An integrated semiconductor device enabling non-optical genome sequencing.

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing.

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.

Analysis of one million base pairs of Neanderthal DNA.

Neanderthals are the extinct hominid group most closely related to contemporary humans, so their genome offers a unique opportunity to identify genetic changes specific to anatomically fully modern humans. We have identified a 38,000-year-old Neanderthal fossil that is exceptionally free of contamination from modern human DNA. Direct high-throughput sequencing of a DNA extract from this fossil has thus far yielded over one million base pairs of hominoid nuclear DNA sequences. Comparison with the human and chimpanzee genomes reveals that modern human and Neanderthal DNA sequences diverged on average about 500,000 years ago. Existing technology and fossil resources are now sufficient to initiate a Neanderthal genome-sequencing effort.

Simultaneous gene expression analysis of steady-state and actively translated mRNA populations from osteosarcoma MG-63 cells in response to IL-1alpha via an open expression analysis platform.

Pro-inflammatory cytokines play a key role in various forms of metabolic bone diseases, including osteopenia and osteoporosis. Human MG-63 cells treated with IL-1alpha were used as a model system to identify potential marker genes that are differentially expressed. This study is designed to quantitate gene expression of actively translated mRNAs as compared to the steady-state mRNA population. Both steady-state mRNAs and actively translated mRNAs from control MG-63 cells and MG-63 cells treated with IL-1alpha were isolated and converted to cDNA. The gene expression analysis from these samples was then quantitated with an open expression analysis platform with no requirement for a priori knowledge of sequence information. As a result, many differentially regulated genes were discovered via IL-1alpha treatment. Some of the genes have been described previously as playing important roles in the regulation of inflammation and cell adhesion. These comparisons provided a panoramic overview of gene expression at both the total transcript and post-transcriptional levels. In addition, the quantitation of actively translated mRNAs associated with polysomes also provided a better estimation of protein expression levels. This methodology allows for the identification of genes acutely regulated during translation. Furthermore, the process may aid in the identification of new drug targets or biomarkers.

"One plate/three-reporter" assay format for the detection and validation of yeast two-hybrid interactions.

We describe a novel assay format for the Gal4-based yeast two-hybrid-system, in which the readout from three different reporter genes is measured sequentially in a single microplate. Activation of the URA3, MEL1, and lacZ reporters in response to a protein-protein interaction is monitored by measuring sequentially: (i) growth in medium lacking uracil, (ii) alpha-galactosidase activity, and (iii) beta-galactosidase. The data thus generated permit elimination of many false positive signals and provide a preliminary measurement of reporter activation-strength that may be confirmed by further analysis. The assay procedure is inexpensive and requires few liquid-handling steps. It is appropriate for automated high-throughput interaction mating assays, validation of putative interactor strains and hybrid-protein self-activator tests.

The Melanoma Vascular Mimicry Phenotype Defined in Gene Expression and Microsome Sequencing Analysis.

The phenomenon of vasculogenic mimicry in melanoma has been recently described to be an important factor relating to melanoma progression. Large scale gene expression profiling by real-time quantitative RT-QPCR of a panel of 40 normal tissues and 54 cancer cell lines revealed that two genetically related melanoma cell lines, one derived from a primary lesion Hs.688(A) and one derived from a lymph node metastasis Hs.688(B), displayed a unique expression pattern when compared to other cancer cell lines and tissue samples in the panel. Quantitative-RT-PCR data indicated that these melanoma cells expressed a number of activated endothelial cell-associated genes such as tissue inhibitors of matrix metalloproteinases TIMP-2, matrix metalloproteinase (MMP-1, MMP-2), thrombospondin 1 (TSP1), proto-oncogene c-MET and vascular endothelial growth factor (VEGF). To examine the gene expression profile of these unique melanoma cells in greater depth, cDNA libraries were made from isolated microsome complexes to enrich those transcripts that were destined to be translated into cell surface or secreted proteins. High throughput sequencing analysis revealed that this library contained over 7000 cDNAs and was enriched by over 80% of secreted or membrane-bound proteins. The presence in the cDNA library of genes such as acetyl LDL receptor, tumor endothelial markers-1, 5 and 8 (TEMs), flow-induced endothelial G protein coupled receptor-1 and VEGF-related protein (VRP), all of which are known to be expressed uniquely by endothelial cells, supported the hypothesis that Hs.688(A) and Hs.688(B) cells were mimicking an activated vascular phenotype. Ultimately the goal is to investigate the biological roles of endothelial cell-associated genes in the behavior of Hs.688(A) and Hs.688 (B) melanoma cells.