In the mid-1980s in California,Mullis and coworkers developed a method, the polymerase chain reaction (PCR), to amplify exponentially target sequences of DNA.11 As the name suggests,the method is a DNA polymerase-mediated chain reaction of nucleic acid amplification. Arguably, it is the single most important "invention" that has led to development of a new discipline in clinical laboratory medicine, that is, molecular pathology. Both PCR and Southern blotting are techniques used to investigate specific genomic targets. However, PCR is orders of magnitude more sensitive and much faster, permitting turnaround time of 24 hours or less. PCR lends itself to much higher test volumes than Southern blotting, a crucial point in its acceptance in the clinical laboratory setting. Opportunity for high test volumes, excellent specificity and sensitivity, and the rapid turnaround times of PCR are the principal reasons this technology has spread so quickly in clinical molecular laboratories.
In PCR, a unique sequence of the target nucleic acid of interest is chosen for amplification, for example, oncogene, invading pathogen DNA, genetic mutation. The inherent specificity of the ensuing reaction is provided by two short oligonucleotides, called PCR primers (see Figure 2-1). These short oligonucleotides serve as primers for DNA polymerase-mediated DNA synthesis using denatured target DNA as a template. The two primers are complementary to opposite strands and opposite ends of the targeted DNA template region. Usually the primers bracket
Primer 1 Cycle 1
Figure 2-1. The polymerase chain reaction. (Reprinted with permission from Tsongalis GJ, Coleman WB. Molecular Diagnostics—A Training and Study Guide. Washington, DC: AACC Press, 2002.)
the area of interest, but one type of PCR (allele-specific PCR; see below) uses primers that overlap the area of interest. Successful PCR depends on temperature cycling, and in the first step of PCR the reaction temperature is raised to 95°C to 98°C to denature the target DNA. After 10 to 60 seconds at this temperature, the temperature is reduced to about 50°C to 70°C, depending on the specific protocol, and held there for usually 10 to 60 seconds. This facilitates hybridization (annealing) between the now-denatured target and the PCR primers, and is thus called the annealing step. This hybridization event is favored over target reannealing because the PCR primers are small and present in vast molar excess, and move more rapidly in solution than larger DNA molecules.
The hybridized PCR primers form local areas of double strandedness with the template DNA, thereby serving as primers for DNA polymerase to bind and synthesize a new strand of DNA, using the target DNA as a template. Subsequent to the initial discovery of PCR, the opportunity for automating the cyclical nature of PCR was realized by using DNA polymerase from hot-spring living bacteria, Thermophilus aquaticus (hence the term "Taq polymerase"). T. aquaticus thrives at very high temperatures, and so its proteins do not denature at the high temperatures needed to denature DNA in the first step of PCR. Catalysis by Taq polymerase of a new strand of DNA proceeds at a temperature intermediate to the near-boiling temperature used for denaturation and the relatively lower temperature used for annealing. DNA polymerization occurs during this extension step, typically at 65°C to 75°C. Taken together, these three steps (denaturation, annealing, and extension) define one PCR cycle.
Temperature cycling is automated through the use of an instrument called a thermal cycler. Thermal cyclers hold small capped tubes containing the reagents needed for PCR and cycle between the temperatures needed for the different steps of the PCR.12 A single PCR tube contains template DNA (<1ng to 1 |ig), Taq DNA polymerase, two PCR primers (~15 to 30 nucleotides long), all four dNTPs, Mg2+, and buffer to maintain an elevated pH (~8.4) optimal for Taq.
The repetition of the cycles generates exponential amplification of the target DNA because each double-stranded target DNA molecule, theoretically even if there is only one, is replicated after one PCR cycle. Both the original and replicated DNA molecules are then available to function as templates for cycle 2, in true "chain reaction" style, generating another doubling, or four copies of the original target. Cycle 3 ends with eight molecules, and doubling continues with completion of each new cycle. This doubling plateaus in later cycles since reagents, usually dNTPs, become limiting. Additionally, the enzyme may not function at 100% efficiency, and so true exponential amplification is theoretical, although there is a true exponential phase of amplification.
Greater than one billion identical copies of the original target DNA region are generated after 32 cycles of PCR: 232 or more than four billion, the difference owing to the fact that unit-length amplicons are not generated until the end of the second cycle of PCR. Amplicons (PCR products) are defined as replicated target molecules created by PCR. Unit-length amplicons are those whose ends are defined by the primers. During the first cycle, the primers are extended by Taq polymerase using template DNA. The termination of this extension is undefined and a function of how far the polymerase moves down the template during the time allotted. The enzyme, therefore, moves beyond the ends of the primer-binding site on the complementary strand. After completion of the first cycle, therefore, the newly synthesized DNA molecules are greater in length than the sequence bracketed on each strand by the primers. In the second cycle, DNA molecules are synthesized from the products of the first cycle whose ends are defined by the two primers. These are so-called unit-length ampli-cons. While all of the above is true, the practical clinical laboratory difference between one- and four-billion-fold amplification is irrelevant because either number is sufficient for detection of the target, often by elec-trophoresis with EtBr visualization.
Several factors affect the specificity and sensitivity of PCR. The production of specific PCR amplicons is a function of both the complementarity of the primers to the target DNA and the annealing temperature of the PCR cycle. Heating will denature the primer from its target DNA. The temperature at which the primer melts from the target DNA varies directly with the length of the primer and the guano-sine-cytosine (GC) content of the primer, and inversely with the degree of mismatch between the primer and the target DNA. The melting temperature (Tm) of the primer is the temperature at which 50% of the primer is denatured from the target DNA. If the thermal cycler is programmed to reach an annealing temperature higher than the primer Tm, the efficiency of PCR is compromised and sensitivity decreased. In contrast, if the annealing temperature is substantially less than the primer Tm, the primer can bind to both complementary and noncomplementary DNA, resulting in reduced PCR specificity as nontarget DNA is amplified (and potentially decreased sensitivity as reaction components are used nonspecifically). Therefore, the ideal annealing temperature is slightly less than the Tm of both primers, and the primers should be designed to have a very similar Tm. The annealing temperature can be decreased with subsequent cycles during PCR in a process called "touchdown" PCR. This allows the initial cycles to produce specific products at high annealing temperatures, while later cycles amplify previously generated amplicons more efficiently using lower annealing temperatures, thereby increasing sensitivity (see also the use of touchdown PCR in multiplex PCR, below).
Taq polymerase is very sensitive to mismatches between the primer and the target DNA at the 3' end of the primer but can withstand considerable noncomplementarity at the 5' end of the primer. Numerous PCR variations have been designed to take advantage of both these facts. Taq polymerase also requires Mg2+ as a cofactor for stabilization of primer annealing. Insufficient Mg2+ decreases PCR efficiency,while too much Mg2+ stabilizes nonspecific primer annealing. Primers with a high GC content may show a narrow range of tolerance for variation from ideal PCR conditions, leading to decreased amplification or nonspecific products. This may be alleviated by using PCR additives such as dimethyl sulfoxide (DMSO) or glycerol, but the success of these additives may need to be determined empirically for different primer pairs. Another strategy to improve specificity is the use of "hot-start" PCR, in which a crucial PCR reactant such as Taq is either physically or chemically sequestered from other PCR reagents until denaturation begins. This prevents the generation of nonspecific amplification products by inhibiting the activity of Taq until after the initial PCR denaturation step.
PCR is more sensitive than Southern blot hybridization because of the amplification of the target sequence. However, the specificity of the amplified PCR product must be verified. Simple agarose gel electrophoresis coupled with EtBr staining may be used to observe the PCR product(s). When a clinical PCR protocol is established, such gels may be subjected the first time to blot hybridization with a specific probe complementary to the internal, non-primer sequence of the amplicon(s). This exercise proves that the PCR-generated band not only is the correct size and highly likely therefore to be the correct target, but also is a DNA fragment that has high or perfect homology with a known probe. For example, hybridization of a particular 302 bp PCR product band detectable on an agarose gel with a defined cytomegalovirus (CMV) DNA probe confirms that the oligonucleotide primers synthesized based on the CMV sequence and used in the PCR are recognizing CMV-specific DNA and that the PCR is indeed specific for detection of CMV. An alternative method to validate the specificity of the PCR product is to sequence the PCR product. Following this one-time validation analysis, electrophoresis alone, as opposed to blot hybridization or sequencing, may be the assay endpoint.
There have been significant commercial endeavors to automate or semiautomate high-volume PCR-based clinical tests. For example, denatured aliquots of completed PCRs can be added to microtiter plates with wells to which specific DNA probes are bound. In the presence of amplicon, for example, if the patient is infected with the pathogen of interest or a specific mutation is present, the amplicons hybridize to the bound probe and are retained in the well during washing. Subsequent biochemical reactions are used to detect labeled moieties in the amplicons ("built in" to the PCR components), facilitating colorimet-ric detection of a positive patient reaction by an automated plate reader. Absence of colored product in a well indicates a negative result for that patient specimen, provided that all positive and negative controls are within tolerance limits. This scheme has gained US Food and Drug Administration (FDA) approval for clinical PCR-based detection kits for Chlamydia trachomatis, Neisseria gonorrhoeae, HCV (qualitative) and HIV.13 (For a complete list of FDA-approved tests, go to the Resources section at http://www.amp.org/, the home page for the Association for Molecular Pathology). Subsequent generations of automated PCR instrumentation are now available that completely automate the amplification and detection process.14 The field is moving toward real-time PCR detection (described below).
Another aspect of PCR that is attractive for the clincal molecular laboratory is the ability to use relatively crude extractions of patient specimens rather than highly purified DNA. Cell lysis and subsequent DNA liberation accomplished by boiling or treatment with detergent may be sufficient to process a specimen in preparation for PCR.15 Conventional PCR-based tests may be completed with turnaround times of as short as 2 to 4 hours, while realtime PCR can be completed in 30 minutes, making this technique attractive for stat testing.
Examples of Applications of PCR
1. Detection of the diagnostic BCL2/IGH gene rearrangement in follicular lymphoma
2. Detection of Chlamydia trachomatis in urine
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