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Applications & Demonstrations

Chip based DNA Resequencing
The DOC has demonstrated the ability to synthesize Affymetrix style arrays with up to 200,000 features (oligos).  We have demonstrated re-sequencing, array CGH, methylation analysis, and double-stranded oligo features (for proteomics) on the array and are preparing for production of chips for rapid re-sequencing of large genomic regions and discrete genes important to cancer biology.  The primary advantage of this technology is the ability to reconfigure the array on a daily basis to take advantage of emerging data about the genome.

Array Comparative Genomic Hybridization 
Genetic deletions and copy number increases are associated with many diseases.  For example, the transcriptional effects of deletion of tumor suppressor genes or their regulatory elements are associated with cancer.  Copy number increases of oncogenes contribute to tumorigenesis. Comparative genomic hybridization (CGH) is a technique that provides a genome-wide map of DNA sequence copy number as a function on the chromosome.  CGH is typically carried out by co-hybridization of two labeled genomic samples to array of large genomic DNA clones (e.g., BACs) or cDNAs.  We are utilizing oligonucleotide arrays made with DOC for CGH.  Oligonucleotide arrays have the advantages of high sequence resolution of chromosomal abnormalities and the ability to probe specifically for abnormalities in non-coding regions.

Transcript profiling and array CGH for 8000 human genes in a lung
cancer cell line (H1299) with a DOC array.

Expression analysis
 An array was designed to probe 8000 human genes. To select probes that were specific for the individual genes, four 23mers were selected from within the sequences of 70-nucleotide probes available from Operon for this gene set. Of the 47 possible probes for each gene, the four with the most favorable predicted free energy of RNA/DNA heteroduplex formation were chosen for inclusion on the array. Total RNA (20 µg) from a lung tumor cell line was labeled by reverse transcription, second strand synthesis, and transcription with T7 RNA polymerase with incorporation of biotin-labeled monomers.

DNA Packing Assay
One mechanism for epigenetic regulation in cancer that is being extensively studied by many groups, including us, is methylation.  Methylation alone cannot explain all epigenetic affects observed in cancer, but there are potentially other mechanisms, such as DNA packing.   The level of chromatin compression has been shown to be correlated with transcription.  The utility of expression analysis has been limited to the examination of a fraction of the available genes on an array due to the limited sensitivity for genes with low or transient expression levels (or none).  We have developed and demonstrated using DOC oligonucleotide arrays and spotted oligonucleotide arrays a new genome wide assay for the packing status of the chromatin in the nucleus. 

Double stranded (proteomics) applications
Sequence-specific binding of proteins to double-stranded DNA regulates gene expression and DNA replication by a variety of mechanisms. Transcription factors with a diversity of structural motifs, methylases, topoisomerases, and polymerases act to create the metabolic condition of the cell. Thus, description of cellular states includes the relative levels of DNA binding factors and their sequence specificities. To access this abundance of cellular information by investigating DNA-protein interactions in a highly parallel format, we are developing arrays of double-stranded DNA oligomers. Arrays of single-stranded oligonucleotides synthesized with DOC can be converted to double-stranded arrays by hybridizing a mixture of complementary oligonucleotides to the array. More generally for large numbers of features, double-stranded arrays can be produced by creating the array with a short constant sequence at the 3’ end of every probe and annealing and enzymatically extending a complementary primer. The resulting array of short duplexes can be configured as an array of defined probes for sequence-specific DNA binding proteins. Protein binding to similar oligonucleotide duplex arrays has recently been demonstrated by others.

Chip based resequencing to detect methylated DNA promoter regions

It  has become apparent that DNA hypermethylation is a contributing factor or result of gene expression abnormalities in cancer.  Many researchers have found it important to determine the methylation state of genes of interest.  A number of approaches to studying the methylation states of gene promoter regions have been devised, including restriction endonuclease digestion, Sanger sequencing of chemically modified DNA, and site specific PCR.  We have achieved an order of magnitude improvement in quality, throughput and quantitativeness over existing technologies by adapting DOC to measure the methylation state by re-sequencing each desired base and inspecting for changes of an unmethylated C to a U following sodium bisulfite treatment

A portion of our p16 promoter methylation chip.  Each vertical set of 4 features interrogates one base of the p16 promoter sequence.  This two color experiment shows that for bases converted from C to U (T after amplification/labeling) have high green intensity and high red intensity, respectively, which indicates the presence of a protected (methylated) C, instead of yellow which indicates the base calls are the same for both labeled samples.  The insert shows a base that in one sample is methylated and in another it is not.

To demonstrate the methylation analysis mode, we show here data from two cell lines, with different methylation states in the p16 promoter.  DNA was purified from H1618 and H69 lung cancer cell lines and treated with sodium bisulfite.  The p16 promoter region of sodium bisulfite treated DNA from the H69 (no bases methylated) cell line was amplified using Cy5 labeled primer while the H1618 cell lines p16 promoter region was amplified using Cy3 labeled primer.  Our array was designed to include probes to measure converted Cs within the region, by including all possible probe variants.  Seven of the 144 bases of the H1618 cell line were found to be methylated.  Correct identification of the methylation state of all queried CpGs  (16 of them) in the promoter of p16 has been reproduced with at least seven different samples. Correct identification of the methylation state of all queried CpGs in a region of the promoter of RASSF1A has been reproduced with two different samples.  Two other major findings (see appendix material) are:  1) Spiking experiments confirm that we can detect methylation quantitatively, down to 20% of the sample content and 2) we resolve the methylation status of each base, and can resolve differing patterns within a given region inspected.  Our ongoing experiments entail optimizing the amplification and labeling of the promoter regions of 50 cancer genes.  This table summarizes the current list of working genes on the assay.

The DOC methylation chip currently being produced has over 50 genes on it.  As mentioned, the development of the multiplexed assay (amplification and labeling reaction) is the most difficult.  We currently have 40 gene regions working, and it is our intention to expand this soon to over 100 genes.  Note that this list contains a number of genes that have been implicated as methylated in cancer research, and some that yet to be linked (we identified with IRIDESCENT software), and we have included a few other genes (FIZZ3, for example) that are being used measure the methylation status in the genes of diabetes type II patients (to test a theory advanced by our code IRIDESCENT).