Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray

Abstract

We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP. Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use of 60-200 bp amplicons for SNP typing analysis on the NanoChip platform. In a comparative study, we typed the 5 Y chromosome loci M173, 92R7, P25, SRY1532, and M9 in 400 males using the NanoChip SNP typing protocol and the SNaPshot kit. Concording results were obtained for all samples demonstrating the accuracy of the NanoChip SNP typing protocol.

Low-volume amplification on chemically structured chips using the PowerPlex16 DNA amplification kit

“…The major detection platform for fragment length analysis of fluorescence-labeled PCR fragments in forensic DNA testing is capillary gel electrophoresis. Supplementary technologies, such as mass spectrometry or microchip devices [23,27], rely on completely different methodologies and equipment and are unlikely to replace capillary gel electrophoresis. In contrast, use of the chemically structured chip presented in this study requires only slight modifications of pre-PCR processing.…”

“…Initial developments have been made of lab-on-chip solutions that combine sample preparation, DNA amplification and detection, and determination and visualization of results [26]. Various approaches in (micro)miniaturization of PCR reactions, using microchips, capillaries, and surfaces of microarrays, exist [18,20,[27][28][29]. Electronic microarrays also integrate the amplification and detection of multiple samples [30].…”

“…These methods are aimed at automated high-throughput processing of samples and have been introduced to forensic single nucleotide polymorphism typing, rather than to STR typing [31]. However, many chip-based approaches require considerable expansion or even change of laboratory equipment, especially when analyzing STRs [27,32,33].…”

Hierarchical analysis of 30 Y-chromosome SNPs in European populations

“…In particular, Y-chromosome SNPs, because of their abundance, simplicity and low mutation rate, are becoming an extensively used marker set. Forensic laboratories are starting to implement Y chromosome SNP analysis, in order to investigate the forensic usefulness of these markers (Sanchez et al 2003;Borsting et al 2004). …”

Electronic microarray analysis of 16S rDNA amplicons for bacterial detection

“…Electronic microarrays provide speed, convenience and full automation. It has been used in different applications including on-chip strand displacement amplification Westin et al, 2000Westin et al, , 2001, single nucleotide polymorphism (SNP) and mutation detection (Gilles et al, 1999;Santacroce et al, 2002;Cooper and Goering, 2003;Borsting et al, 2004), gene expression profiling (Weidenhammer et al, 2002), immunoassay (Yang et al, 2002) and simple sequence repeat discrimination (Radtkey et al, 2000).…”