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Multiple PCR cycles

Fig. 1 Principle of PCR technology. Technology for the amplification of a single-stranded DNA target is outlined. Start: After heat denaturation (95°C), primers are annealed at a lower temperature (e.g., 50°C). Initially, only primer A can anneal to its complementary sequence (thick red line) in the target DNA. First elongation: The optimal reaction temperature for the thermostable Taq DNA polymerase is 72°C, and the annealed primer A is elongated, in principle, all the way to the end of the target DNA, including the sequence (thick green line) complementary to primer B. Second elongation: After heat denaturation and annealing, this step generates the first specific PCR product with a defined length, containing only the sequence stretch flanked by the two primer sequences (boxed product with green arrow, blue stretch, and thick red line). Finish: Multiple copies of the specific PCR product are generated (shown as smaller schemes). Primer A (red arrow), primer B (green arrow), target DNA (black), and PCR-generated DNA (blue). (View this art in color at www.dekker.com.)

DNA polymerase. The thermostable DNA polymerases have a high temperature optimum but they are not completely inactive at room temperature, and this can lead to elongation of misannealed primers or excessive primer dimer formation. These low-temperature products can be avoided by hot-start PCR: An essential component such as polymerase or magnesium solution is added only after the ''initial template denaturation'' step, during the ''primer annealing'' step (Tables 2 and 3). Another, more convenient option is the use of inactivated DNA polymerase enzyme (commercially available, using heat-labile chemical modification, e.g., from Qiagen; or by antibodies, e.g., from Roche Diagnostics). The enzyme activation requires an extended heat treatment and, for this purpose, the ''initial template denaturation'' at 95°C can be extended to 10 min.

After solving the basic problems of the PCR procedure, one serious practical problem is the occurrence of PCR inhibition, leading to false-negative results. This can be avoided by the inclusion of a separately amplified PCR target as internal control (Fig. 2).[5] A further complication derives from the required high precision in diagnostics. This means that the occurrence of an amplicon with the expected size is not sufficient proof for the positive detection of the targeted pathogen, considering the far-reaching consequences. Therefore in all diagnostic procedures, a sequence-specific identification was and still is a mandatory additional requirement to confirm the amplification of the desired target. Two basic procedures can be applied:[6] 1) Southern hybridization involves transfer (blotting) of the PCR products (with or without prior gel electrophoresis) to nitrocellulose or nylon membranes, followed by hybridization with a labeled probe. Stringent washes remove all nonspecifically bound probes. Finally, the specifically bound, labeled probe is detected. Different types of labels can be used: direct labels are radioactive isotopes (detected with X-ray film, or phosphoimager) or fluorescent dyes (detected with flourimager). Indirect labels are biotin or digoxigenin, combined with enzymes (phosphatase or peroxidase), which carry the corresponding haptens—streptavidin for biotin or antidigoxigenin antibodies for digoxigenin. A wide variety of enzyme substrates are available, which can be converted to fluorescent or nonfluorescent dyes, or produce a chemiluminescent reaction. The multiple turnover of substrates results in much higher sensitivity of these enzyme-coupled detections. Products and detailed protocols are available from many commercial suppliers (e.g., Amersham and Roche Diagnostics). 2) Higher throughput and automation are possible by a combination of capture and detection probes. Both probe sequences must be present in the PCR amplicons. First, amplicons

Table 1 General PCR conditions

Useful range of template copies

Primers dNTP/MgCl2

Thermostable DNA polymerase

Additives

Theoretical lower limit: one single copy (30 cycles can generate up to 1 billion copies) Upper limit: 100,000 to 1 million copies

5-50 pmol 20-300 mM/1.5 mMa

0.2-5 U Taq DNA polymerase (from Thermus aquaticus; no proofreading)

Pfu DNA polymerase (from Pyrococcus furiosus; with proofreading)

Increased annealing stringency: 2-5% DMSO, 5% formamide

Increased efficiency and specificity: 1-10% glycerol, or nonionic detergents such as Tween, Nonidet, CHAPS, Triton aA surplus of free Mg + is required. Therefore higher dNTP concentrations require higher Mg + concentrations.

are hybridized to an immobilized capture probe (on membrane or paramagnetic beads), followed by hybridization with a labeled detector probe, stringent washes, and detection, as described above.

This means that the nowadays fully automated and straightforward PCR is complicated by these extra steps after PCR completion, which include the risk of spreading the multimillion-fold amplified DNA segment. If high numbers of samples are analyzed in parallel, laboratory contamination can lead to many false-positive results. Therefore in routine diagnostics, conventional PCR is now widely replaced by sequence-specific real-time PCR techniques (see ''Real-Time PCR Technology'').

Exciting applications in molecular biology derive from the use of composite primers (principle in Fig. 3), combining a 3'-terminal segment with the usual target-specific primer sequence and an unrelated 5' track that can introduce functional DNA sequence recognition sites. Examples are cleavage sites for restriction enzymes in cloning applications, or a promoter sequence of an RNA polymerase for the production of large amounts of PCR-designed RNA transcripts,[7] useful for in vitro translation, RNA processing, tRNA charging, ribozyme studies, etc. For cloning applications, it is important to keep in mind that the most widely used thermostable Taq DNA polymerase has an error rate of about 1:1000.[1] If the PCR-generated mixture of millions of amplicon molecules is used directly (e.g., in sequencing, or for the generation of RNA transcripts), the mutated molecules can be ignored. However, if PCR products are cloned, these clones are derived from individual amplicon molecules. This means that, if only one clone is analyzed, the derived sequence can have one or several mutations. Therefore multiple clones have to be sequenced, and a functional PCR construct (e.g., for overexpression of a protein) with the correct sequence has to be selected.

The aim to perform quantitative PCR analysis is a major challenge in conventional PCR. In principle, the amount of PCR product (P) is directly related to the

Table 2 General thermoprofile for three-step PCR

Number

Temperature

Time

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Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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