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

Polymerase chain reaction (PCR) amplification has become themethod of choice for preparing the DNA template in mutationanalysis from complex mixtures of DNA or RNA molecules. Thisstrategy is optimal for small genes or genes with mutational hotspots.However, PCR-based mutation detection is neither labour nor costeffective when large or multiple genes, with many target fragments,are involved. In addition, such an approach is limited by samplequantity. A typical example is the identification of all possiblemutations, scattered along the length of disease genes. The problemis compounded by the necessity of processing large numbers ofsamples.It has been known for some time that multiple target sequencesin complex (higher animal) genomes can be amplified simulta-neously, i.e., by multiplex PCR. In multiplex PCR, different DNAfragments are co-amplified under identical conditions, in the samereaction. When the aim is simply to amplify many fragmentssimultaneously, it is possible to overcome limiting primer kineticsand fragment competition to design optimal conditions for amultiplex system. However, when other constraints are alsopertinent, the design of a set of conditions that allows multiplexingof a large number of gene fragments is not trivial (1). The firstextensive multiplex reactions of nine fragments for the dystrophingene were described by Chamberlain et al . ( 2) and Beggs et al . ( 3).These are exceptions: most multiplex systems do not involve morethan about five amplicons. The obvious reason for this is that witheach primer set added, the permissive reaction conditions allowingeach fragment to reach its annealing temperature while evadingspurious amplification products become increasingly less flexible.Ultimately, this lack of flexibility is due to the complexity of thegenomic sequence environment which allows ample opportunityfor non-specific priming (1).When primers must be selected according to specific criteria,such as in mutation analysis, special constraints greatly lower theflexibility in experimental design of the multiplex system. A goodexample is denaturing gradient gel electrophoresis (DGGE) inwhich primers must be designed to encompass fragments withoptimal melting behaviour. In such experiments, one of theprimers is usually coupled to a GC-rich ‘clamp’ sequence ( 4). Inan optimal situation, this will generate a two-domain structurewith the GC-clamp as the higher melting domain, a configurationwhich will allow the detection of all possible mutations in thetarget sequence (5). It will often turn out that primers positioned