Primers are complementary to existing sequences necessitating that some flanking sequence information is known
Figure 2.2 The location of PCR primers: PCR primers designed to sequences adjacent to the region to be amplified, allowing a region of DNA (eg. a gene) to be amplified from a complex starting material of genomic template DNA
DNA products in addition to the specific target or may not produce any amplified products at all. The annealing step allows the hybridization 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.
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 the original strands will 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 denatura-tion, 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.
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, so it is still active after each of the denaturation steps. The widespread utility of the technique is also due to the ability to automate the reaction and, as such, many thermal cyclers have been produced in which it is possible to program in the temperatures and times for a particular PCR reaction.
Polymerase chain reaction primer design and bioinformatics
The specificity of the PCR lies in the design of the two oligonucleotide primers. These have to be complementary to sequences flanking the target DNA but must not be self-complementary or bind each other to form dimers since both prevent DNA amplification. They also have to be matched in their GC content and have similar annealing temperatures. The increasing use of bioinformatics resources such as Oligo, Generunner and Primer Design Assistant (Chen et al., 2003) in the design of primers makes the design and selection of reaction conditions much more straightforward. These resources allow the sequences to be amplified, the primer length, product size, GC content, etc. to be input 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.
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, thus enabling the genetic material of a single cell to be analysed. This property of the PCR has been exploited by forensic scientists through the use of low copy number (LCN) DNA profiling. The sensitivity of the LCN profiling process depends on an increased number of PCR cycles, typically 34. Unlike many manipulation methods used in current molecular biology, the PCR technique is sensitive enough to require very little template preparation. The PCR may also be used to amplify RNA, a process termed reverse transcriptase PCR (RT-PCR) (Gill et al., 2000).
The enormous 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 contaminating DNA, such as that contained in dust particles, may be amplified to significant levels and may give misleading results. Hence a rigorous sample-handling protocol is essential for casework when carrying out the PCR and dedicated equipment and even laboratories are preferable. All precautions must be taken to prevent previously amplified products (amplicons) from contaminating the PCR.
Many traditional methods in molecular biology have now been superseded by the PCR and the applications for the technique appear to be unlimited. The success of the PCR process has given impetus to the development of other amplification techniques, which are based on either thermal cycling or non-thermal cycling (isothermal) methods. The most popular alternative to the PCR is termed the ligase chain reaction (LCR). This operates in a similar fashion to the PCR but a thermostable DNA ligase joins sets of primers together that are complementary to the target DNA. Following this a similar exponential amplification reaction takes place, producing amounts of DNA that are similar to the PCR.
One of the most useful PCR applications is quantitative PCR (Q-PCR) (see Chapter 4). Quantitative PCR is gaining popularity in forensic science mainly because of the rapidity of the method compared to conventional PCR amplification whilst simultaneously providing a lower limit of detection and greater dynamic range. Another advantage is that Q-PCR enables a rigorous analysis of PCR problems as they arise. Early quantitative PCR methods involved the comparison of a standard or control DNA template amplified with separate primers at the same time as the specific target DNA (Higuchi et al., 1993). These types of quantitation rely on the reaction being exponential and so any factors affecting this may also affect the result. Other methods involve the incorporation of a radiolabel through the primers or nucleotides and their subsequent detection following purification of the amplicon. An alternative automated real-time PCR method is the 5' fluorogenic exonuclease detection system or TaqMan assay (Holland et al., 1991). In its simplest form a DNA binding dye such as SYBR Green is included in the reaction. As amplicons accumulate, SYBR Green binds the double-stranded DNA proportionally. Fluorescence emission of the dye is detected following excitation. The binding of SYBR Green is non-specific, therefore in order to detect specific amplicons an oligonucleotide probe labelled with a fluorescent reporter and quencher molecule at either end is included in the reaction in the place of SYBR Green. When the oligonucleotide probe binds to the target sequence the 5' exonuclease activity of Taq polymerase degrades and releases the reporter from the quencher. A signal is thus generated that increases in direct proportion to the number of starting molecules. Thus a detection system is able to induce and detect fluorescence in real-time as the PCR proceeds. In addition to quantitation, real-time PCR systems may also be used for genotyping and for accurate determination of amplicon melting temperature using curve analysis. This allows accurate amplicon identification and also offers the potential to detect mutations and SNPs (see Chapter 6). Further developments in probe-based PCR systems have also been used and include scorpion probe systems (Solinas et al., 2001), amplifluor and real-time LUX probes.
2.7 Nucleotide sequencing of DNA Concepts of nucleic acid sequencing
The determination of the order or sequence of bases along a length of DNA is one of the central techniques in molecular biology and has a key role to play in the development of polymorphic systems and analysis of the mitochondrial genome in forensic science (see Chapter 8). The precise usage of codons, information regarding mutations and polymorphisms and the identification of gene regulatory control sequences are also only possible by analysing DNA sequences. Two techniques have been developed for this, one based on an enzymatic method, frequently termed Sanger sequencing after its developer, and a chemical method called Maxam and Gilbert, named for the same reason. At present Sanger sequencing is by far the most popular method and many commercial kits are available for its use. However, there are certain occasions, such as the sequencing of short oligonucleotides, where the Maxam and Gilbert method is more appropriate (Kieleczawa, 2005).
One absolute requirement for Sanger sequencing is that the DNA to be sequenced is in a single-stranded form. Traditionally this demanded that the DNA fragment of interest be inserted and cloned into a specialized bacteri-ophage vector termed M13, which is naturally single-stranded. Although M13 is still universally used the advent of the PCR has provided the means to not only amplify a region of any genome or complementary DNA but also very quickly to generate the corresponding nucleotide sequence. This has led to an explosion in the accumulation of DNA sequence information and has provided much impetus for gene discovery and genome mapping.
The Sanger method is simple and elegant and mimics in many ways the natural ability of DNA polymerase to extend a growing nucleotide chain based on an existing template. Initially the DNA to be sequenced is allowed to hybridize with an oligonucleotide primer, which is complementary to a sequence adjacent to the 3' side of DNA within a vector such as M13 or in an amplicon. The oligonucleotide will then act as a primer for the synthesis of a second strand of DNA, catalysed by DNA polymerase. Since the new strand is synthesized from its 5' end, virtually the first DNA to be made will be complementary to the DNA to be sequenced. One of the deoxyribonucleoside triphosphates (dNTPs) that must be provided for DNA synthesis is radioactively labelled with 32P or 35S, and so the newly synthesized strand will be labelled.
The reaction mixture is then divided into four aliquots, representing the four dNTPs A, C, G and T. In addition to all of the dNTPs being present in the A tube, an analogue of dATP is added (2',3'-dideoxyadenosine triphosphate, ddATP) that is similar to A but has no 3' hydroxyl group and so will terminate the growing
chain because a 5' to 3' phosphodiester linkage cannot be formed without a 3'-hydroxyl group. The situation for tube C is identical except that ddCTP is added; similarly the G and T tubes contain ddGTP and ddTTP, respectively.
Since the incorporation of ddNTP rather than dNTP is a random event, the reaction will produce new molecules varying widely in length, but all terminating at the same type of base. Thus four sets of DNA sequence are generated, each terminating at a different type of base, but all having a common 5' end (the primer). The four labelled and chain-terminated samples are then denatured by heating and loaded next to each other on a polyacrylamide gel for electro-phoresis. Electrophoresis is performed at approximately 70°C in the presence of urea, to prevent renaturation of the DNA, since even partial renaturation alters the rates of migration of DNA fragments. Very thin, long gels are used for maximum resolution over a wide range of fragment lengths. After electro-phoresis, the positions of radioactive DNA bands on the gel are determined by autoradiography. Since every band in the track from the ddATP sample must contain molecules that terminate at adenine, and those in the ddCTP that terminate at cytosine, etc., it is possible to read the sequence of the newly synthesized strand from the autoradiogram, provided that the gel can resolve differences in length equal to a single nucleotide, hence the ability to detect and characterize point mutations (Figure 2.4). Under ideal conditions, sequences up to about 300 bases in length can be read from one gel.
Rapid PCR sequencing has also been made possible by the use of pyrosequencing. This is a sequencing by synthesis whereby a PCR template is hybridized to an oligonucleotide and incubated with DNA polymerase, ATP sulphurylase, luciferase and apyrase. During the reaction the first of the four dNTPs is added and, if incorporated, it releases pyrophosphate (PPi). The ATP sulphurylase converts the PPi to ATP, which drives the luciferase-mediated conversion of luciferin to oxyluciferin in order to generate light. Apyrase degrades the resulting component dNTPs and ATP. This is followed by another round of dNTP addition. A resulting pyrogram provides an output of the sequence. The method provides short reads very quickly and is especially useful for the determination of mutations or SNPs (Ronaghi et al., 1998).
It is also possible to undertake nucleotide sequencing from double-stranded molecules such as plasmid cloning vectors and PCR amplicons directly. The double-stranded DNA must be denatured prior to annealing with primer. In the case of plasmid an alkaline denaturation step is sufficient, however for amplicons this is more problematic and a focus of much research. Unlike plas-mids, amplicons are short and re-anneal rapidly, thereby preventing the re-annealing process or biasing the amplification towards one strand by using a primer ratio of 100 : 1 to overcome this problem to a certain extent. Denaturants such as formamide or dimethylsulphoxide (DMSO) have also been used with some success in preventing the re-annealing of PCR strands following their separation.
It is possible to physically separate and retain one PCR strand by incorporating a molecule such as biotin into one of the primers. Following PCR one strand with an affinity molecule may be removed by affinity chromatography with strepavidin, leaving the complementary PCR strand. This affinity purification provides single-stranded DNA derived from the PCR amplicon and although it is somewhat time-consuming it does provide high-quality single-stranded DNA for sequencing.
One of the most useful methods of sequencing PCR amplicons is termed PCR cycle sequencing. This is not strictly a PCR since it involves linear amplification with a single primer. Approximately 20 cycles of denaturation, annealing and extension take place. Radiolabelled or fluorescent-labelled dideoxynucleotides are then introduced in the final stages of the reaction to generate the chain-terminated extension products. Automated direct PCR sequencing is increasingly being refined, allowing greater lengths of DNA to be analysed in one sequencing run, and provides a very rapid means of analysing DNA sequences (Dugan et al., 2002).
Advances in fluorescent dye terminator and labelling chemistry have led to the development of high-throughput automated sequencing techniques. Essentially most systems involve the use of dideoxynucleotides labelled with different fluorochromes. The advantage of this modification is that since a different label is incorporated with each ddNTP it is unnecessary to perform four separate reactions. Therefore the four chain-terminated products are run on the same track of a denaturing electrophoresis gel. Each product with their base-specific dye is excited by a laser and the dye then emits light at its characteristic wavelength. A diffraction grating separates the emissions, which are detected by a charge-coupled device (CCD), and the sequence is interpreted by a computer. The advantages of these techniques include real-time detection of the sequence. In addition, the lengths of sequence that may be analysed are in excess of 500 bp. Capillary electrophoresis is increasingly being used for the detection of sequencing products (Plate 2.1). This is where liquid polymers in thin capillary tubes are used, obviating the need to pour sequencing gels and requiring little manual operation. This substantially reduces the electrophoresis run times and allows high throughput to be achieved. A number of large-scale sequence facilities are now fully automated using 96-well microtitre-based formats. The derived sequences can be downloaded automatically to databases and manipulated using a variety of bioinformatics resources. Developments in the technology of DNA sequencing have made whole genome sequencing projects a realistic proposition within achievable time-scales, and a number of these have been or are nearing completion (Kline et al., 2005).
Sanger sequencing is by far the most popular technique for DNA sequencing, however an alternative technique developed at the same time may also be used. The chemical cleavage method of DNA sequencing developed by Maxam and Gilbert is often used for sequencing small fragments of DNA such as oli-gonucleotides, where Sanger sequencing is problematic. A radioactive label is added to either the 3' or the 5' ends of a double-stranded DNA. The strands are then separated by electrophoresis under denaturing conditions, and analysed separately. DNA labelled at one end is divided into four aliquots and each is treated with chemicals that act on specific bases by methylation or removal of the base. Conditions are chosen so that, on average, each molecule is modified at only one position along its length; every base in the DNA strand has an equal chance of being modified. Following the modification reactions, the separate samples are cleaved by piperidine, which breaks phosphodiester bonds exclusively at the 5' side of nucleotides whose base has been modified. The result is similar to that produced by the Sanger method, since each sample now contains radioactively labelled molecules of various lengths, all with one end in common (the labelled end) and with the other end cut at the same type of base. Analysis of the reaction products by electrophoresis is as described for the Sanger method.
The impact of molecular biology on forensic science has been massive and far-reaching. The combined information content of molecular polymorphisms has literally revolutionized the aim of the scientists to the extent that exclusion probabilities have given way to positive identification of individuals matched with evidential material (see Chapter 12). Bioinformatics and greater emphasis on mapping complex trait genes could lead to the identification of DNA markers for many common characteristics, enabling crime detection at the levels of the genotype and phenotype simultaneously. The future is also likely to witness the widespread introduction of genotyping microchips for both nucleic acids and proteins. Proteomics (see Chapter 13) offers the significant potential of utilizing gene products for the advancement of forensic analysis.
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