Processing of nucleotide sequences

The invention relates to a method and an apparatus (100) for the processing of nucleotide sequences. An apparatus (100) according to an embodiment of the invention comprises an array of electrodes (120a,120b,...), wherein at least one nanoball (NB) comprising replications of a nucleotide sequence (1,2) of interest is attached to an electrode to which only one nanoball (NB) of that size can be attached at the same time. Thus a unique association of electrodes (120a,120b,...) to nucleotide sequences (1,2) of interest can be achieved. The nanoballs (NB) are preferably produced by rolling circle amplification. Application of attractive and/or repulsive electric potentials to the electrodes (120a,120b,...) can be used to control the attachment of nanoballs (NB). The measurement of changes in the capacitance of electrodes (120a,120b,...) can be used to detect and monitor the incorporation of mono-or oligonucleotides provided sequentially by different solutions (A, T, G, C) into strands that are replicated in a nanoball (NB) at an electrode

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor