Primer Information and Courses from MediaLab, Inc.

The polymerase chain reaction (PCR) copies DNA, utilizing repeated cycles of three basic steps. This reaction utilizes Taq DNA polymerase enzyme, which is a recombinant, thermostable DNA polymerase from the organism Thermus aquaticus. Step 1: DenaturationAfter an extraction process designed to release DNA from cellular material, an aliquot of the extracted sample is added to a reaction mixture which contains polymerase enzyme, forward and reverse primers for the target of interest, and nucleotides. During the first step, this mixture is heated (generally to 95°C). This causes complementary strands of DNA to separate (denaturation).Step 2: AnnealingThe reaction mixture is cooled to 55°C. During this annealing phase, if the target of interest is present in the patient sample, the primers will bind to their complementary sequences of DNA. Primers are short sequences of single stranded DNA that mark both ends of the target sequence. Two primers are utilized, one for each of the complementary single strands of DNA released during denaturation. The forward primer attaches to the start codon of the template DNA (the sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the anti-sense strand). The 5' ends of both primers bind to the 3' end of each DNA strand.Step 3: Synthesis at 72°CThe temperature is raised, typically to a temperature of 72°C. At this temperature the polymerase enzyme begins the process of DNA synthesis. Free nucleotides, complementary to the bases in each strand, are added sequentially to both the sense and anti-sense strands. Synthesis always occurs from the 5' to the 3' direction on each primer. This results in the simultaneous synthesis of two new strands of DNA: in the direction from the start codon to the stop codon from the forward primer, and in the direction from the stop codon to the start codon from the reverse primer.These steps are repeated in cycles, resulting in a geometric doubling of the target sequence at the end of each cycle. A typical assay will utilize around 45 cycles. Once amplification is completed, detection and identification of the multiplied target occurs.

In real-time PCR, amplification and detection occur simultaneously, within the same reaction tube. Amplification occurs in the presence of a reporter: a probe specific for the target to be amplified, which is typically bound to some type of fluorogenic compound. Although the principles of real time reactions vary depending on the type of probe utilized, the basic premise is that the instrument platform combines a thermocycler with a fluorimeter, and utilizes light of specific wavelengths to excite the reporter molecules. If the target is present, probes bind to the target during each cycle of amplification. A fluorescent signal of increasing intensity is generated as the target is amplified, which is measured by the real time instrumentation. This signal intensity is directly proportional to the amount of amplified nucleic acid. When the signal exceeds a threshold, amplification of the target can be demonstrated.Once the sample is prepared and placed on the instrument, results are available in approximately 1 to 2 hours, depending on the assay and platform. In addition to reduced turnaround times, real time methodologies are more adaptable to automation and require significantly reduced handling of amplicons, and reduced risk of cross contamination. In some ways, the introduction of real time PCR accelerated the integration of molecular methods into the routine clinical diagnostic laboratory setting. Melt curve analysis Real time PCR methods can also specifically identify amplified products through melt curve analysis. The melting temperature of double stranded DNA depends on its base composition and length. All PCR products for a specific primer pair should have the same melting temperature, unless there is contamination, primer-dimer pairs, or some other problem. Real time instruments can be programmed to perform an analysis of the melting temperature of the PCR product. When hybridization probes are utilized, after the last PCR cycle, the samples are denatured, and then cooled to a temperature approximately 10o C below the expected melt temperature (Tm), as determined by previous testing of known samples. (This cooling protocol maximizes the formation of probe-target duplexes.) Then the temperature is incrementally raised while the fluorescence is continually monitored. At the melting point, the probe separates from the target strand, and the fluorescence rapidly decreases. The instrumentation software plots the rate of change in fluorescence with time on the Y axis, versus temperature on the X axis. The temperature at which the peak rate of change occurs is the Tm, and can be compared to known controls or established ranges to identify the PCR product.

Polymerases are enzymes that synthesize DNA from an existing template. Polymerase requires a primer, nucleotides, and magnesium in order to function. In early PCR methods, the DNA polymerase was inactivated during the denaturation step. This required new polymerase to be added during each cycle of PCR. This problem was solved by the discovery of an enzyme termed Taq polymerase. Taq polymerase is isolated from the thermophilic organism Thermus aquaticus, a natural bacterium found in thermal springs. The Taq polymerase has optimal activity at 72°C but it can survive in temperatures up to 95°C. Today there are several other kinds of thermostable enzymes that are available for PCR, such as Pfu and Tli DNA polymerase. However, Taq is the DNA polymerase that is used most often in PCR procedures.

There are two main methods for detection of the real-time PCR products: non-specific fluorescent dyes and sequence-specific DNA probes.Non-specific dyesThese are dyes that bind to all pieces of double-stranded DNA and result in fluorescence. SYBR green is an example of a non-specific dye that is commonly used in real-time PCR. Because these dyes bind to all double-stranded DNA sequences, the fluorescence intensity increases after each cycle and allows for the concentrations to be determined. Since there is no associated unit of measure, only a fraction or ratio to a standard dilution can be determined. One problem with this method is that the dyes are non-specific and will even bind with primer dimers. This can potentially cause inaccurate quantification of the intended target sequence. However, if the specificity is not required, this is a more cost-effective method than the sequence-specific DNA probe method. Sequence-specific DNA probesAs the name implies, this method of detection is more specific than non-specific dyes. Probes can be designed to bind only to certain DNA sequences and are labeled with a fluorescence reporter that permits detection only after hybridization. The use of these sequence-specific probes allows for the detection of only the specific DNA product and utilizes fluorescence resonance energy transfer (FRET). These probes are also used for multiplexing. Multiplexing is the process of assaying several different genes in the same reaction. In this process, each probe has a different color reporter, which allows for the quantification of several different sequence products at the same time.