby quencher, the fluorescent molecule can now emit detectable light when", "num": "35"}, "36": {"seconds": 212, "content": "stimulated.", "num": "36"}, "37": {"seconds": 214, "content": "Each time another amplicon is produced, another fluorescent marker is released from its neighboring", "num": "37"}, "38": {"seconds": 221, "content": "quencher.", "num": "38"}, "39": {"seconds": 223, "content": "Therefore, just as the number of amplicons doubles in each PCR cycle, the amount of emitted", "num": "39"}, "40": {"seconds": 230, "content": "fluorescent energy also doubles.", "num": "40"}, "41": {"seconds": 232, "content": "This light generation can be monitored during the PCR reaction thermocycler that is equipped", "num": "41"}, "42": {"seconds": 240, "content": "with a fluorometer.", "num": "42"}, "43": {"seconds": 242, "content": "So, if you begin with a clinical sample that had only one copy of the target DNA, it could", "num": "43"}, "44": {"seconds": 248, "content": "take 40 or more cycles before the amplicons are detected by a fluorometer in a specialized", "num": "44"}, "45": {"seconds": 255, "content": "thermocycler.", "num": "45"}, "46": {"seconds": 256, "content": "However, if the original sample contained 32 times more copies of the target DNA, then", "num": "46"}, "47": {"seconds": 264, "content": "the fluorometric detection would occur after 5 fewer rounds of PCR.", "num": "47"}, "48": {"seconds": 270, "content": "And if there were 1,024 more target DNA sequences in the original sample, then the fluorescent", "num": "48"}, "49": {"seconds": 278, "content": "signal would be detected 10 rounds earlier.", "num": "49"}, "50": {"seconds": 282, "content": "So, the amount of specific DNA in the clinical sample is determined by a reference to the", "num": "50"}, "51": {"seconds": 288, "content": "round of PCR in which the amount of fluorescence first crosses the threshold of detection.", "num": "51"}, "52": {"seconds": 296, "content": "RTPCR is most commonly used to quantify the burden of viruses in the blood of patients", "num": "52"}, "53": {"seconds": 304, "content": "with HIV, Hepatitis B, and other viruses.", "num": "53"}, "54": {"seconds": 308, "content": "But HIV is an RNA virus; it has no DNA, and the RNA that it possesses is single stranded.", "num": "54"}, "55": {"seconds": 319, "content": "So, how can this method work?", "num": "55"}, "56": {"seconds": 323, "content": "The answer is that RNA, from an RNA virus, can be quantified after it has been copied", "num": "56"}, "57": {"seconds": 330, "content": "and converted to double-stranded DNA.", "num": "57"}, "58": {"seconds": 334, "content": "This animation shows how this is accomplished. First, the viral RNA is released from the", "num": "58"}, "59": {"seconds": 342, "content": "virion.", "num": "59"}, "60": {"seconds": 343, "content": "Then, a complimentary DNA strand is synthesized from the viral RNA using purified reverse", "num": "60"}, "61": {"seconds": 351, "content": "transcriptase, just as it does in natural replication.", "num": "61"}, "62": {"seconds": 356, "content": "In some protocols, a specialized RNAse enzyme is then added to make the RNA and allow it", "num": "62"}, "63": {"seconds": 366, "content": "to be degraded.", "num": "63"}, "64": {"seconds": 368, "content": "Whether or not this is part of the procedure, the next key step occurs when a DNA polymerase", "num": "64"}, "65": {"seconds": 375, "content": "and a primer generate a complimentary DNA strand, just as in the PCR reaction.", "num": "65"}, "66": {"seconds": 382, "content": "At the end of this reaction, a single strand of viral RNA has been converted to a double-stranded", "num": "66"}, "67": {"seconds": 391, "content": "DNA that has the same sequence of nucleotide bases.", "num": "67"}, "68": {"seconds": 397, "content": "The qualitative PCR reaction can proceed as described previously.", "num": "68"}, "69": {"seconds": 402, "content": "Subtitles by the Amara.org community", "num": "69"}}