Genomic Services
EST’s (expressed sequence tags) are sequencing projects that are in process, often require arrays designed for them before all of the cluster builds have been completed. This is an ideal application for the CombiMatrix CustomArray®, as the list of transcripts can easily evolve from build to build. In addition, different sets of EST’s are often collected from different tissue types surveyed. Sequence lists are collected from the investigator and augmented with sequences from Unigene, dbEST, or TIGR. Clustering is performed to create groups of similar sequences. From these clusters, putative assemblies can be generated. These can then be submitted to the Probe Design Suite. Please contact our technical services department for more information on clustering services we can provide.

Transcript Discovery
The prevailing gene structures encountered in simple organisms consist of coding regions with few or short intervening regions. In more complex organisms, short exons are often interspersed with very large introns, making the identification of coding regions more complex. Genome tiling arrays offer the opportunity to comprehensively investigate the RNA coding regions of any chromosomal region. CustomArray approaches that have been applied to assess genome-wide transcription in genomes have identified previously unannotated features, discovering much novel transcription beyond that expected from existing gene annotation data.

SNP Discovery
SNP (single nucleotide polymorphism) genotyping and resequencing help researchers study individual sequence variation at high resolution. This application can be used to look for sequence variation in a given gene or chromosomal region. There are two ways to look for sequence variation. One can determine the sequence by differential hybridization to a perfect match and mismatched probe. Alternatively, there are methods that perform discrimination by enzymatic methods. Design-on-Demand supports both types on this platform. CombiMatrix has developed a proprietary enzymatic method for mutation detection called Hyb&Seq® that allows hundreds of base calls to be made from a single amplification product, using a single set of primers. This method and design strategy is available to all our customers.

Pathogen Genotyping
Typing of organisms is used to specifically identify an organism in a complex mixture. This method is critical for detection of pathogens in environmental samples. The investigator supplies a database of organism sequences. This is often expanded to include related organisms. Once an amplification strategy has been determined, the expanded database is used to design a set of probes that will allow typing of each member of the list. Depending on what the investigator wishes to do, probes are chosen based on different attributes. Probes that are unique to a member of a set will allow typing of organisms that are part of the investigator’s original set. Probes that have more general reactivity are chosen as positive controls as well as to type organisms that were not part of the original starting set, but which may appear in the course of the investigation. This approach has many advantages. Foremost is its ability to type novel organisms that have yet to be sequenced. Secondly, this approach can be extremely robust as it makes its case for the classification of an unknown organism from the simultaneous hybridization to multiple probes, that each contribute to the final picture.

An alternative approach is to use the 16S rRNA sequences or conserved regions that can be uniformly amplified and to use Hyb&Seq for the accurate enzymatic discrimination of the organism. The choice of either method depends entirely on the scope of the experiment and the intuition of the investigator.

Comparative Genomic Hybridization
Gene expression profiling of mRNA provides insight into the molecular basis of cancer or of other disease states; however, RNA profiling has some limitations: RNA is not a stable molecule, and the physiological state of the cell can vary leading to significant variations in gene expression profiles. DNA is a far more stable component of a cell. Cancers and genetic diseases are often accompanied by lesions of chromosomal regions. Array based comparative genomic hybridization allows the identification of genomic lesions, of gains and losses of genomic regions that lead to copy number changes, and sequence rearrangements in chromosomes. This powerful technique allows investigators to pinpoint genomic regions that have undergone significant change. Genomic Tiling arrays can be generated with capture probes designed for any interval of a sequenced region of a genome. Probes are selected for overall quality, as well as for uniqueness over the genomic sequence space. Hybridization is performed using a reference chromosome in one channel and the test channel in the other. Variation in the log ratios of the test and reference sample at each capture probe indicates a putative chromosomal lesion.

Chromosomal Complexity Reduction
Chromosomal representations are reproducible samplings of DNA populations in which the resulting DNA has a reduced complexity. Representations can be prepared in large amounts from very little starting material. The representations preserve gene ratios between genomes and they can reflect genetic polymorphism in the original genome. Cleaving the starting DNA with a restriction enzyme and ligation of universal linkers will usually lead to the preferential amplification of small (<1kb) fragments. The degree of complexity reduction is determined in large part by the restriction enzyme used. These fragments can be predicted from the starting genomic sequence and a determination can be made whether the restriction enzyme choice is appropriate for the experiment at hand. Furthermore, representational difference analysis can also be applied. An advantage is given to regions of sequence difference between two populations. There is an advantage to designing experiments to use lower complexity target mixes. It allows more precise targeting of capture probe design and should ultimately lead to more predictable results. CombiMatrix supports many of the techniques involved in creating reduced representations. We can analyze the strategy for coverage of the region of interest, and we can use the strategy to direct probe design towards the regions we expect to see in the final hybridization product.

ChIP on Chip
With many genomes sequenced, we now face the task of determining the structural and functional components encoded in the sequence space. A more complete picture of the chromosome would include more than just coding and non-coding regions, but would also elucidate transcriptional regulatory elements, sequences that mediate chromosome structure as well as regions of chemical modification. Chromatin Immunoprecipitation (ChIP) allows one to rapidly identify chromosomal features on binding sites of DNA-binding proteins, such as transcription factors, histones, chromatin remodeling enzymes and polymerases, as well as regions of DNA methylation. This assay relies on the ability of proteins or antibodies to preferentially precipitate DNA fragments containing features of interest. It is a powerful new technique that has revolutionized the field of genomics. CombiMatrix design specialists can work with the investigator to design experiments toward the regions of interest.