10 Apr


Figure 5.1 The forward and reverse primers that are used to amplify the STR locus D16S539 in the Promega PowerplexTM 1.2 kit are shown and the position that they bind to within the sequence is indicated by the arrows. The target sequence is shown in bold – the position of the primers around either side of the target determines the length of the PCR product. The example shown contains eleven repeats of the core GATA sequence is inactive when it is first added to the PCR – it only becomes active after incubation at 95 ◦C for approximately 10 minutes [21]. This ‘hot start’ enzyme allows the PCR to start at an elevated temperature and minimizes the non-specific binding that can occur at lower temperatures [22].

The primers used in PCR define the region of the genome that will be analysed. Primers are short synthetic pieces of DNA that anneal to the template molecule either side of the target region. The primer sequences are therefore limited to some degree by the DNA sequence that flanks the target sequence. Figure 5.1 illustrates the sequence and positioning of two primers that are used to amplify the D16S539 locus in one of the commercially available kits for analysing STR loci (Promega Corporation). When designing primers for forensic analysis it is important that they will bind to conserved regions of DNA and therefore effectively amplify human DNA from all populations [23] while at the same time not binding to the DNA of other species. When designing a multiplex PCR, the allelic size ranges are also important considerations for the position of the primer binding sites. There are a number of basic guidelines for primer design. Primers are normally between 18 and 30 nucleotides long, and have a balanced number of G/C and A/T nucleotides. A primer should not be self complementary or be complementary to any of the other primers that are in the reaction. Self complementary regions will result in the primer pairing with itself to form a loop, whereas primers that are complementary will bind to each other to form primer dimers. The temperature at which primers anneal to the template DNA depends upon their length and sequence – most primers are designed to anneal between 50 and 65 ◦C. A basic rule of thumb can be used when designing primers to estimate the melting temperature: for each A or T in the primer 2 ◦C is added to the melting temperature and foreachCorG4◦Cisadded-CsandGswillbindtothecomplementarynucleotidewith three hydrogen bonds and are therefore more thermodynamically stable. To estimate the annealing temperature 5 ◦C is subtracted from the melting temperature. PCR primers can be designed manually or with help using software such as Oligo [24] and Primer3 [25].

Magnesium chloride, nucleotide triphosphates and reaction buffer
Magnesium chloride is a critical component of the PCR. The primers bind to the template DNA to form a primer-template duplex: magnesium chloride stabilizes the interaction. The concentration of MgCl2 is typically between 1.5 mM and 2.5 mM; the template-primer stability increases with higher concentrations of MgCl2. The Taq polymerase also requires magnesium to be present in order to function. The building blocks for the PCR are deoxynucleotide triphosphates, which are in- corporated into the nascent DNA strand during replication. The four nucleotides are in the PCR in equal concentration, normally 200 μM. The reaction buffer maintains optimal pH and salt conditions for the reaction.

The PCR process
The PCR amplifies specific regions of template DNA. The power of the technique is illustrated in Table 5.1 In theory, a single molecule can be amplified one billion-fold by 30 cycles of amplification; in practice the PCR is not 100% efficient but does still produce tens of millions of copies of the target sequence [19]. The amplification of DNA occurs in the cycling phase of PCR, which consists of three stages (Figure 5.2): denaturation, annealing and extension. In the denaturation stage the reaction is heated to 94 ◦C; this causes the double stranded DNA molecule to ‘melt’ forming two single stranded molecules. DNA melts at this temperature be- cause the hydrogen bonds that hold the two strands of the DNA molecule together are relatively weak. As the temperature is lowered, typically to between 50 and 65 ◦C, the oligonucleotide primers anneal to the template. The primers are in molar excess to the template strands and bind to the complementary sequences before the template DNA reassociates to form double stranded DNA. After the primers have annealed the temperature is increased to 72 ◦C, which is in the optimum temperature range for the Taq polymerase. Nucleotides are added to the nascent DNA strand at the rate of approx- imately 40-60 per second [26, 27]. The enzyme catalyses the addition of nucleotides to the 3′ ends of the primers using the original DNA strand as a template; it has a high pro- cessivity, catalysing the addition of approximately 50 nucleotides to the nascent DNA strand before the enzyme dissociates – several Taq polymerase enzymes will associate and disassociate during the extension phase of longer PCR products. The normal range of cycles for a PCR is between 28 and 32. In extreme cases, where the amount of target DNA is very low the cycle number can be increased to up to 34 cycles. It has been demonstrated that going above this cycle number does Table 5.1 is not 100% efficient but is still extremely powerful. (The AmpFlSTR® and PowerPlex® STR kits are
described in Chapter 6)

Random Posts

Comments are closed.