GENOMICS
To simultaneously study most if not all the possible genes of a particular genome (Genomics) for detection of genome variability or changes in gene expression under a variety of normal and abnormal biological or environmental conditions, or due to exposure to particular vaccines or therapeutic drugs.DNA ARRAYS
There are two major types of DNA arrays. Arrays prepared by in situ synthesis of oligos or by printing or spotting of DNA preparations which could be either PCR products or synthetic oligos. Whereas In situ synthesis of oligos has its own advantages, it does not allow custom design of microarrays, or independent evaluation and purification of oligos before their attachment to microarray. PCR product or pre-synthesized oligo (50-70mers) preparations avoid these limitations and in addition provide a larger region of inquiry.PCR products: Clients can supply their own purified DNA preparations with authenticity of each preparation having been fully established by them. Alternatively, we provide a service for PCR product sample preparation from bacterial clones. This includes amplification of Plasmid DNA from bacterial clones, purification of amplified DNA preparation; determine the authenticity of the amplified material, quantitation and reconstitution of purified DNA at the desired concentrations of 200-300ng/ul for printing.
Oligos: Oligo arrays are gaining popularity due to availability of gene sequences for increasing number of species, and wide spread availability of high throughput synthesis of long oligos (50-70mers) at a relatively low cost, high reproducibility and fast turn around time. The latter includes quality check of each oligo during synthesis, capillary electrophoresis or mass spectroscopy of each oligo after synthesis, and well to well normalization of each preparation. Clients can provide sequences of the oligos to be printed or they can provide already synthesized 50-70mer oligos reconstituted at the required concentrations in printing buffers of their choice and distributed in 384 well plates.
GENE CHIP DESIGN
We at KamTek work with our clients to custom design their arrays, before the sample plates are prepared. Design features common to all biochips are described in detailed printing section. Those specific to DNA arrays may include:
- Organizing functional groups of genes in specific sub-arrays. It is especially useful for custom arrays of few genes.
- Placement of positive and negative controls (known PCR products or Oligos always used for printing) at desired places in the grids.
- Accommodate other features required by a particular experimental design.
CUSTOM GENE CHIP PRINTING
- Both PCR product DNA and 50-70mer oligo-nucleotides are custom printed as DNA microarray on a variety of substrates.
- Printing on ultra clean, ultra flat slides with super-amine chemistry from Telechem and Corning (UltraGAP) gives best results for DNA arrays in our hands. In most cases, these slides do not need blocking before hybridization.
- Immobilization of DNA printed on glass slides is achieved either by UV or heat as desired by the client.
- We offer printing of high-density arrays (up to15000 spots/slide) as well as low density printing in large batches (up to 100 slides/batch).
- The system generates ‘gal-file’ for information on the location of each gene sequence on the array and it is accepted by majority of scanners.
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SAMPLE PREPARATION AND LABELING
Sample preparation involves isolation of RNA from test and control samples, cDNA synthesis and labeling steps. RNA isolation procedure uses Qiagen kits. The cDNA synthesis and follow up steps are performed according to Genispheres instructions for their 3DNA Array kits. We have adopted Qiagen's RNA isolation procedures because of the following features:- Ability to isolate RNA from a variety of samples.
- Ease of use
- Speed
- Excellent yield and quality of RNA.
HYBRIDIZATION
KamTek has evaluated various labeling and hybridization protocols using reagents from a variety of commercial sources and found that 3DNA Array kits of Genisphere are best both in performance sensitivity and efficiency. Choice of this protocol is based on the following features:- User-friendly procedure.
- Designed for both PCR product and oligo arrays.
- Does not require incorporation of fluorescent dye during cDNA synthesis.
- Does not require additional amplification steps.
- Signal amplification by use of dendrimers with 50 to 900 fluors per cDNA.
- Independent of signal from base composition and length of the transcript.
- Time effective assay compared to other available assay systems.
- Produces minimal background.
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SCANNING AND QUANTITATION
We provide custom scanning services to our customers at reasonable rates, which comprise:- Single or dual wavelength scanning of hybridized arrays with ScanArray Express scanner, which has capability to scan images at 10-micron resolution. The dual wavelength images are scanned one after the other.
- Quantitation of scanned images in TIFF format using QuantArray software. The gal file generated by our Microarray Printer is compatible with the ScanArray Express scanner for quantitation purposes.
- Conversion of raw data generated by the system into Excel Spread sheet and scatter-plots for further analysis.
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DATA ANALYSES
Currently our major emphasis is on the Validity and Biological Relevance of Data. Details provided later. DNA Microarray services are complimented with RELATED MOLECULAR BIOLOGY services such as:- Plasmid growth, Bacterial growth media, agar growth media plates
- DNA isolation
- PCR amplification,
- PCR Product detection and preparation for chip printing
- RNA isolation
- RT/PCR amplification
- Nucleic acid hybridization experiments
- In-situ-hybridization experiments on tissue arrays
