Ind) that have the ability to detect multiple targets (multiplex PCR) by using different probes labeled with specific fluorescent dyes that each have their own "unique emission spectra.
Some real-time PCR instruments also have the ability to perform melting curve analysis. This type of analysis of amplified products confirms the identification (Le., specificity) of the amplified products and/or identifies nonspecific products. Melting curve analysis can be performed with assays using hybridization probes and molecular beacons, but not-hydrolysis probes because hydrolysis probes are destroyed during the amplification process. The underlying basis of melting curve analysis is the ability of the double-stranded DNA to become single-stranded upon heating (referred to as melting or denaturation). The melting temperature, or Tm, is the temperature at which the DNA becomes single-stranded ("melts") and is dependent on its base sequence (stretches of double-stranded DNA with more cytosines and guanines require more heat [energy] to break the three hydrogen bonds between these two bases in contrast to adenine and thymidine base pairing, which has only two hydrogen bonds). By definition, the Tm is the temperature at which 50% of the DNA is single-stranded. Because the Tm of the probe from its target is specific being primarily based on probe-target base composition, amplification products can be confirmed as correct by its melting characteristics or Tm. Of significance, the Tm can also be used to distinguish base pair differences (e.g., genotypes, mutations, or polymorphisms) in target DNA thus forming the basis for many genetic testing assays because base pair mismatches due to mutations lower the Tm. With respect to real-time PCR assays, because fluorescence of single-labeled probes is reversible by breaking the hydrogen bonds between the probe and target (i.e., denaturation), the Tm can be determined by measuring fluorescence. In real-time PCR thermal cyclers, melting curve analysis is performed once amplification is finished. The temperature of the reaction vessel is lowered below the established annealing temperature of the hybridization probe or molecular beacon; this step allows the probe or beacon to anneal to its target as well as other similar DNA sequences in the reaction. As the temperature is slowly raised, the hybridization probes or molecular beacon that were hybridized to the target will separate (melt) and the fluorescent signal will decrease (Figure 8-12).
Finally, as with conventional PCR, real-time PCR assays also have the ability to quantitate the amount of target in a clinical sample. For quantitative analysis, amplification curves are evaluated. As previously discussed, amplification is monitored either through the fluorescence of double-stranded DNA-specific dyes like SYBR Green 1 or by sequence-specific probes; thus during amplification, a curve is generated. During real-time PCR, there are at least three distinct phases for these curves: (1) an initial lag phase in which no product is detected, (2) an exponential phase of amplified product detected, and a (3) plateau phase. The number of targets in the original specimen can be determined with precision when the number of cycles needed for the signal to achieve an arbitrary threshold (the portion of the curve where the signal begins to increase exponentially or logarithmically) is determined. This segment of the real-time PCR cycle is within the
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