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AUTOMATED NUCLEIC ACID EXTRACTIONSDNA: MSM I (CHEMAGEN)
In the Chemagic system, magnetic separation is achieved through the use of an electromagnet and separation heads with rods that can be magnetized.
The metal rods are immersed into the magnetic bead suspension. When the electromagnet is switched on, the rods become magnetic and the beads are separated.
To resuspend separated magnetic bead pellet, the magnet is switched off and a stirring motor to which all rods are connected is switched on. This leads to a
very effective and gentle resuspension process resulting in separation products with both highest yields and purities.
Chemagen MSM I RNA: QIACUBE (QIAGEN)
This is an advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep. All steps in the purification
procedure are fully automated and up to 12 samples can be processed per run. It is designed around spin-column technology and includes an integrated self-positioning
centrifuge, a heatable shaker and a pipette system with a robotic column gripper. The robot mimics exactly the same procedure from lysis, binding and washing through
to the elution without a change in the chemistry or protocol.
QIAcube SNP GENOTYPING
Genotyping provides a measurement of the genetic variation between members of a species of which Single Nucleotide Polymorphisms (SNP) are the most common type of
genetic variation. A SNP is a single base pair variation at a specific locus, usually consisting of two alleles (where the rare allele frequency ≥ 1%). SNPs are often
found to be the etiology of many human diseases and are becoming of particular interest in pharmacogenomics. Because SNPs are evolutionarily conserved, they have been
proposed as markers for use in association studies in place of microsatellites. The use of SNPs is extended in the HapMap project to provide the minimal set of SNPs
needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing.
SINGLE ASSAY: Quantstudio 12K Flex (LIFE TECHNOLOGIES)
Taqman Genotyping Assay combines PCR amplification and detection in the same reaction. The method is based on the 5' nuclease activity of Taq DNA polymerase.
A PCR is performed using primers that will amplify the DNA region containing the SNP of interest. Included in the reaction are two allele-specific fluorogenic probes,
each consisting of a different fluorescent reporter dye and a fluorescent quencher. In the intact probe, the proximity of the quencher to the fluorphore causes fluorescence
resonance energy transfer (FRET), reducing the fluorescence from the reporter dye. During PCR, the 5' nuclease activity of Taq digests the allele-specific probe bound to
the region of the SNP, releasing the fluorescent dye from the quencher and allowing generation of a fluorescence signal. Depending on which dye signal is generated,
the SNP alleles are determined. If only one dye signal is detected, the SNP is homozygous for the allele corresponding to the allele-specific probe, and if both dyes
are detected, then the SNP is heterozygous.
The ABI reactions contain 5 ng of genomic DNA, specific PCR primers, two allele-specific probes, dNTPs, Taq DNA polymerase and buffer according to the manufacturer's
specifications. Reactions are set up in 384-well plates using a Biomek FX (Beckman Coulter), and cycled in ABI 7900 HT. SNP alleles are called using ABI software that
provides scatter plots of allelic calls.
Quantstudio 12K Flex MULTIPLE ASSAYS: BIOMARK HD (FLUIDIGM)
Recent advances in nanofluidics technology have made possible the use of integrated fluidic circuits (IFCs) for high-throughput real-time PCR. Nanoliter-scale quantities of
samples and reagents are channeled into thousands of nanoliter-scale chambers in which distinct real-time PCRs can be run. Fluidigm Dynamic Array integrated fluidic circuits
(IFCs) deliver a new level of efficiency and throughput for real time PCR and genotyping that is compatible with existing TaqMan genotyping assays. Potentially,
this technology allows up to 9216 individual TaqMan reactions to be run in a single experiment, with the promise of considerable reagent and time savings achievable
from using nanofluidics arrays, compared with standard TaqMan genotyping on ABI PRISM 7900HT. Dynamic Array IFCs have an on-chip network of microfluidic channels,
chambers, and valves that automatically assemble individual PCR reactions, decreasing the number of pipetting steps required by up to 100 fold. The available chip
formats that can be used in our facility are:
Fluidigm 96.96 Array GENOME WIDE ASSAY
Affymetrix is a leading provider of microarray solutions for targeted and genome-wide applications. Their powerful portfolio includes arrays,
reagents, instruments, and informatics tools that enable you to detect common and rare single nucleotide polymorphisms (SNPs), copy number variants, and other genetic
variations that can contribute to complex diseases.
MICROARRAY ASSAY: GENE TITAN (AFFYMETRIX)
The Axiom Genotyping Solution is for genome-wide association studies (GWAS), replication studies, and candidate gene association studies.
It includes predesigned and customized array plates with validated genomic content from the Axiom Genomic Database. This end-to-end solution also includes complete
reagent kits, data analysis tools, and a fully automated workflow utilizing the Gene Titan Multi-Channel (MC) Instrument.
Key Features and Benefits of the Assay
Gene Titan Multi Channel Instrument MICROARRAY ASSAY: GENECHIP INSTRUMENT SYSTEM (AFFYMETRIX)
The Array 6.0 features 1.8 million genetic markers, including more than 906,600 single nucleotide polymorphisms (SNPs) and more than 946,000 probes for the detection of
copy number variation. The SNP Array 6.0 is the only platform with analysis tools to truly bridge copy number and association, including a new, high-resolution reference
map and a copy number polymorphism (CNP) calling algorithm developed by the Broad Institute. The SNP Array 6.0 demonstrates industry-leading performance and represents
more genetic variation on a single array than any other product, providing maximum panel power and the highest physical coverage of the genome.
The high price-performance value of the SNP Array 6.0 enables researchers to design association studies with larger sample sizes in the initial scan and replication phases,
thereby significantly increasing the overall genetic power of their studies.
The SNP 6.0 Assay is based on a whole-genome sampling assay. In this assay, genomic DNA (500 ng) is digested with Nsp I and Sty I restriction enzymes and ligated to
adaptors that recognize the cohesive 4 bp overhangs. All fragments resulting from restriction enzyme digestion, regardless of size, are substrates for adaptor ligation.
A generic primer that recognizes the adaptor sequence is used to amplify adaptor-ligated DNA fragments. PCR conditions have been optimized to preferentially amplify
fragments in the 200 to 1,100 bp size range. PCR amplification products for each restriction enzyme digest are combined and cleaned using a bead based or isopropanol
precipitation method. Amplified DNA is then fragmented, labeled, and hybridized to the array.
Genome-Wide Human SNP 6.0 Array AGILENT aCGH ASSAY: CGH & CGH+SNP Microarrays
Agilent CGH microarrays are commonly used for genome-wide chromosomal analysis. Agilent offers catalog products for a variety of
CGH applications as well as most affordable and flexible custom content options.
Comparative Genomic Hybridization (CGH) is a technique used for measuring copy number changes over an entire genome between two DNA samples—a
sample and a control. With the copy number information one can visualize large or small chromosomal aberrations, including very small DNA segments
such as microdeletions and microduplications. Using Agilent microarray technology based on 60-mer oligo array probes the genome can be scanned for
imbalances on a massively parallel scale, thus increasing the coverage and resolution compared to other microarray technologies.
Agilent's 2-color CGH array provides superior copy number data as opposed to 1-color because sample and reference DNA are directly compared on the same array.
Agilent's long 60-mer probes are more specific than probes that are shorter in length, allowing one to use fewer probes to make accurate calls.
Agilent offers three types of cytogenetic microarrays: CGH, CGH+SNP, and CNV, which are optimized for the most challenging sample types including FFPE and single cells.
The microarrays are available for human, mouse, rat and other model organisms. The targeted microarrays include those designed for exon level coverage of various
disease associated and cancer associated regions of the genome.
Agilent Scanner GENE EXPRESSION
Gene expression is the most fundamental level at which the genotype gives rise to the phenotype. The genetic code stored in DNA is "interpreted" by gene expression,
and the properties of the expression give rise to the organism's phenotype. Such phenotypes are often expressed by the synthesis of proteins that control the organism's
shape, or that act as enzymes catalyzing specific metabolic pathways characterizing the organism. In general gene expression is regulated through changes in the number
and type of interactions between molecules that collectively influence transcription of DNA and translation of RNA.
REAL TIME PCR
In conventional PCR, the amplified product, or amplicon, is detected by an end-point analysis, by running DNA on an agarose gel after the reaction has finished.
In contrast, real-time PCR allows the accumulation of amplified product to be detected and measured as the reaction progresses, that is, in "real time".
SINGLE ASSAY: Quantstudio 12K Flex (LIFE TECHNOLOGIES)
Real-time detection of PCR products is made possible by including in the reaction a fluorescent molecule that reports an increase in the amount of DNA with a
proportional increase in fluorescent signal. The fluorescent chemistries employed for this purpose include DNA-binding dyes and fluorescently labeled sequence
specific primers or probes. Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence as amplification occurs.
The measured fluorescence reflects the amount of amplified product in each cycle.
Key Features and Benefits of the Assay
Quantstudio 12K Flex MULTILPLE ASSAYS: BIOMARK HD (FLUIDIGM)
Recent advances in nanofluidics technology have made possible the use of integrated fluidic circuits (IFCs) for high-throughput real-time PCR. Nanoliter-scale quantities
of samples and reagents are channeled into thousands of nanoliter-scale chambers in which distinct real-time PCRs can be run. Fluidigm DELTAgene Assays are high-quality,
rapid turnaround, custom qPCR gene expression assays; also the platform is compatible with existing TaqMan Gene Expression Assays.
MULTIPLEXED ASSAY: FLEXMAP 3D (LUMINEX)
Luminex xMAP technology is built on proven, existing technology-flow cytometry, microspheres, lasers, digital signal processing and traditional chemistry-that have been
combined in a unique way. Featuring a flexible, open-architecture design, xMAP technology can be configured to perform a wide variety of bioassays quickly, cost-effectively
and accurately.
Luminex color-codes tiny beads, called microspheres has 100 distinct sets. Each bead set can be coated with a reagent specific to a particular bioassay, allowing the
capture and detection of specific analytes from a sample. Within the Luminex compact analyzer, lasers excite the internal dyes that identify each microsphere particle
and any reporter dye captured during the assay. Many readings are made on each bead set, further validating the results.
The QuantiGene Plex (QGP) assay (Panomics/Affymetrix) is the most accurate and precise assay for multiplexed gene expression quantitation. Using the Luminex xMAP technology,
QGP assays allow the simultaneous measurement of 3 to 80 mRNA species in every well of a 96-well plate. The QGP assay incorporates the exclusively-licensed and clinically
proven branched DNA technology from Siemens that is used in the Versant® viral load assays for HIV, HBV and HCV. Branched DNA assays allow for the direct measurement of RNA
transcripts by using signal amplification rather than template amplification. The assay is simple and easy to use; QGP does not require RNA extraction, cDNA synthesis or PCR
amplification. The QuantiGene Plex assay is ideal to validate GeneChip or Next Generation Sequencing data and to validate biomarkers for translational research.
Luminex xMAP 3D MICROARRAY ASSAY: GENE TITAN (AFFYMETRIX)
The Gene Titan family of instruments and proven GeneChip Array Plates from Affymetrix are the first to provide a hands-free, automated solution for monitoring gene expression.
The Gene Titan Multi-Channel (MC) Instrument automate array processing from target hybridization to data generation by combining a hybridization oven, fluidics processing,
and state-of-the art imaging device into a single bench-top instrument.
The imaging device in the Gene Titan MC Instrument uses an external, high-intensity xenon lamp and dual excitation and emission filters to capture images from array plates for
use in genome-wide expression and genotyping studies.
Key Features and Benefits of the Assay
GeneChip Array Plate MICROARRAY ASSAY: GENECHIP INSTRUMENT SYSTEM (AFFYMETRIX)
Gene expression profiling or microarray analysis has enabled the measurement of thousands of genes in a single RNA sample. GeneChip microarrays are manufacture by
photochemical synthesis. Because of this manufacturing technology, more than a million different probes can be synthesized on an array roughly the size of a thumbnail.
These numbers allow the inclusion of multiple probes to interrogate the same target sequence, providing statistical rigor to data interpretation. Over the years the
GeneChip platform has proven to be a reliable and robust system, enabling many new discoveries and breakthroughs to be made by the scientific community.
After hybridization of the biotinylated sample, the chip is stained with streptavidin-phycoerythrin and read with a confocal scanner. Control and experimental samples
are hybridized to separate chips. Comparison of the two chips is performed to determine the differential gene expression levels of the two compared samples.
Affymetrix GeneChip System DIGITAL PCR
Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while
others do not (negative). Following PCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample,
without reference to standards or endogenous controls.
MULTIPLE ASSAY: BIOMARK HD (FLUIDIGM)
To meet the need for absolute quantitation and detection of target sequences Fluidigm qdPCR 37K™ IFC has the ability to perform four-plex detection.
Since other platforms are limited to two-plex capabilities up to 80% savings per sample is expected when an experiment demands higher-plex capabilities,
without sacrificing accuracy.
Key Features and Benefits of the Assay
Fluidigm System MULTIPLEXED AMPLICON LIBRARY PREPARATION FOR NEXT GENERATION SEQUENCINGACCESS ARRAY SYSTEM (FLUIDIGM)
This is the first high-throughput, target-enrichment system designed to work with all of the major next-generation sequencing instruments. The Access Array System
enables the user to enrich multiple unique targets (such as exons) from a large number of samples, all at one time. The system combines the cost and throughput benefits
of microfluidics with the proven performance and flexibility of PCR. Custom designed primer sets amplify (and tag) PCR products for sequencing.
Key Features and Benefits of the Assay
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