DNA microarrays are a primary tool for DNA genotyping because of their capacity to detect multiple targets simultaneously. However, DNA microarrays require fluorescent labeling and long incubation steps which limit their applicability. Furthermore, DNA microarrays are not capable of detecting DNA targets in the presence of excess background DNA. This capacity is essential in multiple biomedical applications, such as tumor genotyping, microbiology testing and non-invasive prenatal genetic analysis. Here, we propose development of a novel microarray based on Twist-Biosensor (TBS) technology that will be label-free and more than 20 times faster than standard microarrays. Additionally, the new technique will be capable of detecting DNA in the presence of 20-fold excess background DNA, making it ideal for difficult genotyping situations. In this application, we propose the development of novel microarrays to dramatically improve and simplify tumor genotyping for research and clinical applications. Available techniques used to detect somatic mutations are either limited in their multiplexing capacity, such as real-time PCR and mass spectroscopy, or time consuming and labor intensive such as Next-Gen, Sanger and Pyro-sequencing. Twist-Biosensor is a novel microarray technique in which hybridization is detected with single molecule resolution. In addition, Twist- Biosensor applies disrupting torsional stress to DNA hybrids, a novel strategy that gives the microarrays extremely high sequence selectivity. Using these unique properties, we will develop highly multiplexed microarrays to detect somatic mutations that will be accurate, rapid, and cost- effective. The overall aim of this proposal is to demonstrate the capabilities of Twist-Biosensor microarrays for the detection of multiple mutations in the presence of excess background DNA. We will develop prototype devices to detect mutations of the BRAF and KRAS genes and test them using tumor samples. Aim 1 focuses on demonstrating rapid (3 hours) and accurate detection of a single mutation in samples containing 20-fold excess background DNA. Aim 2 focuses on improving TBS detection by integrating the biosensors in a digital sensor array. The new system will have high multiplexing capability, will use inexpensive equipment and will be easy to automate. Our ultimate goal is to produce a flexible platform for biomedical research and clinical applications.
Public Health Relevance Statement: Public Health Relevance: We propose development of DNA microarrays based on Twist-Biosensor technology to dramatically simplify the genetic analysis of samples with excess background DNA, such as tumors. The new technology will facilitate the evaluation of new genetic makers and the application of personalized medicine.
Project Terms: Antineoplastic Agents; base; Biomedical Research; Biosensor; BRAF gene; cancer type; clinical application; Clinical Research; Codon Nucleotides; Colorectal; cost; cost effective; Detection; Development; Devices; Diagnostic Procedure; digital; DNA; DNA Fingerprinting; DNA Microarray Chip; Electronics; Equipment; Evaluation; fetal; flexibility; Formalin; Genes; Genetic; genetic analysis; Genomic DNA; Genotype; Goals; Hour; Hybrids; improved; KRAS2 gene; Label; Lung; Malignant Neoplasms; Mass Spectrum Analysis; Medicine; melanoma; Microbiology; multiplex detection; mutant; Mutate; Mutation; new technology; novel; novel strategies; Nucleotides; Oligonucleotides; Oncogenes; Pancreas; Paraffin Embedding; Performance; Phase; Physicians; prenatal; Preparation; Property; prototype; public health relevance; Reproducibility; Research; Resolution; Sampling; Sensitivity and Specificity; sensor; single molecule; Small Business Innovation Research Grant; Somatic Mutation; Stress; synthetic construct; System; Techniques; Technology; Testing; Time; tool; tumor; Tumor Cell Line
