Services

Gene Discovery

DNA Precision specializes in developing genes and gene information.  Our skill set  includes library construction, sequencing and PCR primer design and validation.  We can carry these processes out with or without GLP controls.

Gene discovery usually starts with library construction, is followed by sequencing and BLAST and may include PCR as an integral or separate step in the process.  We have worked with 16 species and have done library construction for xx species, primarily aquatic vertebrates and invertebrates. 

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Gene Libraries

As described below, libraries may be general (cDNA) or focused (SSH).   Either form may be normalized (to reduce the number of commonly-occuring genes) or non-normalized (to show the actual expression profile).

cDNA Library Contruction

cDNA libraries identify all expressed genes in a tissue.  Gene expression, also called protein expression or often simply expression is the process by which a gene's DNA sequence is converted into the structures and functions of a cell.  Gene expression is a multi-step process that begins with DNA, which genes are made of, into messenger RNA. It is then followed by post transcriptional modification and translation into a gene product, followed by folding, post-translational modification and targeting.  The amount of protein that a cell expresses depends on the tissue, the developmental stage of the organism and the metabolic or physiologic state of the cell. Thus, each library will represent a “snapshot” in time of the activity in the particular cells. 
Each library starts with 1.0 ug of high quality total RNA.  Steps involved in the creation of the libraries include double-strand cDNA preparation and amplification, purification to remove primer excess, etc., hybridization and fractionation of the double-strand cDNA, and normalization (if requested) followed by two amplifications.

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Suppression Subtractive Hybridization (SSH) Library Construction

Suppression subtractive hybridization (SSH) is one of the most powerful and popular PCR-based methods for isolating differentially expressed transcripts. Using a small quantity of either total RNA or poly A+ mRNA (2.5 ug) from each of two populations, the SSH procedure simultaneously subtracts and partially normalizes the abundance of target cDNAs in the subtracted population.  Two subtraction libraries, forward (“experimental”) and reverse (“control”), are generated by each subtraction protocol.  The clones are ligated into a vector, plated, picked, and sequenced.  The customer decides how many clones will be sequenced (in batches of 96), although we generally recommend sequencing 96 forward and 96 reverse clones (n=192).  These genes are then available for analysis and further investigation by means of follow-on projects (e.g., creating a custom nylon membrane array or running real-time PCR). 

DNA Precision can begin the suppression subtractive hybridization the procedure with tissue, blood, total RNA, or poly A+ mRNA that you provide.  Pricing is based on how much of the preparation is done by the customer, how much starting material is provided, and how many clones are sequenced.  We provide two options for suppressive subtraction hybridization:

• Standard subtraction: In this less expensive option, the customer provides (for each population to be subtracted):
• Enough tissue or blood to obtain 500 ug of total RNA, or
• 500 ug of high quality total RNA total, or
• 2.5 ug of high quality poly A+ mRNA
• Limited starting material:  When the tissue is at a premium and it is not possible to obtain any of the quantities listed for the standard subtraction, we can start with 1 ug of high quality total RNA.  This process costs more as it takes several additional days to remove the excess ribosomal RNA and amplify the cDNA corresponding to the poly A+ fraction.

Normalized Library

Each cell is known to express from about 10,000 to 50,000 genes, and transcript abundance varies from 200,000 copies to 1 or fewer copies per cell. As a rule, each cell expresses 10-20 highly abundant genes (several thousands of mRNA copies per cell), several hundreds of genes of medium abundance (several hundreds of mRNA copies per cell), and several thousands of rare genes (from one to several dozens of mRNA copies per cell).  Hence, direct random sequencing of clones from standard cDNA libraries is inefficient for discovering rare transcripts, because cDNAs of medium and high abundance are sequenced repeatedly instead. Normalization performed before cDNA library sequencing decreases the prevalence of clones representing abundant transcripts. Thus, normalization increases the efficiency of random sequencing dramatically, and is essential for rare gene discovery.

Non-normalized Library

In the non-normalized library, high and medium abundance genes have a greater chance of being sequenced than rare transcripts.  This approach may be useful for initial gene discovery using pooled samples of several different tissues, when little is known about an organism.  Both libraries (normalized and non-normalized) can be provided simultaneously for an additional fee. 

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Sequencing

DNA Precision contracts this work, typically with the University of  Florida Interdisciplinary Center for Biotechnology Research.  We manage the process for our customers from sample preparation through sequencing and bioinformatics.  The type of sequencing we recommend depends on the number of genes to be processed and the nature of the discovery effort.

Sanger sequencing. If a small number of clones are to be sequenced, we clone the library into a vector.  The clones are picked, grown and sequenced.  The number of clones to be sequenced is determined by the customer, but sequencing must be done in batches of 96. 
454 sequencing. Preparation includes amplification of sufficient quantities and purification of the library. A titration run is made, then 1-4 production runs may be specified.
FLX Titanium. Higher volume sequencing, producing xx to xx reads per production run.  Preparation includes amplification of sufficient quantities and purification of the library. A titration run is made, with production runs following.

Upon completion of the library, the customer will receive:

• An Excel listing of all genes sequenced.  All gene sequences belong to the customer.  If an academic discount has been provided, DNA Precision reserves the right to use these sequences for commercial use. 
  
• Glycerol stocks of the library.

Pricing:  Please contact us by email or get a quote online for academic or commercial price.

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Real Time PCR

Real-time polymerase chain reaction is perhaps the most sensitive and accurate way to measure relative gene expression in an organism.  Real-time PCR is a natural follow-on to microarray analysis.  Genes that microarrays show to be differentially regulated are subjected to RT-PCR to look at fold changes and to evaluate the statistical significance of the change.  
DNA Precision’s involvement can be limited to designing and validating primers for your use or can include full processing, as follows:

• Design custom primers and probe sets using state of the art software and design parameters
• Optimize the custom primers and probe sets.  Each primer set is validated for specificity and efficiency by running dissociation and standard curves, respectively.  Standard curves include data from a minimum of four serially diluted cDNA samples. The efficiency of amplification will be >95% for each primer set including 18S rRNA, which is used as a normalizing gene. If there is reason to suspect 18S does fluctuate, another gene can be used for normalization.
  
• Set up and run the real-time PCR reactions.  High quality total RNA samples are DNase-treated, reverse transcribed to single-strand cDNA, and run in duplicate alongside no-RT controls.
  
• Analyze the data.  The averaged value is normalized to measured 18S rRNA values for each sample.  To evaluate the results a  ΔΔCt method of analysis is used to compare changes in gene expression between controls and treated samples. 
  

Each customer receives the primer design sequences, raw data, dissociation and standard curves, and the analyzed data file including all fold-change information and p-values. 

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Microarray Processing

DNA Precision processes microarrays manufactured by Agilent Technologies, a leading supplier of genomics and proteomics products.  DNA Precision’s parent, EcoArray, has developed several custom arrays using the Agilent platform, and we have processed hundreds of Agilent arrays in both one-color and two-color protocols.  We have refined our own and Agilent’s protocols to make certain that we can get the very best results the starting material will allow.   Agilent uses its SurePrint inkjet deposition technology to synthesize 60-mer oligonucleotides in situ.  Agilent arrays are manufactured on superior quality 1” x 3” (24.2 mm x 76.2 mm) treated glass slides for overall excellent performance.  This process minimizes variability across each production lot (batch to batch) and provides the highest consistency in slide-to-slide comparisons.  All of the glass slide microarrays are designed for either a two-color  (cyanine-3 and cyanine-5) or one-color (cyanine-3) labeling reaction, the most reliable and sensitive methods for detecting differentially expressed genes. 

• We will run a minimum of one slide (one, four, or 8 arrays, depending on format).
• Sample shipping, receipt and preparation:  DNA Precision can arrange for shipment to our Gainesville, FL laboratory, or
• We can give you instructions as to packing and labeling requirements for your package.

Labeling, Hybridization, and Washing:  This includes preparation and quality checks of the RNA samples, labeling with cyanine dyes, and hybridization.  We use recommended Agilent protocols for processing arrays.   The best results are achieved using their optimized reagents and kits. For a complete table or required and recommended protocols and equipment, click here.
Scanning and evaluation:  After the microarray is prepared and processed, it is scanned using an Agilent scanner.  Agilent’s Feature Extraction software provides quantitative estimates of gene expression in a very large data file.  For each array processed, we carry out a basic statistical analysis with GeneSpring software. You will receive an DNA Precision CD with the scanned images and metrics, raw data, a basic analysis, instructions on use of the databases, and customer support.

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Bioinformatics

Bioinformatics is the statistical analysis of high-throughput data generated by microarray technology.  Bioinformatics is usually a custom-designed activity, since any in-depth treatment depends on the design of the experiment being analyzed and the goals of the research.  With that said, there are standard approaches to analysis that work for many experiments.  EcoArray can analyze microarrays of any species (e.g., human, rat, mouse) and most platforms, including Agilent, Affymetrix and Illumina.  Each project is accompanied a check sheet which is included with final results.  The check sheet both insures high quality work and explains the process step-by-step

• Basic Analysis.   For Agilent arrays, analysis begins in the Feature Extraction software that processes the data produced by the microarray scanner. Once good-quality scanned data are identified from a set of arrays and quality control filters have been applied, we start the next stage of analysis.  We typically use Agilent’s GeneSpring microarray analysis software for basic analysis because it includes powerful data preparation protocols and visualization tools.  GeneSpring carries out the processing of array data that initially begins with array normalization. The process of array normalization is used to minimize systematic variations in the gene expression levels to allow the comparison of gene levels across multiple slides. Normalization methods take advantage of the fact that when log transformed, gene expression data approach a normal distribution. Next, the specifications of experimental parameters are designated to help define the grouping and replicate structure of the experiment. Outliers are detected using a variety of filtering tools, and Principal Components Analysis (PCA) is employed to test for meaningful differential expression.  We then analyze variances using t-tests or ANOVA (one or two -way) based on your experiment. Specifications of this statistical test included asymptotic selection for p-value computation and the determination if whether a  Multiple Testing Correction is needed or not. Genes are considered differentially regulated if they exhibit a p-value of 0.05 or lower (for our standard analysis, we consider this statistically significant) as well as demonstrating a fold change >2.0 (or your specification). This includes the Gene Ontology information when available. This basic analysis is included in our microarray processing package.
• Custom Analysis. This complements the basic analysis. We may use GeneSpring alone or in combination with other software for custom analysis.  If you want to dig deeper into your data, perform multiple analyses based on several different parameters, analyze the data experimenting with the different  Multiple Testing Correction options , change the stringency criteria (e.g., p<0.01), or the p-value computation,  then this is the way to do it. We can perform cluster analysis to organize your genes and conditions in the dataset into clusters based on the similarity of their expression profiles. A variety of clustering algorithms that can be employed include: K-Means, Hierarchical, Self Organizing Maps (SOM), along with a variety of other specifications that go along with clustering that include ways of defining similarity measures or distance (e.g., Pearson Absolute, Euclidean, Manhattan, Chebychev, etc.). This custom analysis is priced per hour.

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