Obtaining a nicely balanced multiplex PCR reaction with each PCR product having a similar yield is a challenging task. With the widespread availability of commercial kits, individual forensic laboratories rarely perform PCR optimization experiments any more. Rather, internal validation studies (see Chapter 16) focus on performance of the multiplex with varying conditions around the optimal parameters supplied with the kit protocol. For example, PCR product yields in the form of STR peak heights produced by a commercial kit might be evaluated at the optimal annealing temperature (e.g., 59°C) and 2°C and 4°C higher and lower (e.g., 55, 57, 61, and 63°C). Differences, if any, would then be noted relative to the optimal annealing temperature supplied in the kit protocol.
The development of an efficient multiplex PCR reaction requires careful planning and numerous tests and efforts in the area of primer design and balancing reaction components (Shuber et al. 1995, Henegariu et al. 1997, Markoulatos et al. 2002). A range of thermal cycling parameters including annealing temperatures and extension times are often examined in developing the final protocol. Primer concentrations are one of the largest factors in a multiplex PCR reaction determining the overall yield of each amplicon (Schoske et al. 2003). Repeated experiments and primer titrations are usually performed to achieve an optimal balance. Concentrations of magnesium chloride and deoxynucleotide triphosphates are typically increased slightly relative to single-plex reactions. A thorough evaluation of performance for a multiplex will also involve addition and removal of primer sets to see if overall balance in other amplification targets are affected.
Co-amplification of 25 PCR products followed by simultaneous detection of 35 Y chromosome single nucleotide polymorphism markers contained within the 25 amplicons has been performed in one of the most impressive demonstrations of multiplexing to date (Sanchez et al. 2003). The availability of 5-dye detection systems has enabled development of multiplexes capable of amplifying and analyzing in excess of 20 short tandem repeat loci (Butler et al. 2002, Hanson and Ballantyne 2004).
REAL-TIME (QUANTITATIVE) PCR
Instruments and assays are now available that can monitor the PCR process as it is happening enabling 'real-time' data collection. Real-time PCR, which was first described by Higuchi and co-workers at the Cetus Corporation in the early 1990s (Higuchi et al. 1992, Higuchi et al. 1993), is sometimes referred to as quantitative PCR or 'kinetic analysis' because it analyzes the cycle-to-cycle change in fluorescence signal resulting from amplification of a target sequence during PCR. This analysis is performed without opening the PCR tube and therefore can be referred as a closed-tube or homogeneous detection assay.
Several approaches to performing real-time PCR homogeneous detection assays have been published (see Foy and Parkes 2001, Nicklas and Buel 2003a). The most common approaches utilize either the fluorogenic 5' nuclease assay -better known as TaqMan® - or use of an intercalating dye, such as SYBR® Green, that is highly specific for double-stranded DNA molecules (see Chapter 13). The TaqMan approach monitors change in fluorescence due to displacement of a dual dye-labeled probe from a specific sequence within the target region while the SYBR Green assay detects formation of any PCR product.
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