Technology
Rubicon’s pre-analytical platforms substantially improve nucleic acids for downstream analysis by standardizing the quality and quantity of the nucleic acids, increasing the amount of nucleic acid that can be analyzed, and reducing the background from unwanted sequences. These improvements enable qPCR, microarray, and next-gen sequencing platforms to achieve higher sensitivity, specificity, and robustness. This has been validated for genotyping, aCGH, sequencing, and methylation analysis in research and diagnostic applications.
GenomePlex, TransPlex, and MethylPlex pre-analytical platforms are schematically displayed below. After conventional isolation of the DNA or RNA, the molecules are converted to amplifiable libraries consisting of total DNA, cDNA, or methylated DNA sequences. Each type of library is then amplified >1,000-fold using a universal-primer PCR reaction to produce DNA or cDNA of standardized concentration, molecular weight and quality, ready to be loaded onto any of the microarray, qPCR, or next-gen sequencing platforms for analysis. Any impurities such as oligonucleotides, unwanted salts, and PCR inhibitors are diluted >1,000-fold in the process. As a result of nucleic acid pre-amplification and standardization, the analytical platforms are operated under ideal conditions to produce more consistent results than could be produced from the unamplified DNA or RNA. For non-invasive testing, as little as 2 mL of plasma is sufficient for detection of an unlimited number of mutation or methylation biomarkers. For specialized analysis of specific parts of the genome, a proprietary method of sequence enrichment is used to reduce the sequence complexity of the amplified materials by ~100 times, which is especially important in microarray and next-gen sequencing applications. Synthesis, amplification, and enrichment of these libraries is rapid, inexpensive, and automatable on existing general-purpose instruments.
GenomePlex is a patented method that employs quasi-random, non-self-complementary primers to make libraries for amplifying total genomic DNA with high fidelity. Highly representative and reproducible amplification can be achieved on intact or degraded DNA, as found in most clinical samples such as plasma, serum, urine, formalin-fixed, and necrotic frozen tissue. GenomePlex can also amplify DNA from single cells and single isolated chromosomes. GenomePlex is more robust and reproducible than any of the other total DNA amplification products that have been brought to market over the years. In fact, the National Cancer Institute has chosen GenomePlex as the best method to amplify the cancer and normal DNA samples for the Cancer Genome Atlas Project. Other organizations, such as GSK, Genome Institute of Singapore, Sanger Institute, Johns Hopkins University, have published results showing that the GenomePlex amplified DNA performs exceptionally well for genotyping, sequencing, and gene copy number analysis using qPCR and microarray platforms.
TransPlex is a patented method that uses quasi-random primers to make libraries for amplifying total cellular or viral RNA with high fidelity. Highly representative and reproducible amplification can be achieve from intact RNA, or degraded RNA as found in most clinical samples. TransPlex is very robust and reproducible and seems to be the only effective method to amplify RNA from fixed tissue. The NCI-sponsored National Surgical Adjuvant Breast and Bowel Program and several diagnostic companies and universities have chosen TransPlex as the standard pre-analytical platform for discovery of novel expression biomarkers for cancer.
MethylPlex represents several patented methods for selective amplification of the methylated portions of any genome utilizing very efficient quasi-random primers or ligation. MethylPlex is unique and elegant, and results in increasing the amount of DNA that is specific for the presence and status of the tumor. MethylPlex is achieved in a simple, enzymatic process that completely avoids the bisulfite conversion chemical reactions used in other methylation assays. MethylPlex enables the discovery and validation of diagnostic biomarkers faster and more accurately than bisulfite methods and is the only method capable of measuring multiple methylated DNA analytes from a single sample of plasma or serum. MethylPlex has been chosen by several diagnostic companies to discover new cancer biomarkers and will be used as a pre-analytical step in patient tests. Not only is MethylPlex processing simple, rapid, and inexpensive, but it has also been automated on sophisticated diagnostic platforms.
Platform Combinations are useful for achieving maximal multiplexing among genetic, epigenetic, and expression assays. For example, total DNA and RNA can be amplified in a single reaction in a single tube for purposes of single-array detection of DNA and RNA viruses. Another example is the combination of methylation and gene copy number profiling on the same arrays. In principle, all three amplified libraries from a single patient samples can be combined and hybridized to a single array for simultaneous genetic, epigenetic, and expression analysis.
Derivative Rubicon Technologies are patented processes that have value independent of the amplification platforms. These include:
- Oligonucleotide compositions for whole genome amplification of bisulfite-converted DNA
- Oligonucleotide compositions to greatly reduce background of MDA WGA
- Oligonucleotide compositions for hot-start PCR with any polymerase
- Non-enzymatic random fragmentation to improve labeling for microarrays and FISH
- Non-enzymatic random fragmentation to increase reproducibility of conventional qPCR
- Enrichment procedures to reduce background on microarrays and next-gen sequencing
- Single-tube preparation of amplimers for next-gen sequencing
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