Increasing the size of PCR products without redesigning primer binding sequences.

Short tandem repeat (STR) loci in the human genome can be used for individual identification (1). Simultaneous PCR-amplification and size determination at several STR loci is desirable in order to: (i) increase the information gained when only very small amounts of template DNA are available, (ii) maximise throughput of samples and (iii) minimise costs. One means of increasing the number of loci that can be analysed together is to use fluorescently labelled primers (2), with the same dye label used on more than one STR system, provided the alleles from those systems do not overlap. During our development of an STR-PCR octoplex system for use in routine forensic casework, we realised even from preliminary database studies (-100 individuals) that alleles from two of the constituent loci (D6S5O2 and D2OS85) were separated from each other by <2 bp. As these loci had originally carried the same tetrachlorofluroscein (Tet) fluorescent label (3) there was the potential that rarer alleles than those currently observed could result in overlap of the two systems. However, simple re-assignation of the fluorescent labels carried on the eight different loci was not possible due to extensive overlapping of the PCR products from several of the systems. The alternative of redesigning the primers to increase the separation between the D6 and D20 loci may have entailed a lot of work to re-optimise the PCR conditions of the octoplex, assuming the redesigned primers worked at all. It was more attractive to retain the various primer sequences which were known to co-amplify with each other under the same PCR conditions. An increase in the size of the D6 PCR products was achieved by attaching a Tag' tail onto the 5' end of one of the D6 primers. This tail had to be inert in its capacity to anneal to genomic template, and should not bind any of the other primers in the octoplex reaction mixture. The M13 reverse sequencing primer *1233 (New England Biolabs, Hertfordshire, UK) which derives from Escherichia coli, was used to achieve the desired increase in product size. This approach may have a wider application to reposition PCR products for more convenient analysis. Multiplex PCR was carried out using primers specific for the STR loci D18S51, D21S11, FGA, HUMTHO1, VWA, D6S502 and D20S85 (Table 1, and references therein). Primers which flank a 6 bp deletion in the amelogenin gene on the X chromosome were also included to provide a simple sex test Reactions (50 uJ) contained lx PE buffer (Perkin-Elmer, Warrington, UK), 200 uM dNTPs (Pharmacia, St Albans, UK), 250 nM primers for VWA, D21Sll,D6S502andD2OS85,160 nM primers for HumThoL 60 nM primers for D18S51,50 nM primers for FGA and Amelogenin. Reaction mixtures lacking Tag DNA polymerase and template Table 1. Genetic loci and prifrter sequences used in the multiplex PCR reaction