Novel high-throughput technology for gene expression profiling based on AFM

基于AFM的基因表达谱高通量新技术

• 批准号: 7684145
• 负责人: James K Gimzewski
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7684145
• 关键词: Applications Grants; Benchmarking; Biochemistry; Bioinformatics; Biological; Biological Process; Biology; Biopsy Specimen; Cells; Chemistry; Clinical; Code; Collaborations; Complementary DNA; Data Analyses; Detection; Development; Diagnostic; Digestion; Disease; Engineering; Enzymes; Exploratory/Developmental Grant; Funding; Future; Gene Expression; Gene Expression Profiling; Genomics; Health; Image; In Situ; Individual; Location; Mathematics; Measures; Medicine; Methods; Molecular; Molecular Profiling; Nanotechnology; New York; Polynucleotides; Principal Investigator; Publications; Research; Resolution; Sampling; Signal Transduction; Software Tools; Sorting - Cell Movement; Specific qualifier value; Speed; Surface; Technology; Testing; Time; United States National Institutes of Health; Universities; Vertebral column; Work; advanced disease; base; computer science; cost; cost efficient; depression; design; digital; drug discovery; high throughput technology; interdisciplinary collaboration; mathematical model; meetings; nanoscale; nanoscience; nanosystems; novel; programs; response; restriction enzyme; scale up; single molecule; technology development; tool

DESCRIPTION (provided by applicant): We will develop an AFM-based, single-molecule, gene expression analysis technology. This technology meets a pressing need for order of magnitude improvements in detection sensitivity, and three-to-four orders of magnitude lower cost. This will allow for the first time, routine, high-resolution expression profiling of clinical biopsy samples, and single cells. Our technology is ultra sensitive and extremely low cost. This approach utilizes nanoscale, 'coded' cDNA molecular 'tracks' that are interrogated, molecule by molecule, using AFM. Each cDNA molecule is 'coded' by in situ digestion with sequence-specific restriction enzymes, that produce narrow, detectible, breaks in individual polynucleotide backbones whose locations correspond to particular 4bp or 6bp enzyme recognition sequences. These slight depressions or dimples in the molecule backbone serve as digital 1's and 0's, uniquely identifying each cDNA. In the future these molecular 'tracks' can be arrayed on inexpensive 'cDNA disks', analogous to today's DVDs, for high-speed readout. This technology requires no signal amplification (PCR, etc), and can uniquely identify up to 107 distinct cDNA species in an individual sample, with single molecule resolution. We estimate that throughput of 1,000+ cDNAs per second is technically achievable, at a cost 104 times less than exiting methods. Our integrated team incorporates expertise in nanotechnology, biochemistry, and bioinformatics, in collaboration between the UCLA Department of Chemistry and Biochemistry (Gimzewski group) and the New York University Departments of Computer Science and Mathematics (Mishra group). Our team has worked together on this concept for two years prior to this RFA. We have explored the concept with a mathematical model, AFM imaging and tests of the basic surface/biochemistry. Our Preliminary Studies clearly specify quantitative, experimental benchmarks that will validate the concept, and which are necessary for engineering scale-up. The Specific Aims of this Proposal are to achieve those benchmarks. R21 Proposal: Single Molecule cDNA profiling with AFM Achieving the Specific Aims of this proposal will establish complete proof-of-concept, and a technology development path for Single Molecule cDNA profiling with AFM. After engineering scale-up, this technology will allow precise and cost efficient gene expression profiling of thousands of individual cells. Routinely conducting such analysis would significantly advance disease prediction, diagnostics and treatment, as well as drug discovery.

描述（由申请人提供）：我们将开发基于AFM的单分子，基因表达分析技术。这项技术满足了检测灵敏度提高数量级的迫切需求，成本降低了三到四个数量级。这将首次允许临床活检样品和单个细胞的常规，常规，高分辨率的表达分析。我们的技术非常敏感，成本极低。这种方法利用纳米级，“编码为“ cDNA分子”轨道”，这些cDNA分子“轨道”使用AFM通过分子进行询问，分子。每个cDNA分子都通过原位消化为“序列特异性限制酶”“编码”，这些酶会在单个多核苷酸骨架中产生狭窄，可检测的酶，其位置对应于特定的4BP或6BP酶识别序列。分子主链中的这些轻微凹陷或凹痕用作数字1和0，独特地识别每个cDNA。将来，这些分子“曲目”可以在廉价的“ cDNA磁盘”上驱动，类似于当今的DVD，以进行高速读数。该技术不需要信号扩增（PCR等），并且可以在单个样品中以单分子分辨率唯一识别多达107种不同的cDNA物种。我们估计，每秒1,000多种cDNA的吞吐量在技术上是可以实现的，比退出方法低104倍。我们的综合团队在UCLA化学和生物化学系（Gimzewski Group）与纽约大学计算机科学与数学学院（Mishra Group）（Mishra Group）之间合作，纳入了纳米技术，生物化学和生物信息学方面的专业知识。在此RFA之前，我们的团队已经共同研究了这一概念。我们已经通过数学模型，AFM成像和基本表面/生物化学的测试探索了这个概念。我们的初步研究清楚地指定了定量的实验基准，这些基准将验证该概念，并且对于工程规模而言是必要的。该提案的具体目的是实现这些基准。 R21提案：AFM实现该建议的特定目的的单分子cDNA分析将建立完整的概念验证，以及与AFM进行单分子cDNA分析的技术开发路径。在工程扩展之后，该技术将允许数千个单个细胞的精确和高效的基因表达分析。通常进行这种分析将显着提高疾病预测，诊断和治疗以及药物发现。