ArrayXpress™ brings the latest, most accurate, and most efficienct technologies to your research or commercialization project.
 We continuously monitor the market for technologies and products that bring either incremental improvement or radical innovation to the field. Sometimes new technology allows us to do things a little more accurately or a little more cost efficiently. Other times disruptive technology completely shifts the paradigm for how discovery or development can be approached. In both cases, the methods and techniques must be adapted and optimized for the capabilities afforded. It also goes without saying however that many times new technology is just that, new technology, and may not be appropriate for your needs. Here we have summarized the products we have found to be of significant value. It is difficult to keep this up to date as we are evaluating so many technologies on a continuous basis.
NextGeneration Sequencing
Recently, revolutionary approaches to DNA sequencing have significantly impacted our ability to query large amounts of nucleic acid sequence. These platforms, collectively known as massive parallel DNA sequencing, encompass a combination of sophisticated enzymology, chemistry, high-resolution optics, hardware, and software engineering. The performance levels are impressive, generating vary from several hundred thousand reads (Roche/454) to tens of millions of reads (Illumina and Applied Biosystems SOLiD). A brief discussion of the two platforms offered by ArrayXpress and their research applications is provided below.
Illumina Genome Analyzer (Solexa) – extracted from Illumina website:
Illumina Sequencing technology relies on the attachment of randomly fragmented genomic DNA to a planar, optically transparent surface. Attached DNA fragments are extended and bridge amplified to create an ultra-high density sequencing flow cell with hundreds of millions of clusters, each containing ~1,000 copies of the same template. These templates are sequenced using a robust four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescent dyes. This novel approach ensures high accuracy and true base-by-base sequencing, eliminating sequence-context specific errors and enabling sequencing through homopolymers and repetitive sequences. High-sensitivity fluorescence detection is achieved using laser excitation and total internal reflection optics. Sequence reads are aligned against a reference genome and genetic differences are called using specially developed data analysis pipeline software. Alternative sample preparation methods allow the same system to be used for a range of applications including gene expression, small RNA discovery, and protein-nucleic acid interactions. After completion of the first read, the templates can be regenerated in situ to enable a second 75+ bp read from the opposite end of the templates, for a total of > 20 Gb of paired-end data per run.
Applications:
- Full (Whole) Genome Sequencing (de novo sequencing and resequencing)
- Transcriptome Sequencing
- Gene Regulation (protein-nucleic acid interactions) & Epigenetic Analysis
- Paired-end and Mate Pair Sequencing
- Multiplexed Sample Sequencing
454 Sequencing (Roche)
This large-scale, parallel pyrosequencing system generates 400-600 megabases of DNA per 10-hour run using the Genome Sequencer FLX with GS FLX Titanium series chemistry. This system is compatible with a variety of starting materials, including genomic DNA, PCR products, BACs, and cDNA. Samples consisting of large molecules (genomic DNA and BACs) are fractionated into small, 300 to 800 bp fragments. For smaller sized samples (small non-coding RNA or PCR amplicons), this step is omitted. The workflow comprises four main steps, starting from purified DNA and resulting in analyzed results. Initially, short adaptors are added to each fragment at both the 3' and 5' ends that are later used for the purification, amplification, and sequencing steps. The result is a library composed of single-stranded DNA immobilized onto the DNA capture beads with each bead carrying a unique DNA library fragment. This bead-bound library is emulsified with amplification reagents in a water-in-oil mixture and amplified in the resulting in microreactors. The entire collection is amplified in parallel generating millions of copies per bead/each fragment. The bead-bound amplicons are enriched and loaded onto a PicoTiterPlate that allows for only one bead per well for sequencing. Individual nucleotides flow across the hundreds of thousands of wells. The resulting chemiluminescent signal is recorded by the Genome Sequencer FLX Instrument. With the combination of signal intensity and positional information, the software determines the sequence of more than 1,000,000 individual reads per 10-hour instrument run. Three different bioinformatics tools are available for data analysis aimed to the applications below.
Applications:
- Full (Whole) Genome Sequencing (de novo sequencing and resequencing)
- Transcriptome Sequencing
- Metagenomics
- Amplicon (Ultra Deep) Sequencing
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Microarrays
DNA Microarrays are small sized, solid supports used for the parallel investigation of thousands of different genes. DNA sequences corresponding to all genes under investigation are immobilized at fixed locations directly onto the support surface either by printing, spotting, or actual molecule synthesis technology. By using such powerful tools, a large number of genes can be quickly surveyed at once in a scale previously unthinkable, comprising a significant advance in genetic studies.
Microarrays work by exploiting the principle of sequence complementarity. Nucleic acids have the property of forming anti-parallel, double-stranded molecules in which one strand specifically binds to a complementary one. This complementary sequence guided, binding process, is called hybridization. Scientists utilize microarrays to measure the amount of nucleic acid that binds at each site on the array, taking a snap shot of the nucleic acid content of a specific cell or tissue at a particular moment.
The use of microarrays for basic research and R&D has increased enormously throughout its initial development. Nowadays, microarrays have become a central technology to genomics studies and critical to the successful investigation of many biological processes. Using such tools, investigators can, for instance, assign probable functions to new genes according to their expression pattern and in comparison to genes of known function. This more traditional use of microarrays for gene expression profiling has been further expanded for the investigation of variations in the DNA sequence, comparative genomic hybridization studies, pathway discovery, genome wide studies of chromatin structure such as identification of histone and transcription factors binding sites, methylation patterns and selection of target sequences for deep re-sequencing.
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Real-Time Quantitative PCR
The fluorescence-based quantitative real-time PCR (qPCR) monitors the progress and collect the data of the PCR in real time, as it occurs. This powerful technology has the ability to detect and measure minute amounts of nucleic acids and has become the yardstick in the areas of molecular diagnostics, agriculture, medicine and life sciences. For instance, it is widely used as a research and applied tool to determine microbial load, gene dosage, GMO/transgene detection, forensics applications, and biomarker-based disease risk assessment and prognosis.
Currently, there is diversity of reagents, protocols, and analysis methods for Real-time qPCR. In addition, there is a range of factors that can significantly impact the final outcome of the assay, including sample quality, primers and chemistry performance, and statistical analysis of the data. That, composed with a lack of consensus and consistency on how to best perform and analyze the data, can significantly impair the analysis and result in misleading information.
To unlock the real power of this technology, ArrayXpress will work with you on an individual basis in order to provide accurate target identification and quantification and maximize the statistical power and biological meaning of your data. a well-optimized reaction is therefore essential for accurate results. We understand that your project is unique and your samples are invaluable. We strictly adhere to the highest standards, closely following the established MIQE guidelines. Prior to performing your experiment, for every target, we initially conduct a thorough PCR optimization and evaluate all key parameters of assay performance: efficiency, dynamic range, linearity, reproducibility, capacity, sensitivity and specificity. In the case of a gene expression experiment, the efficiency of the reverse transcription step is also evaluated, followed by an assay validation experiment to evaluate the relative amplification efficiency of the target(s) and the reference(s) (endogenous control). Such steps are critical to proper target quantification and aim to provide you with the best data integrity, relevance, accuracy, interpretation, repeatability, and transparency.
Our scientific team has extensive experience with both absolute and relative quantification using Real-time qPCR for DNA (e.g., plasmid and gene copy number) and cDNA/RNA quantification (e.g., gene expression) and also end-point analysis for Allelic Discrimination and Plus/Minus assays; working with both Taq Man and SYBR Green chemistries. We will be glad to assist you in choosing the best real-time qPCR approach for your experiment.
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