We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics.
The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of ϳ53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 ؉ 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH 4 Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH 4 Cltreated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.
'MegaPlex PCR' is a robust technology for highly multiplexed amplification of specific DNA sequences. It uses target-specific pairs of PCR primers that are physically separated by surface immobilization. Initial surface-based amplification cycles are then coupled to efficient solution-phase PCR using one common primer pair. We demonstrate this method by coamplifying and genotyping 75 unselected human single-nucleotide polymorphism (SNP) loci.The key challenge in developing a high-multiplex amplification procedure is preventing excessive off-target priming by the many primers in the reaction [1][2][3] . Several previously developed methods use surface immobilization of primer pairs as a way to separate reactions and thus limit primerdimer formation [4][5][6] , but drawbacks of these methods include inefficient amplification, loss of reactants from the surface and considerable primer-dimer formation within pairs of primers.MegaPlex PCR likewise uses physical separation of primer pairs to prevent their interaction, but it additionally includes 'common sequences' at the 5′ ends of the surface primers so that early-stage amplicons can be moved into the solution phase for co-amplification by highly efficient and unbiased PCR using a single common primer pair [6][7][8] (Fig. 1). To reduce primer-dimer formation, MegaPlex PCR uses surface primers that are made partially double-stranded by base-pairing their common 5′ sequences with complementary 'barrier oligonucleotides'. A detailed methods description is available in Supplementary Methods online.During the development of MegaPlex PCR we evaluated a range of targets, input DNAs and reaction conditions. We used membrane arrays and microbeads as binding surfaces, with the beads now routinely attached to microtiter plate wells to provide a platform compatible with automation.Using the microbead support we found MegaPlex PCR to be effective with as little as 200 ng of human genomic DNA, and in various 15-plex reactions it recovered many different target sequences of up to at least 500 bp in size, with little bias against larger amplicons ( Supplementary Fig. 1 online). As occasional targets (~1/10 in preliminary studies) generated severe primer-dimer artifacts, regardless of the multiplex level of the experiment, optimization of MegaPlex PCR was required. One option was the exclusion of these targets by empirical or computational prefiltering, but we selected a modification that avoided the need for target preselection.This modification involves enriching the input genomic DNA for sequences of interest via a crude solution-phase multiplex PCR using specific primers for all the amplification targets. The primer sequences for this can match the specific portions of those on the solid surface, or they can be designed to prime slightly outside these sites in the genomic DNA. This strategy is used routinely in digital molecular counting and genome mapping studies that use limiting-dilution samples, with multiplexing up to at least 1,200 (ref. 9). Although this is less specific ...
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