Real-Time PCR Method

Real-time PCR is able to detect sequence-specific PCR products as they accumulate in "real-time" during the PCR amplification process. As the PCR product of interest is produced, real-time PCR can detect their accumulation and quantify the number of substrates present in the initial PCR mixture before amplification began.

There are a few different variations of the procedure, but the one illustrated here is called molecular beacon <>. Molecular beacons are short segments of single-stranded DNA (Figure 1). The sequence of each molecular beacon must be customized to detect the PCR product of interest. In figure one, you can see there are nine bases on one end of the molecular beacon that can base pair with nine bases on the other end of the beacon. This complementation permits the molecular beacon to form a hairpin structure. The loop portion of the molecular beacon is composed of bases (shown as pink lines) that are complementary to one strand of the PCR product the investigator wants to detect and quantify.

Attached to opposite ends of the beacon are a fluorescent reporter dye and a quencher dye. When the molecular beacon is in the hairpin conformation, any fluorescence emitted by the reporter is absorbed by the quencher dye and no fluorescence is detected.

Figure 1. Diagram of molecular beacon. This beacon is 33 nucleotides long with a reporter dye attached to the 5' end and a quencher attached to the 3' end. The nine 5' bases are able to form base pairs with the nine 3' bases which brings the reporter and quencher in very close proximity. Therefore, when the reporter is excited by the appropriate light, its emission is absorbed by the quencher and no fluorescence is detected. The pink lines represent nucleotides that can form base pairs with the PCR product under investigation.

The PCR portion of real-time PCR is standard. Two PCR primers are used to amplify a segment of DNA (Figure 2).

Figure 2. PCR product of interest. The two primers are show as purple arrows and the base pairing between the two strands are shown in pink.

As the PCR continues, the newly synthesized PCR products are denatured by high temperatures. As each strand of the product are separated, the molecular beacon also is denatured so the hairpin structure is disrupted. As the temperatures cool for the next round of primer annealing, the molecular beacon is capable of forming base pairs with the appropriate strand of the PCR product (Figure 3). Any molecular beacons that do not bind to PCR product reform the hairpin structures and thus are unable to fluoresce. However, molecular beacons that bind to PCR product remove the ability for the quencher to block fluorescence from the reporter dye. Therefore, as PCR product accumulates, there is a linear increase in fluorescence.

Figure 3. Detection of PCR product by molecular beacon. When the beacon binds to the PCR product, it is able to fluoresce when excited by the appropriate wavelength of light. The amount of fluorescence is directly proportional to the amount of PCR product amplified.

Real-time PCR can be performed in a "multiplex" format which means that more than one PCR product can be detected in a single reaction tube. For each sequence, there is a unique color of fluorescent dye and therefore, each PCR product is associated with its own color which is detected by the real-time PCR machine.

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