Pcr Components

3.1. NUCLEIC ACID TEMPLATES FOR PCR Polymerase chain reaction amplifies specific sequences from DNA templates (either genomic DNA or cDNA derived from RNA) that can be prepared from various sample sources (Fig. 2). This DNA could be genomic DNA isolated directly from experimental or patient material or it could be cDNA that has been synthesized from DNA or RNA templates by polymerase or reverse transcriptase enzymes. Various sources of biological material can be utilized for the preparation of PCR templates. In the research lab, PCR templates can be derived from cultured cells or tissue sections. Clinical specimens could be derived from various bodily fluids (such as blood and amniotic fluid), as well as surgical samples (such as frozen tumors) (7). Forensic specimens could be derived from hair samples, blood, or semen (8). In addition to fresh specimens, DNA derived from fixed tissues (paraffin-embedded specimens) can be used routinely (with rare exception) in PCR applications (9). In fact, PCR analysis has been applied to prehistoric DNA derived from fossilized biological materials (10).

Most PCR reactions amplify small targets in the DNA sequence (200-1000 bp). Thus, high-molecular-weight DNA is not necessary, and highly fragmented DNA (like that obtained from paraffin tissue sections) can be effectively utilized. However, certain tissue fixatives (like Bouin's solution, which contains picric acid) and treatments (like tissue decalcification) can harm DNA and render tissues useless for molecular analysis. These simple guidelines should be kept in mind when decisions are made regarding sources of DNA templates, especially when archived specimens are being considered for use in a study. Preparation of RNA typically requires fresh or frozen tissues, although techniques are being developed that promise to yield analyzable RNA from paraffin tissue sections.

3.2. DNA POLYMERASE ENZYMES IN PCR A DNA polymerase enzyme is required for DNA synthesis during the primer extension step of PCR. The contemporary PCR employs Taq DNA polymerase (isolated from T. aquaticus) (11). Taq polymerase exhibits 5'^3' polymerase activity, 5'^3' exonu-clease activity, thermostability, and optimum performance at 70-80°C (5,12). Temperature, pH, and ion concentrations (Mg2+) can influence the activity of Taq polymerase. The halflife of Taq activity at 95°C is approx 40-60 minutes (13,14), and extremely high denaturation temperatures (>97°C) will significantly reduce its active lifetime. Because time at temperature represents the critical parameter for maintenance of Taq activity, lowering of the denaturation temperature or reduction in the denaturation time can prolong the activity of the enzyme during PCR. The optimum pH for a given PCR will be between 8.0 and 10.0, but it must be determined empirically. The typical PCR will be carried out in a buffer (usually Tris-Cl) of pH 8.3. Taq polymerase requires divalent cations in the form of Mg2+. Lower divalent cation (Mg2+) concentrations decrease the rate of dissociation of enzyme from the template by stabilizing the enzyme-nucleic acid interaction (15). Most

Archival Material Surgical Samples

(paraffin-embedded) (frozen tissues)

I Cultured j.

I ^Cells^RNA

DNA ^Reverse Transcription cDNA

Fig. 2. Sources of nucleic acid templates for PCR and derived analytical methods. PCR analysis can be performed using DNA or cDNA (RNA), which can be prepared from a variety of sources, including those shown and others (such as forensic samples). PCR forms the basis numerous analytical techniques, some of which are based on differential amplification of target sequences. Other PCR-based methods involve advanced applications for analysis of PCR products (such as electrophoretic methods for mutation detection).

Fig. 2. Sources of nucleic acid templates for PCR and derived analytical methods. PCR analysis can be performed using DNA or cDNA (RNA), which can be prepared from a variety of sources, including those shown and others (such as forensic samples). PCR forms the basis numerous analytical techniques, some of which are based on differential amplification of target sequences. Other PCR-based methods involve advanced applications for analysis of PCR products (such as electrophoretic methods for mutation detection).

PCR mixtures will contain at least 1.5 mM MgCl2. However, a MgCl2 titration is recommended for any new template-primer combination.

Although Taq DNA polymerase is ideal for routine PCR, there are several other thermostable DNA polymerases with unique qualities (16) that make them useful for special PCR applications such as amplification of long pieces of DNA or high-fidelity amplification. Taq polymerase displays an error rate of approx 1 x 10-4 to 2 x 10-5 errors/bp (including both base substitutions and frameshift errors) (17,18). Vent polymerase (also known as Tli polymerase from Thermococcus litoralis) exhibits an error rate of 2 x 10-5 to 5 x 10-5 errors/bp (19,20). In addition to its increased fidelity over Taq polymerase, Vent polymerase has a half-life of approx 400 min at 95°C (19), making it a very hardy enzyme. The highest-fidelity polymerase for PCR applications is Pfu polymerase (from Pyrococcus furiosus), which displays an error rate of 1.5 x 10-6 errors/bp (21). The increased fidelity observed with Vent and Pfu polymerases (compared to Taq) is related to the 3'^5' exonuclease proofreading activity that is inherent to these enzymes. Taq polymerase lacks 3'^5' exonuclease proofreading activity. For high-fidelity applications, such as PCR amplification of DNA for sequence determination, the use of a higher-fidelity poly-merase is preferred. Some manufacturers also produce blends of DNA polymerases for specialized PCR applications. One of these is referred to as Expand High Fidelity polymerase blend, which contains Taq (for polymerase activity) and Pwo (for proofreading). This blend is used in long PCR applications that require a higher fidelity than Taq can provide when used alone.

3.3. DESIGN OF OLIGODEOXYNUCLEOTIDE PRIMERS FOR PCR Design of oligonucleotide primers could be the most critical factor in determining the success of a PCR. Effective oligonucleotide primers for PCR are highly specific, free of secondary structure, and form stable duplexes with target sequences. Basically, four parameters need to be considered when designing a set of oligonucleotide primers: (1) size of the target sequence to be amplified, (2) the location of the target sequence within the overall genomic DNA (or cDNA) sequence, (3) secondary structure within the target and flanking regions, and (4) specificity of amplification. The size of the target sequence should be selected such that the PCR products produced range from 400 to 2000 bp in length. Products less than 400 bp in length are difficult to resolve using standard agarose gel electrophoresis techniques and might be obscured by excess primers or PCR artifacts. Products larger than 2000 bp might be amplified less efficiently because of the limited processivity of Taq polymerase (22). Primer length can influence target specificity and efficiency of hybridization. A long oligonucleotide primer might be more specific for the target sequence, but it is less efficient at hybridization, whereas a short oligonucleotide primer is efficient at hybridization but less specific for the target sequence (23). As a general guideline, oligonucleotide primers should be 17-30 nucleotides in length. Whenever possible, both primers should be of the same length because oligonucleotide primer length influences the calculated optimal annealing temperature for a specific primer. The base composition of the oligonucleotide primers is also important, because annealing temperature is governed in part by the G + C content of the primers. Ideally, G + C content should be 50-60%, and the percent G + C should be the same or very similar for both oligonucleotide primers in any given primer pair. The 3'-terminus of an oligonucleotide primer should contain a G, C, GC, or CG. Given the tighter hydrogen-bonding between G: C pairs, the presence of these nucleotides at the 3' end of the oligonucleotide primer reduces the possibility for excessive breathing of the target-primer duplex, increasing the efficiency of primer extension. However, runs of C or G at the 3' end of an oligonucleotide primer should be avoided, as these can cause nonspecific hybridization with GC-rich sequences. Repetitive or palindromic sequences should be avoided in an oligonucleotide primer, and primer pairs should not contain sequences that are complementary to each other. Likewise, oligonucleotide primer pairs should not hybridize elsewhere in the gene being amplified or in other sequences contained within the genome. Web-based tools are available for easily analyzing the characteristics and properties of selected oligonucleotide primers (http://www.basic.nwu.edu/biotools/oligocalc.html).

When designing oligonucleotide primers for reverse tran-scriptase (RT)-PCR applications, all of the general guidelines for oligonucleotide primer design apply, but a few additional special considerations are needed. The precise location of the target sequence within the gene of interest must be chosen carefully. This is related to the fact that reverse transcription of mRNA templates using oligo(dT) as a primer often fails to generate full-length cDNA transcripts. Thus, amplification of target sequences that are distant from the 3' end of the mRNA might be difficult to accomplish. Selection of target sequences that are within 1 kb of the 3'-terminus of the mRNA will ensure successful PCR amplification in most cases. However, if amplification of target sequences in the 5' region of a gene is required, there are specialized techniques that can facilitate this amplification. One option is to use a gene-specific oligonu-cleotide primer during the reverse-transcription step, which will give rise to a cDNA template that includes the target sequence of interest. Another method for obtaining 5'-terminal sequence from mRNA is referred to as 5' RACE (for rapid amplification of cDNA ends) (24). RACE techniques can be applied to either the 3' or 5' ends of the mRNA transcript and are particularly useful when the mRNA sequence in that region of the gene is not known. mRNA secondary structure is another reason for the failure of RT to synthesize full-length cDNA transcripts, so regions known to have bulky secondary structure should be avoided if possible. RNA folding algorithms capable of predicting secondary structure within regions of known sequence are available (25).

The optimal annealing temperature (Tm) for a given oligonu-cleotide primer set is very important for correctly setting up an effective PCR. The melting temperature of an oligonucleotide primer is most accurately calculated using nearest-neighbor thermodynamic calculations represented in the following formula: Tm = H[S + R ln(c/4)] - 273°C + 16.6 log10[K+], where H is the enthalpy and S is the entropy for helix formation, R is the molar gas constant, and c is the concentration of primer. Using such a formula is most easily accomplished using software for primer design and analysis, including some Web-based programs (such as http://www.rnature.com/oligonucleotide.html or http://alces.med.umn.edu/rawtm.html). However, an excellent working approximation for primer melting temperature can be calculated using simplified formulas that are generally valid for oligonucleotide primers that are 18-24 bp in length. One such simple formula for calculating annealing temperature for any given primer is Tm = 69.3 + 0.41(%G + C) - (650/L), where L is the primer length in bases (26). Another formula for this calculation is T = 2(A + T) + 4(G + C), where A + T and G + C refer to the number of bases in each group. Primer annealing temperatures between 55°C and 72°C are preferred, but many standard PCR primers will have annealing temperatures of 55-65°C.

Several computer algorithms and programs have been developed to facilitate the design of primers with appropriate characteristics for PCR applications. Numerous Web-based oligonucleotide primer design programs are available, including OligoPerfect Designer (http://www.invitrogen.com), Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), and DoPrimer (http://doprimer.interactiva.de/index.html). Web-based tools are also available for designing primers that are specific for exonic and intronic sequences of genes of interest (http://ihg.gsf.de/ihg/ExonPrimer.html) and for making degenerate oligonucleotide primers (http://bioinformatics.weizmann. ac.il/blocks/codehop.html).

3.4. REACTION MIXTURES FOR PCR A typical PCR mixture will include a reaction buffer, oligonucleotide primers, Taq polymerase, and an appropriate DNA template. The PCR buffer consists of 50 mM KCl, 1.5 mM MgCl2,10-50 mM Tris-Cl (pH 8.3), and 50-200 pM dNTPs. Concentrations of KCl that are higher than 50 mM can inhibit Taq polymerase and should be avoided. However, the presence of KCl is necessary to encourage primer annealing to the template DNA. Likewise, excessive NaCl concentrations in a PCR mixture can adversely affect the activity of Taq polymerase. The amount of MgCl2 that is optimal for a given PCR must be determined empirically. However, most standard PCR can be accomplished using 1.5-2 mM MgCl2. The final concentration of dNTPs is 200 pM for a typical PCR, but some applications can be accomplished using much lower concentrations. Higher concentrations of dNTPs (or MgCl2) can encourage errors related to dNTP misincorpo-ration by Taq polymerase and should be avoided. The typical PCR mixture will include 0.2-1 pM of each oligonucleotide primer. The concentration of primers should not exceed 1 pM unless the primers employed contain a high degree of degeneracy. Taq polymerase is provided from the supplier at 5 U/pL. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol of dNTP into acid-insoluble material in 30 min under standard reaction conditions. The amount of Taq polymerase included in a PCR will depend on the reaction size (20-50 pL). A 50-pL reaction will typically require 2.5 U of enzyme activity. The amount of DNA template included in a PCR will vary with the nature of the template source and the target sequence. Amplification from genomic DNA might require as much as 100 ng of DNA for a 50-pL reaction, whereas amplification from a plasmid template might only require 5 ng of DNA. Likewise, amplification of a target sequence that corresponds to a single allele might require more template, whereas amplification of a repetitive sequence (like Alu) will require substantially less template.

The various components of the PCR mixture can be prepared in the laboratory or can be purchased from commercial sources. A prepared reaction buffer can be obtained for use with Taq polymerase (Perkin-Elmer Cetus or other suppliers of the enzyme) and other thermostable polymerase enzymes. Likewise, commercially prepared dNTP stock solutions can be purchased from several sources. Complete PCR mix-in-a-tube reaction mixtures can be purchased that contain all of the required PCR components except DNA template, oligonucleotide primers, and Taq polymerase (such as the EasyStart Micro 50 PCR mix-in-a-tube from Molecular BioProducts, http://www.mbpinc.com/). In fact, some commercially prepared PCR mixtures are supplied in a form that includes the Taq polymerase (such as PCR Master Mix from Promega, http://www.promega.com), requiring the addition of only oligonucleotide primers and DNA template. These commercially prepared reaction mixtures are extremely consistent and reliable and they work for many routine PCR applications.

3.5. REACTION MIXTURE ADDITIVES FOR PCR The inclusion of gelatin or bovine serum albumin (BSA), which could be included at concentrations up to 100 |ag/mL, can enhance the efficiency of PCR. These agents act to stabilize the polymerase enzyme. The addition of helix destabilizing chemicals might be necessary if the target sequence for PCR is known to be of high G + C content (27). For example, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), formamide, or urea could be included for this purpose. In most cases, these additives are included in the reaction mixture at 10% (w/v or v/v). These additives are thought to lower the Tm of the target sequence. Care must be used when these additives are incorporated into a PCR because high concentrations of these chemicals can adversely affect polymerase activity. For instance, concentrations of DMSO that exceed 10% can decrease Taq polymerase activity by as much as 50%. Many PCR mixtures will include nonionic detergents such as Tween-20, Triton X-100, or Nonident P40 at 0.05-0.1%. The inclusion of these detergents can increase enzyme activity for certain polymerase preparations by preventing enzyme aggregation.

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