Basic concept of the PCR

The polymerase chain reaction or PCR is one of the mainstays of molecular biology. One of the reasons for the wide adoption of the PCR is the elegant simplicity of the reaction and relative ease of the practical manipulation steps. Indeed, combined with the relevant bioinformatics resources for its design and for determination of the required experimental conditions, it provides a rapid means for DNA identification and analysis. It has opened up the investigation of cellular and molecular processes to those outside the fi eld of molecular biology.

The PCR is used to amplify a precise fragment of DNA from a complex mixture of starting material usually termed the template DNA and in many cases requires little DNA purification. It does require the knowledge of some DNA sequence information flanking the fragment of DNA to be amplified (target DNA). Using this information, two oligonucleotide primers may be chemically synthesised, each complementary to a stretch of DNA to the 3′ side of the target DNA, one oligonucleotide for each of the two DNA strands (Figure 1). It may be thought of as a technique analogous to the DNA replication process that takes place in cells since the outcome is the same: the generation of new complementary DNA stretches based upon the existing ones. It is also a technique that has replaced, in many cases, the traditional DNA cloning methods, since it fulfils the same function: the production of large amounts of DNA from limited starting material. However, this is achieved in a fraction of the time needed to clone a DNA fragment. Although not without its drawbacks, the PCR is a remarkable development that has been changing the approach of many scientists to the analysis of nucleic acids and continues to have a profound impact on core biosciences and biotechnology.

Fig1. The location of PCR primers. PCR primers designed for sequences adjacent to the region to be amplified allow a region of DNA (e.g. a gene) to be amplified from a complex starting material of genomic template DNA.

Stages in the PCR

The PCR consists of three defined sets of times and temperatures, termed steps: (i) denaturation , (ii) annealing and (iii) extension . Each of these steps is repeated 30–40 times, each repetition being termed a cycle (Figure 2). In the first cycle, the double-stranded template DNA is denatured by heating the reaction mixture to above 90 °C. Upon denaturation, the region to be specifically amplified (target) is made accessible within the complex DNA. The reaction mixture is then cooled to a temperature between 40 and 60 °C. The precise temperature is critical and each PCR system has to be defined and optimised. One useful technique for optimisation is touchdown PCR, where a programmable thermal cycler is used to incrementally decrease the annealing temperature until the optimum is derived. Reactions that are not optimised may give rise to other DNA products in addition to the specific target or may not produce any amplified products at all. The annealing step allows the hybridisation of the two oligonucleotide primers, which are present in excess, to bind to their complementary sites that flank the target DNA. The annealed oligonucleotides act as primers for DNA synthesis, since they provide a free 3′-hydroxyl group for DNA polymerase. The DNA synthesis step is termed extension and is carried out by a thermostable DNA polymerase, most commonly Taq DNA polymerase. A range of commercially available DNA polymerases are available that improve on Taq polymerase; benefits include very high fidelity with less than 1 error in 200 000, owing to 3′→5′ exonuclease activity (also called proofreading activity) and an ability to tolerate GC-rich DNA sequences.

Fig2. A simplified scheme of one PCR cycle that involves denaturation, annealing and extension. ds, double-stranded.

DNA synthesis proceeds from both of the primers until the new strands have been extended along and beyond the target DNA to be amplified. It is important to note that, since the new strands extend beyond the target DNA, they will contain a region near their 3′ ends that is complementary to the other primer. Thus, if another round of DNA synthesis is allowed to take place, not only will the original strands be used as templates, but also the new strands. Most interestingly, the products obtained from the new strands will have a precise length, delimited exactly by the two regions complementary to the primers. As the system is taken through successive cycles of denaturation, annealing and extension, all the new strands will act as templates and so there will be an exponential increase in the amount of DNA produced. The net effect is to selectively amplify the target DNA and the primer regions flanking it (Figure3). Note that the original target DNA is also copied in a linear fashion, although this is outpaced by the exponential amplification from the new target strands.

Fig3. Three cycles in the PCR. As the number of cycles increases, the DNA strands that are synthesised and become available as templates are delimited by the ends of the primers. Thus, specific amplification of the desired target sequence flanked by the primers is achieved. Primers are denoted as 5′→3′.

One problem with early PCR reactions was that the temperature needed to denature the DNA also denatured the DNA polymerase. However, the availability of a thermostable DNA polymerase enzyme isolated from the thermophilic bacterium Thermus aquaticus found in hot springs provided the means to automate the reaction. Taq DNA polymerase has a temperature optimum of 72 °C and survives prolonged exposure to temperatures as high as 96 °C; it is therefore still active after each of the denaturation steps. The widespread utility of the technique is not least due to the ability to automate the reaction, and thermal cyclers have therefore been produced in which it is possible to program in the temperatures and times for a particular PCR reaction.

PCR Primer Design and Bioinformatics

The specificity of the PCR lies in the design of the two oligonucleotide primers. These have to not only be complementary to sequences flanking the target DNA, but also must not be self-complementary or bind to each other to form dimers since either scenario prevents DNA amplification. They must also be matched in their GC content and have similar annealing temperatures in order to allow for a an optimum annealing temperature. The increasing use of bioinformatics resources (e.g. Primer3 or NCBI Primer-BLAST) in the design of primers makes the design and selection of reaction conditions usually very straightforward. These resources allow input of the sequences to be amplified, primer length, product size, GC content, etc. and, following analysis, provide a choice of matched primer sequences. Indeed the initial selection and design of primers without the aid of bioinformatics would now be unnecessarily time-consuming.

It is also possible to design primers with additional sequences at their 5′ end, such as restriction endonuclease target sites or promoter sequences. However, modifications such as these require that the annealing conditions be altered to compensate for the areas of non-homology in the primers. A number of PCR methods have been developed where either one or both of the primers are random. This gives rise to arbitrary priming in genomic templates, but interestingly may give rise to discrete banding pat terns when analysed by gel electrophoresis. In many cases, this technique may be used reproducibly to identify a particular organism or species. This is sometimes referred to as random amplified polymorphic DNA (RAPD) and has been used successfully in the detection and differentiation of a number of pathogenic strains of bacteria. In addition, primers can now be synthesised with a variety of labels, such as fluorophores, allowing easier detection and quantification using techniques such as quantitative PCR ( qPCR).

PCR Amplification Templates

DNA from a variety of sources may be used as the initial source of amplification templates. It is also a highly sensitive technique and requires only one or two molecules for successful amplification. Unlike many manipulation methods used in conventional molecular biology, the PCR technique is sensitive enough to require very little template preparation. The extraction from many prokaryotic and eukaryotic cells may involve a simple boiling step. Indeed, the components of many chemical extraction techniques, such as SDS and proteinase K, may adversely affect the PCR. The PCR may also be used to amplify RNA, a process termed RT-PCR (reverse transcriptase-PCR). Initially, a reverse transcription reaction which converts the RNA to cDNA is carried out. This reaction normally involves the use of the enzyme reverse transcriptase, although some thermostable DNA polymerases used in the PCR (e.g. Tth DNA polymerase) have a reverse transcriptase activity under certain buffer conditions. This allows mRNA transcription products to be effectively analysed. It may also be used to differentiate latent viruses (detected by standard PCR) or active viruses that replicate and thus produce transcription products and are therefore detectable by RT-PCR (Figure 4). Additionally, the PCR may be extended to determine relative amounts of a transcription product. To produce long PCR products, so-called long-range PCR has been developed. Here, a cocktail of different DNA polymerases is used in the reaction and allows the production of PCR products of 20–30kb.

Fig4. Reverse transcriptase-PCR (RT-PCR): mRNA is converted to complementary DNA (cDNA) using the enzyme reverse transcriptase. The cDNA is then used directly in the PCR.

Sensitivity of the PCR

 The exquisite sensitivity of the PCR system is also one of its main drawbacks, since the very large degree of amplification makes the system vulnerable to contamination. Even a trace of foreign DNA, such as that contained in dust particles, may be amplified to significant levels and may give misleading results. Hence cleanliness is paramount when carrying out PCR, and dedicated equipment and in some cases dedicated lab oratories are used. It is possible that amplified products may also contaminate the PCR, although this may be overcome by UV irradiation to damage already amplified products so that they cannot be used as templates. A further interesting solution is to incorporate uracil into the PCR and then treat the products with the enzyme uracil N -glycosylase (UNG), which degrades any PCR amplicons with incorporated uracil, rendering them useless as templates. Furthermore, most PCRs are now undertaken using a so-called hotstart. Here, the reaction mixture is physically separated from the template or the enzyme to avoid any mispriming; only when the reaction begins does mixing occur. The separation is achieved by means of a heat-labile chemical moiety or antibody binding to the DNA polymerase.

Alternative Amplification Methods

Many traditional methods in molecular biology have now been superseded by the PCR and the applications for the technique appear to be unlimited. Some of the key areas to which the PCR has been put to use are summarised in Table 1. The success of the PCR process has given impetus to the development of other amplification techniques that are based on either thermal cycling or non-thermal cycling (isothermal) methods. Indeed, the development of isothermal systems such as the LAMP DNA amplification system (loop-mediated isothermal amplification) do away with the need for a thermal cycler and have the advantage of being able to be used outside the laboratory. Two broad methodologies exist that either amplify the target molecules such as DNA and RNA, or detect the target and amplify a signal molecule bound to it (see Table 2).

Table1. Selected applications of the PCR. A number of the techniques are described in this chapter

Table2. Selected alternative amplification techniques to the PCR

Quantitative PCR (qPCR)

One of the most useful PCR applications is quantitative PCR or qPCR. This allows the PCR to be used as a means of identifying the initial concentrations of DNA or cDNA template used. Early qPCR methods involved the comparison of a standard or control DNA template amplified with separate primers at the same time as the specific target DNA. However, these types of quantification rely on the fact that all the reactions are identical and so any factors affecting this may also affect the result. The introduction of thermal cyclers that incorporate the ability to detect the accumulation of DNA through fluorescent dyes binding to the DNA has rapidly transformed this area.

In its simplest form, a DNA-binding cyanine dye, such as SYBR ® Green, is included in the PCR reaction. This dye binds to the minor groove of double-stranded DNA, but not single-stranded DNA; therefore, as amplicons accumulate during the PCR process, SYBR ® Green binds the double-stranded DNA proportionally and fluorescence emission of the dye can be detected following excitation. Thus the accumulation of DNA amplicons can be followed in real time during the reaction run. In order to quantify unknown DNA templates, a standard dilution is prepared using DNA of known concentration. As the DNA accumulates during the early exponential phase of the reaction, an arbitrary point is taken where each of the diluted DNA samples cross. This is termed the crossing threshold (Ct) value. From the various Ct values, a log graph is prepared from which an unknown concentration can be deduced. Since SYBR ® Green and similar DNA-binding dyes are non-specific, most qPCR cyclers have a built-in melting curve function in order to determine if a correctly sized PCR product is present. The cycler gradually increases the temperature of each tube until the double-stranded PCR product denatures or melts and allows a precise, although not definitive, determination of the product. Accurate confirmation of the product can be achieved by gel electrophoresis and DNA sequencing.

The TaqMan System

 In order to make qPCR specific, a number of strategies may be employed that rely on specific hybridisation probes. One ingenious method is called the TaqMan assay or 5′ nuclease assay. Here, the probe consists of an oligonucleotide labelled with a fluorescent reporter at one end of the molecule and a quencher at the other.

During the PCR the oligonucleotide probe binds to the target sequence in the annealing step. As the Taq polymerase extends from the primer, its 5′ exonuclease activity degrades the hybridisation probe and releases the reporter from the quencher. Upon excitation of the reporter, a signal is thus generated that increases in direct pro portion to the number of starting molecules, and fluorescence can be detected in real time as the PCR proceeds (Figure 5). Although relatively expensive in comparison to other methods for determining expression levels, the TaqMan approach is simple, rapid and reliable, and is now in use in many research and clinical areas. Further developments in probe-based PCR systems have also been used and include scorpion probe systems, amplifluor and real-time LUX probes. Quantification is generally undertaken against reference samples, usually housekeeping genes such as GAPDH, rRNA or actin. A further method of quantifying PCR products is digital PCR (dPCR).

Fig5. 5′ nuclease assay (TaqMan assay). PCR is undertaken with a fluorescence reporter/quencher pair (called RQ probe). As the reporter and quencher are in close proximity, fluorescence is quenched. During extension by Taq polymerase, the probe is cleaved owing to the nuclease activity of the polymerase. Due to release of the reporter, there is a detectable increase in fluorescence, which is monitored in real-time PCR experiments.

Here, absolute quantification is achieved without the need for reference samples or standards. The initial DNA or cDNA is separated into many compartments or cells, so only a few molecules are present in each cell. These are then amplified by, for example, the TaqMan process. Compartments are then assessed for fluorescence and are either positive or negative, after which statistics are applied to generate the amount of DNA. This process is also useful for detection of rare alleles.

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مواضيع ذات صلة

Additional Nonhistone Proteins Regulate Transcription and Replication

Nonhistone Proteins Organize Long Chromatin Loops

Modifications of Histone Tails Control Chromatin Condensation and Function

Chromatin Exists in Extended and Condensed Forms

Fetal DNA Sequencing and Fetal Genome Analysis

Globin Gene Clusters

Molecular Analysis of Nucleic Acid Sequences

Genes and Genome Complexity

Structure of Nucleic Acids

Genomics: Genome-Wide Analysis of Gene Structure and Function

Chromosomal Organization of Genes and Noncoding DNA

RNA Interference Causes Gene Inactivation by Destroying the Corresponding mRNA