Multiplex Co-Amplification of 24 Retinoblastoma Gene Exons after Pre-Amplification by Long-Distance PCR

Polymerase chain reaction (PCR) amplification has become the method of choice for preparing the DNA template in mutation analysis from complex mixture of DNA or RNA molecules. This strategy is optimal for small genes or genes with mutational hotspots. However, PCR-based mutation detection is neither labour nor cost effective when large or multiple genes, with many target fragments, are involved. In addition, such as approach is limited by sample quantity. A typical example is the identification of all possible mutations, scattered along the length of disease genes. The problem is compounded by the necessity of processing large numbers of samples. It has been known for some time that multiple target sequences in complex (higher animal) genomes can be amplified simultaneously, i.e., by multiplex PCR. In multiplex PCR, different DNA fragments are co-amplified under identical conditions, in the same reaction. When the aim is simply to amplify many fragments simultaneously, it is possible to overcome limiting primer kinetics and fragment competition to design optimal conditions for a multiplex system. However, when other constraints are also pertinent, the design of a set conditions that allows multiplexing of a large number of gene fragments is not trivial. The first extensive multiplex reactions of nine fragments for the dystrophin gene were described by Chamberlain et al. (2) and Beggs et al. (3). These are exceptions: most multiplex systems do not involve more than about five amplicons. The obvious reason for this is that with each primer set added, the permissive reaction conditions allowing each fragment to reach its annealing temperature while evading spurious amplification products become increasingly less flexible. Ultimately, this lack of flexibility is due to the complexity of the genomic sequence environment which allows ample opportunity for non-specific priming.