|Polymerase Chain Reaction (PCR)|
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.
|Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)|
Reverse transcriptase PCR (RT-PCR) was developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples). Essentially, the method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR.PCR depends on the Taq Polymerase enzyme; RNA is not an efficient substrate for this enzyme. This is why the target of interest (if present) is first transcribed into complementary DNA (cDNA), which can then be amplified. RT-PCR ProcessAfter RNA is released from cellular material through extraction, an aliquot of the extracted sample is added to a reaction mixture which contains reverse transcriptase enzyme, primers specific for the target of interest, and nucleotides.If the target is present, primers anneal to the RNA strand.Reverse transcriptase enzyme synthesizes a complementary DNA strand, extending from the primer.The temperature is raised to 95o C, and the RNA/DNA strands are denatured.The temperatures are lowered, allowing primers to anneal to the newly formed cDNA.Polymerase enzyme synthesizes a new DNA strand, extending from the primer.Multiple cycles geometrically increase the number of copies of DNA.RT-PCR can be performed as one or two step procedures. In a one-step procedure, the reverse transcriptase is performed in the same reaction tube as the polymerase chain reaction. In a two-step procedure, transcription of the RNA to cDNA is performed first. Transcription occurs between 40o C and 50o C, depending on the properties of the reverse transcriptase enzyme utilized; products of that reaction are then amplified in a separate reaction.
|Detection and Identification of Polymerase Chain Reaction (PCR) Products: Advantages of Real-Time PCR|
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.
|Improvements for Influenza Testing|
Public health laboratories were the first provided with the reagents and procedures for the reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed by the Centers for Disease Control (CDC) under the Emergency Use Authorization (EUA). As information was shared between laboratories, other facilities implemented RT-PCR procedures that provided for the detection and differentiation of the H1N1 "swine" strain from previously encountered seasonal strains. Although many facilities utilized laboratory developed procedures, the FDA did grant emergency approval to a handful of commercially developed methods. One example was Prodesse's ProFlu-ST™ assay which became available in October 2009. Employing real time methodology, the kit was also optimized for use with automated extraction platforms, such as Roche's MagNA Pure Systems and Biomerieux's NucliSENS® easyMAG®.The ProFlu-ST™ assay is a multiplex RT-PCR assay utilizing fluorogenic hydrolysis (Taqman) probes for use on the SmartCycler platform. As a multiplex assay, it includes primers and probes for seasonal H1, seasonal H3, and 2009 H1 strains of influenza A. Targets are as follows:Seasonal H1: conserved area of A/H1 hemagglutinin (HA) geneSeasonal H3: conserved area of A/H3 hemagglutinin (HA) gene2009 H1/N1: conserved area of the 2009 nucleoprotein (NP) geneExtraction of RNA from patient samples is followed by a one-step multiplex reverse transcription of RNA targets into complementary DNA (cDNA), which is subsequently amplified in a real time thermocycler. In this process, the probe anneals specifically to the template, followed by primer extension and amplification. The assay utilizes the 5' - 3' exonuclease activity of the Taq polymerase, which cleaves the probe, thus separating the reporter dye of the fluorogenic probe from the quencher. This generates an increase in fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing the fluorescent signal.
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.
|Stages of Real-Time PCR|
The PCR process can generally be divided into three stages and visualized on a graph. The first stage is the exponential amplification where the doubling of the DNA product will occur with each cycle. At this stage there are is an excess of reagents and a limited number of templates, so product renaturation does not compete with primer binding. The second stage is the leveling off stage in which the reaction slows down due to the loss of activity of the DNA polymerase and the consumption of reagents. At this point the product renaturation is competing with product binding. The third stage is the plateau stage in which no more products accumulate due to a complete exhaustion of dNTPs and reagents.
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.