S

DNA Template

The template may be single- or double-stranded DNA. In a clinical sample, depending on the application, the template may be derived from the patient's genomic or mitochondrial DNA or from viruses, bacteria, fungi, or parasites that might be infecting the patient. Genomic DNA will have only one or two copies per cell equivalent of single-copy genes to serve as amplification targets. With robust PCR reagents and conditions, nanogram amounts of genomic DNA are sufficient for consistent results. For routine clinical analysis, 100 ng to 1 ^g of

Advanced Concepts

Reagent systems that are designed to amplify targets optimally with high GC content are available. These systems incorporate an analog of dGTP, deazaGTP, to destabilize secondary structure. Deaza-GTP interferes with EtBr staining in gels and is best used in procedures with other types of detection, such as autoradiography.

cytosine, is added to the synthesis reaction in concentrations sufficient to support the exponential increase of copies of the template. The four dNTP concentrations should be higher than the estimated Km of each dNTP (10-15 mM, the concentration of substrate at half maximal enzyme velocity). Standard procedures require 0.1-0.5 mM concentrations of each nucleotide. Substituted or labeled nucleotides, such as deaza GTP, may be included in the reaction for special applications. These nucleotides will require empirical optimization for best results.

DNA Polymerase

Automation of the PCR procedure was greatly facilitated by the discovery of the thermostable enzyme, Taq polymerase. When Kary Mullis first performed PCR, he used the DNA polymerase isolated from E. coli. Every time the sample was denatured, however, the high temperature denatured the enzyme. Thus, after each round of denaturation, additional E. coli DNA polymerase had to be added to the tube. This was labor-intensive and provided additional opportunities for the introduction of contaminants into the reaction tube. The Taq polymerase was isolated from the thermophilic bacterium, Thermus aquaticus.

Using an enzyme derived from a thermophilic bacterium meant that the DNA polymerase could be added once at the beginning of the procedure and it would maintain its activity throughout the heating and cooling cycles. Other enzymes, such as Tth polymerase from Thermus thermophilus, were subsequently exploited for laboratory use. Tth polymerase also has reverse transcriptase activity so that it can be used in reverse transcriptase PCR (RT-PCR, see below) where the starting material is an RNA template. The addition of proofreading enzymes, e.g., Vent polymerase allows Taq or Tth

Advanced Concepts

Note the nomenclature for the enzymes is derived from the organism from which the enzyme comes, similar to the nomenclature for restriction enzymes. For example, for the Taq polymerase, the "T" comes from the genus name, Thermus, and the "aq"comes from the species name, aquaticus.

polymerase to generate large products over 30,000 bases in length.

Cloning of the genes coding for these polymerases has led to modified versions of the polymerase enzymes, such as the Stoffel fragment lacking the N-terminal 289 amino acids of Taq polymerase and its inherent 3' to 5' exonuclease activity4. The half-life of the Stoffel fragment at high temperatures is about twice that of Taq polymerase, and it has a broader range of optimal MgCl2 concentrations (2-10 mM) than Taq. This enzyme is recommended for allele-specific PCR and for amplification of regions with high GC content. Further modified versions of the Taq enzymes retaining 3' to 5' exonuclease, but not 5' to 3' exonuclease activity, are used where high fidelity (accurate copying of the template) is important. Other variants of Taq polymerase, ThermoSequenase and T7 Sequenase, efficiently incorporate dideoxy NTPs for application to chain termination sequencing (see Chapter 10).

PCR Buffer

PCR buffers provide the optimal conditions for enzyme activity. Potassium chloride (20-100 mM), ammonium sulfate (15-30 mM), or other salts of monovalent cations are important buffer components. These salts affect the denaturing and annealing temperatures of the DNA and the enzyme activity. An increase in salt concentration makes longer DNA products denature more slowly than shorter DNA products during the amplification process, so shorter molecules will be amplified preferentially. The influence of buffer/salt conditions varies with different primers and templates.

Magnesium chloride also affects primer annealing and is very important for enzyme activity. Magnesium requirements will vary with each reaction, because each NTP will take up one magnesium atom. Furthermore, the presence of ethylenediaminetetraacetic acid (EDTA) or other chelators will lower the amount of magnesium available for the enzyme. Too few Mg2+ ions lower enzyme efficiency, resulting in a low yield of PCR product. Overly high Mg2+ concentrations promote misincor-poration and thus increase the yield of nonspecific products. Lower Mg2+ concentrations are desirable when fidelity of the PCR is critical. The recommended range of MgCl2 concentration is 1-4 mM, in standard reaction conditions. If the DNA samples contain EDTA or other chelators, the MgCl2 concentration in the reaction mix ture should be adjusted accordingly. As with other PCR components, the optimal conditions are established empirically. Tris buffer and accessory buffer components are also important for optimal enzyme activity and accurate amplification of the intended product; 10 mM Tris-HCl maintains the proper pH of the buffer, usually between pH 8 and pH 9.5.

Accessory components are sometimes used to optimize reactions. Bovine serum albumin (10-100 ^g/mL) binds inhibitors and stabilizes the enzyme. Dithiothreitol (0.01 mM) provides reducing conditions that may enhance enzyme activity. Formamide (1%-10%) added to the reaction mixture will lower the denaturing temperature of DNA with high secondary structure, thereby increasing the availability for primer binding. Chaotropic agents such as triton X-100, glycerol, and dimethyl sulfoxide added at concentrations of 1%-10% may also reduce secondary structure to allow polymerase extension through difficult areas. These agents contribute to the stability of the enzyme as well.

Enzymes are usually supplied with buffers optimized by the manufacturer. Commercial PCR buffer enhancers of proprietary composition may also be purchased to optimize difficult reactions. Often, the buffer and its ingredients are mixed with the nucleotide bases and stored as aliquots of a master mix. The enzyme, target, and primers are then added when necessary. Dedicated master mixes will also include the primers, so that only the target sequences must be added.

Thermal Cyclers

The first PCRs were performed using multiple water baths or heat blocks set at the required temperatures for each of the steps. The tubes were moved from one temperature to another by hand. In addition, before the discovery of thermostable enzymes, new enzyme had to be added after each denaturation step, further slowing the procedure and increasing the chance of error and contamination.

Automation of this tedious process was greatly facilitated by the availability of the heat stable enzymes. To accomplish the PCR, then, an instrument must only manage temperature according to a scheduled amplification program. Thermal cyclers or thermocyclers were thus designed to rapidly and automatically ramp (change) through the required incubation temperatures, holding at each one for designated periods.

Early versions of thermal cyclers were designed as heater/coolers with programmable memory to accept the appropriate reaction conditions. Compared with modern models, the available memory for recording the reaction conditions was limited, and sample capacity was small. Wax or oil (vapor barriers) had to be added to the reactions to prevent condensation of the sample on the tops of the tubes during the temperature changes. The layer of wax or oil made subsequent sample handling more difficult. Later, thermal cycler models were designed with heated lids that eliminated the requirement for vapor barriers.

There are numerous manufacturers of thermal cyclers. These instruments differ in heating and/or refrigeration systems as well as the programmable software within the units. Samples may be held in open chambers for air heating and cooling or in sample blocks designed to accommodate 0.2-mL tubes, usually in a 96 well format. Some models have interchangeable blocks to accommodate amplification in different sizes and numbers of tubes or slides. A cycler may run more than one block independently at the same time so that different PCR programs can be performed simultaneously. Rapid PCR systems are designed to work with very small sample volumes in chambers that can be heated and cooled quickly by changing the air temperature surrounding the samples. Real-time PCR systems are equipped with fluorescent detectors to measure PCR product as the reaction proceeds. PCR can also be performed in a microchip device in which 1-2 ^L samples are forced through tiny channels etched in a glass chip, passing through temperature zones as the chip rests on a specially adapted heat block.5

For routine PCR in the laboratory, an appropriate amount of DNA that has been isolated from a test specimen is mixed with the other PCR components, either separately or as part of a master mix in 0.2-0.5-mL tubes. Most thermal cyclers take thin-walled tubes, 0.2-mL-tube strips or 96 well plates. Preparation of the specimen for PCR is often referred to as pre-PCR work. To avoid contamination (see below), it is recommended that the pre-PCR work be done in a designated area that is clean and free of amplified products. The sample tubes are then loaded into the thermal cycler. The computer is programmed with the temperatures and times for each step of the PCR cycle, the number of cycles to complete (usually 30-50), the conditions for ramping from step to step, and the temperature at which to hold the tubes once all of the cycles are complete. The technologist starts the run and walks away until it is complete.

After the PCR, a variety of methods are used to analyze the PCR product. Most commonly, the PCR product is analyzed by gel or capillary electrophoresis. Depending on the application, the size, presence, or intensity of PCR products is observed on the gel. An example of the results from a PCR run is shown in Figure 7-11.

Nucleic Acid Amplification Chapter 7 131 Controls for PCR

As with any diagnostic assay, running the correct controls in PCR is essential for maintaining and ensuring the accuracy of the assay. With every PCR run, the appropriate controls must be included. Positive controls ensure that the enzyme is active, the buffer is optimal, the primers are priming the right sequences, and the thermal cycler is cycling appropriately. A negative control without DNA (also called a contamination control or reagent

Molecular weight markers

Reagent blank

PCR product

Reagent blank

Molecular weight markers

PCR product

(misprime)

(primer dimers)

■ Figure 7-11 Example of PCR products after resolution on an agarose gel and staining with ethidium bromide. Molecular weight markers in lane 1 are used to estimate the size of the PCR product. The intended product is 100 bp. Artifactual primer dimers in every lane and a misprimed product in lane 7 can also be observed. Absence of products in lane 8 confirms that there is no contamination in the master mix.

(misprime)

(primer dimers)

■ Figure 7-11 Example of PCR products after resolution on an agarose gel and staining with ethidium bromide. Molecular weight markers in lane 1 are used to estimate the size of the PCR product. The intended product is 100 bp. Artifactual primer dimers in every lane and a misprimed product in lane 7 can also be observed. Absence of products in lane 8 confirms that there is no contamination in the master mix.

blank) ensures that the reaction mix is not contaminated with template DNA or amplified products from a previous run. A negative control with DNA that lacks the target sequence (negative template control) ensures that the primers are not annealing to unintended sequences of DNA. In some applications of PCR, an internal control is included. In this type of control (amplification control), a second set of primers and an unrelated target are added to the reaction mix to demonstrate that the reaction is working even if the test sample is not amplified. Amplification controls are performed, preferably in the same tube with the test reaction, although it is acceptable to perform the amplification control on a duplicate sample. This type of control is most important when PCR results are reported as positive or negative, by which "negative" means that the target sequences are not present. The amplification control is critical to distinguish between a true negative for the sample and an amplification failure (false-negative).

Control of PCR Contamination

Contamination is a significant concern for methods that involve target amplification by PCR. The nature of the amplification procedure is such that, theoretically, a single molecule will give rise to product. This is critical in the clinical laboratory where results may be interpreted based on the presence, absence, size, or amount of a PCR product. With modern reagent systems designed for robust amplification of challenging specimens, such as paraffin embedded tissues or samples with low cell numbers, the balance between aggressive amplification of the intended target and avoidance of a contaminating template is delicate. For this reason, contamination control is of utmost importance in designing a PCR procedure and laboratory setup.

Although genomic DNA is a source of spurious PCR targets, the major cause of contamination is PCR products from previous amplifications. Unlike the relatively large and scarce genomic DNA, the small, highly concentrated PCR product DNA can aerosol when tubes are uncapped and when the DNA is pipetted. This PCR product is a perfect template for primer binding and amplification in a subsequent PCR using the same primers. Contamination control procedures, therefore, are mainly directed toward eliminating PCR product from the setup reaction.

Contamination is controlled both physically and chemically. Physically, the best way to avoid PCR carryover is to separate the pre-PCR areas from the post-PCR analysis areas. Positive airflow, air locks, and more extensive measures are taken by high throughput laboratories that process large numbers of samples and test for a limited number of amplification targets. Most laboratories can separate these areas by assigning separate rooms or using isolation cabinets. Equipment, including laboratory gowns and gloves, and reagents should be dedicated to either pre- or post-PCR. Items can flow from the pre- to the post-PCR area but not in the opposite direction without decontamination.

Ultraviolet (UV) light has been used to decontaminate and maintain pre-PCR areas. UV light catalyzes single-and double-strand breaks in the DNA that will then interfere with replication. Isolation cabinets are equipped with UV light sources that are turned on for about 20 minutes after the box has been used. The effectiveness of UV light may be increased by the addition of psoralens to amplification products after analysis. Psoralens intercalate between the bases of double-stranded DNA, and in the presence of long-wave UV light they covalently attach to the thymidines, uracils, and cytidines in the DNA chain. The bulky adducts of the psoralens prevent denaturation and amplification of the treated DNA.

The efficiency of UV light treatment for decontamination depends on the wavelength, energy, and distance of the light source. Care must be taken to avoid skin or eye exposure to UV light. UV light will also damage some plastics, so that laboratory equipment may be affected by extended exposure. Although convenient, the efficiency of UV treatment may not be the most effective deconta-minant for every procedure.6-8

A widely used method for decontamination and preparation of the workspace is 10% bleach (7 mM sodium hypochlorite). Frequently wiping bench tops, hoods, or any surface that comes in contact with specimen material with dilute bleach or alcohol removes most DNA contamination. As a common practice in forensic work, before handling evidence or items that come in contact with evidence, gloves are wiped with bleach and allowed to air-dry.

Another widely used chemical method of contamination control is the dUTP-UNG system. This requires substitution of dTTP with dUTP in the PCR reagent master

Advanced Concepts

In addition to breaking the sugar phosphate backbone of DNA, UV light also stimulates covalent attachment of adjacent pyrimidines in the DNA chain, forming pyrimidine dimers. These boxy structures are the source of mutations in DNA in some diseases of sun exposure. DNA repair systems remove these structures in vivo. Loss of these repair systems is manifested in diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichoth-iodystrophy.83 Psoralen in combination with UV light is an established treatment for psoriasis and other skin diseases.84,85

mix, which will result in incorporation of dUTP instead of dTTP into the PCR product. Although some polymerase enzymes may be more or less efficient in incorporation of the nucleotide, the dUTP does not affect the PCR product for most applications. At the beginning of each PCR, the enzyme uracil-N-glycosylase (UNG) is added to the reaction mix. This enzyme will degrade any nucleic acid containing uracil, such as contaminating PCR product from previous reactions. A short incubation period is added to the beginning of the PCR amplification program, usually at 50oC for 2-10 minutes to allow the UNG enzyme to function. The initial denaturation step in the PCR cycle will degrade the UNG before synthesis of the new products. Note that this system will not work with some types of PCR, such as nested PCR (discussed below), because a second round of amplification requires the presence of the first round product. The dUTP-UNG system is used routinely in real-time PCR procedures in which contamination control is more important because the contaminant will not be distinguishable from the desired amplicon by gel electrophoresis.

Prevention of Mispriming

As shown in Figure 7-11, PCR products are analyzed for size and purity by electrophoresis. The amplicon size should agree with the size determined by the primer placement. For instance, if two 20 b primers were designed to hybridize to sequences flanking a 100 bp tar get, the amplicon should be 140 bp in size. Much larger or smaller amplicons are due to mispriming or primer dimers or other artifacts of the reaction. For some procedures, these artifacts do not affect interpretation of results and, as long as they do not compromise the efficiency of the reaction, can be ignored. For other purposes, however, extraneous PCR products must be avoided or removed.

Misprimes are initially averted by good primer design and optimal amplification conditions. Even with the best conditions, however, misprimes can occur during preparation of the reaction mix. This is because Taq polymerase has some activity at room temperature. While mixes are prepared and transported to the thermal cycler, the primers and template are in contact at 22o-25oC, a condition of very low stringency (see Chapter 6 for a discussion of stringency). In these conditions, the primers can bind sequences other than their exact complements in the target. These misprimed products, then, are already present before the amplification program begins. Even using well-designed primers and optimizing amplification conditions, however, does not prevent all mispriming. To further prevent mispriming, hot-start PCR can be used.

Hot-start setup is done in three ways. In one approach, the reaction mixes are prepared on ice and placed in the thermal cycler after it has been prewarmed to the denatu-ration temperature. A second way to perform hot-start PCR is to use a wax barrier. A bead of wax is placed in the reaction tubes with all components of the reaction mix except enzyme and template. The tube is heated to 1000C to melt the wax and then cooled to room temperature. The melted wax will float to the top of the reaction mix in the tube and congeal into a physical barrier as it cools. The template and enzyme are then added on top of the wax barrier. When the tubes are placed in the thermal cycler, the wax will melt at the denaturation temperature, and the primers and template will first come in contact at the proper annealing temperature. The wax also serves as an evaporation barrier as the reaction proceeds. After amplification, however, the wax barrier must be punctured to gain access to the PCR products.

The third and most frequently used hot-start method is the use of sequestered enzymes, such as AmpliTaq Gold (Applied Biosystems), Platinum Taq (Invitrogen), JumpStart Taq (Sigma), and numerous others. These enzymes are either supplied in inactive form or the enzyme is inactivated by monoclonal antibodies or by other proprietary methods. Regardless of the inactivation mechanism, the enzyme is inactive until it is activated by heat in the first denaturation step of the PCR program, preventing any primer extension during reagent mix preparation.

PCR Product Cleanup

Even the best procedures sometimes result in extraneous products. Sequence limitations to primer design or reaction conditions may not completely prevent primer dimers or misprimes. These unintended products are unacceptable for analytical procedures that demand pure product, such as sequencing or some mutation analyses (see Chapters 9 and 10). A direct way of obtaining clean PCR product is to resolve the amplification products by gel electrophoresis and then cut the desired bands from the gel and elute the PCR product. The gel slice can be digested with enzymes such as p-agarase (New England BioLabs) or iodine (Fig. 7-12). The agarase enzyme digests the agarose polymer and releases the DNA into solution for further purification.

Residual components of the reaction mix, such as leftover primers and unused nucleotides, also interfere with some post-PCR applications. Moreover, the buffers used for the PCR may not be compatible with post-PCR procedures. Amplicons free of PCR components are most frequently and conveniently prepared using spin columns (Fig. 7-13) or silica beads. The DNA binds to the column, and the rest of the reaction components are rinsed away by centrifugation. The DNA can then be eluted. Although

■ Figure 7-12 After gel electrophoresis, the gel band of PCR product is excised with a clean scalpel or spatula. The gel is disintegrated by centrifugation through a sieve, releasing the DNA. The DNA in solution can then be separated from the gel fragments, precipitated with alcohol and pelleted by a second centrifugation.

■ Figure 7-12 After gel electrophoresis, the gel band of PCR product is excised with a clean scalpel or spatula. The gel is disintegrated by centrifugation through a sieve, releasing the DNA. The DNA in solution can then be separated from the gel fragments, precipitated with alcohol and pelleted by a second centrifugation.

columns or beads provide better recovery than gel elu-tion, they may not completely remove residual primers.

Addition of shrimp alkaline phosphatase (SAP) in combination with exonuclease I (Exol) is an enzymatic method for removing nucleotides and primers from PCR products prior to sequencing or mutational analyses. During a 15-minute incubation at 37°C, SAP dephospho-rylates nucleotides, and Exol degrades primers. The enzymes must then be removed by extraction or inactivated by heating at 80°C for 15 minutes. This method is convenient as it is performed in the same tube as the PCR. It does not, however, remove other buffer components.

In some post-PCR methods, such a small amount of PCR product is added to the next reaction that residual components of the amplification are of no consequence, so that no further clean up of the PCR product is required. The choice of clean-up procedure or whether clean up is necessary at all will depend on the application.

PCR Modifications

PCR today has been adapted for various applications. Several modifications are used in the clinical laboratory. Of the large (and increasing numbers) of PCR modifications, following is a description of those in standard use in the clinical molecular laboratory. These methods are capable of detecting multiple targets in a single run (multiplex PCR), using RNA templates (reverse transcriptase PCR), or such amplified products as templates (nested PCR) and quantitating starting template (quantitative PCR, or real-time PCR).

Multiplex PCR

More than one primer pair can be added to a PCR so that multiple amplifications are primed simultaneously, resulting in the formation of multiple products. Multiplex PCR is especially useful in typing or identification analyses. Individual organisms, from viruses to humans, can be identified or typed by observing a set of several PCR products at once. Pathogen typing and forensic identification kits contain multiple sets of primers that amplify polymorphic DNA regions. The pattern of product sizes will be specific for a given type or individual.

Multiple organisms have been the target of multiplex PCR in clinical microbiology laboratories.911 One respiratory sample, for example, can be used to test for the presence of more than one respiratory virus.12 Organisms

Primer

■ Figure 7-13 PCR product cleanup in spin columns (left) removes residual components in the PCR mix. Amplicon DNA binds to a silica matrix in the column while the buffer components flow through during centrifugation. The column is then inverted, and the DNA is eluted by another centrifugation in low salt (Tris-EDTA) buffer.

Primer

■ Figure 7-13 PCR product cleanup in spin columns (left) removes residual components in the PCR mix. Amplicon DNA binds to a silica matrix in the column while the buffer components flow through during centrifugation. The column is then inverted, and the DNA is eluted by another centrifugation in low salt (Tris-EDTA) buffer.

that cause sexually transmitted diseases can be targeted in multiplex PCR using one genital swab.13 In a slightly different approach to testing for multiple targets, one set of primers can detect an infectious organism, and a second set can detect the presence of a gene that makes that organism resistant to a particular antimicrobial agent. This has been performed and published for methicillin-resistant Staphylococcus aureus.14

Multiplex PCR reagents and conditions require more complex optimization. Often, target sequences will not amplify with the same efficiency, and primers may interfere with other primers for binding to the target sequences. The conditions for the PCR must be adjusted for the optimal amplification of all products in the reaction. This may not be possible in all cases.

Multiplexing primers is useful, not only to detect multiple targets but also to confirm accurate detection of a single target. Internal amplification controls are often multiplexed with test reactions that are interpreted by the presence or absence of product. The control primers and targets must be chosen so that they do not interfere or compete with the amplification of the test region. Internal amplification controls are the ideal for positive/negative qualitative PCR tests.

Reverse Transcriptase PCR

Amplification by PCR requires a double-stranded DNA template. If the starting material for a procedure is RNA, it must first be converted to double-stranded DNA. This is accomplished through the action of reverse transcriptase (RT), an enzyme isolated from RNA viruses. This enzyme first copies the RNA single strand into a RNADNA hybrid strand and then uses a hairpin formation on the end of the newly synthesized DNA strand to prime synthesis of the homologous DNA strand, replacing the original RNA in the hybrid. The resulting double-stranded DNA is called cDNA for copy or complementary DNA. This product is adequate for PCR.

Like other DNA polymerases, reverse transcriptase requires priming. Specific primers, oligo dT primers or random hexamers, are most often used to prime the synthesis of the initial DNA strand. Specific primers will prime cDNA synthesis only from transcripts complementary to the primer sequences. The yield of cDNA will be relatively low using this approach but highly specific for the target of interest. Oligo dT primers are 18-b-long single-stranded polyT sequences that will prime cDNA synthesis only from messenger RNA with poly A tails. Yield of cDNA will be higher with oligo dT primers and should include all mRNA in the specimen. The highest yield of cDNA is achieved with random hexamers or decamers. These are 6-10-b-long single-stranded oligomers of random sequences. The 6-10-b sequences will match sequences in the target RNA with some frequency. Random priming will generate cDNA from all RNA (and DNA) in the specimen. For all strategies of cDNA preparation, the specificity of the final product is still determined by the PCR primers.

RT PCR is used to measure RNA expression profiles, to detect rRNA, to analyze gene regions interrupted by long introns, and to detect microorganisms with RNA genomes. For gene expression analysis, the amount of cDNA reflects the amount of transcript in the preparation. In other applications, genes that are interrupted by long introns can be made more available for consistent amplification using cDNA versions lacking the interrupting sequences. cDNA is often used for sequencing because the sequence of the coding region can be determined without long stretches of introns complicating the analysis. The detection of RNA viruses such as Coronavirus, which is responsible for severe acute respiratory syndrome, can be accomplished using RT PCR.15

RT PCR was originally performed in two steps: cDNA synthesis and PCR. Tth DNA polymerase, which has RT activity and proprietary mixtures of RT and sequestered (hot-start) DNA polymerase, are components of one-step RT PCR procedures.16 These methods are more convenient than the two-step procedure, as RNA is added directly to the PCR. The amplification program is modified to include an initial incubation of 45°-50°C for 30-60 minutes, during which RT makes cDNA from RNA in the sample. The RT activity will then be inactivated in the first denaturation step of the PCR procedure.

Although RT PCR is a widely used and important adjunct to molecular analysis, it is subject to the vulnerabilities of RNA degradation. As with other procedures that target RNA, specimen handling is important for accurate results. Methods have been described for the RT PCR amplification of challenging specimens, such as paraffin embedded tissues; however, fixed specimens are difficult to analyze consistently.17

Nested PCR

Increased sensitivity offered by the PCR is very useful in clinical applications as clinical specimens are often limited in quantity and quality. The low level of target and the presence of interfering sequences can prevent a regular PCR from working with the reliability required for clinical applications. Nested PCR is a modification that increases the sensitivity and specificity of the reaction.18-21

In nested PCR, two pairs of primers are used to amplify a single target in two separate PCR runs. The second pair of primers, designed to bind slightly inside of the binding sites of the first pair, will amplify the product of the first PCR in a second round of amplification. The second amplification will specifically increase the amount of the intended product. In seminested PCR, one of the second-round primers is the same as the first-round primer. Nested and seminested procedures increase specificity and sensitivity of the PCR (Fig. 7-14).

Several variations of nested and seminested PCR have been devised. For example, as shown in Figure 7-14, the first-round primers can have 5' sequences added (5' tails) complementary to sequences used for second-round primers. This tailed primer method is valuable for multi-

First round product

Second round product

■ Figure 7-14 Variations of nested PCR using nested primers and seminested second-round primers (left) and tailed first-round primers (right).

plex procedures in which multiple first-round primers may differ in their binding efficiencies. Due to the tailed primers, sequences complementary to a single set of second-round primers are added to all of the first-round products. In the second round, then, all products will be amplified with the same primers and equal efficiency. Although this tailed primer procedure increases sensitivity in multiplex reactions, it does not increase specificity.

Real-Time (Quantitative) PCR

Standard PCR procedures will indicate if a particular target sequence is present in a clinical sample. For some situations, though, the clinician is also interested in how much of the target sequence is present. Several approaches have been taken to estimate the amount of starting template by PCR. By the nature of amplification, however, calculating direct quantities of starting material becomes complex. Strategies to quantitate starting material by quantitating the end products of PCRs have utilized internal controls, i.e., known quantities of starting material, that are co-amplified with the test template. These types of assays, however, suffer from primer incompatibilities and inconsistent results. Another approach is to add competitor templates at several known levels to assess the amount of test material by preferential amplification over a known amount of competitor.22 These assays are also at times unreliable and inconsistent when test and internal control templates differ by more than 10-fold. They are most accurate with a 1:1 ratio of test and internal control, requiring analysis of multiple dilutions of controls for optimal results.

A very useful modification of the PCR process is realtime or quantitative PCR (qPCR).23,24 This method was initially performed by adding ethidium bromide (EtBr) to a regular PCR. Because EtBr intercalates into double-stranded DNA and fluoresces, it can be used to monitor the accumulation of PCR products during the PCR in real time, i.e., as it is made. The advantage of this method over standard PCR is the ability to determine the amount of starting template accurately. These quantitative measurements are performed with the ease and rapidity of standard PCR without tedious addition of competitor templates or multiple internal controls. A growing number of clinically significant parameters, such as copy numbers of diseased human genes, viral load, tumor load, and the effects of treatment, are measured easily with this method.25-27

The rationale of qPCR is illustrated in Figure 7-15. If the target copy number in a PCR were graphed versus the number of cycles, the results would be an exponential curve where the number of target copies = 2N, N being the number of cycles. If the copy number is measured by detectable fluorescence as shown in the figure, the curve looks similar to a bacterial growth curve, with a lag phase, an exponential (log) phase, a linear phase, and a stationary phase.

In contrast to real-time PCR, analysis of PCR product by the standard method occurs at the end of the PCR stationary phase (endpoint analysis). Exhaustion of reaction components and competition between PCR product and primers during the annealing step slow the PCR product accumulation after the exponential phase of growth until it finally plateaus. In the endpoint analysis, products of widely different starting template amounts are tested at the plateau where they are all the same (observe the ends of the amplification curves shown in Figure 7-15A.). Using the fluorescent signal to detect the growing target copy number during the amplification process, analysis in real-time PCR is performed in the exponential phase of growth where the accumulation of fluorescence is inversely proportional to the amount of starting template. With 10-fold dilutions of known positive standards, a relationship between the starting target copy number and the cycle number at which fluorescence crosses a threshold amount of fluorescence can be established.

The PCR cycle at which sample fluorescence crosses the threshold is the threshold cycle, or CT. Plotting the target copy number of the diluted standards against CT for each standard generates the graph shown in Figure 7-15B. Once this relationship is established, the starting amount of an unknown specimen can be determined by the cycle number at which the unknown crosses the fluorescence threshold.

Advanced Concepts

The optimal threshold level is based on the background or baseline fluorescence and the peak fluorescence in the reaction. Instrument software is designed to set this level automatically. Alternatively, the threshold may be determined and set manually.

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