Isothermal cDNA/mRNA Amplification Technology

cDNA/mRNA 等温扩增技术

• 批准号: 6691793
• 负责人: Roger Sender Lasken
• 金额: $ 9.96万
• 依托单位: MOLECULAR STAGING, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2004-10-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6691793
• 关键词: complementary DNA; genetic transcription; messenger RNA; nucleic acid amplification techniques; polymerase chain reaction; technology /technique development; thermostability

DESCRIPTION (provided by applicant): High throughput mRNA expression profiling studies are useful to evaluate changes in global gene expression under different physiological environments, e.g. diseased vs. normal tissue, drug-treated vs. untreated tissues, carcinogen/toxin-exposed vs. unexposed tissues. Often small quantity of total RNA samples, such as those obtained from cells acquired by aspiration needle biopsies, are available for these studies. Thus, high throughput expression-profiling studies need to be carried out using amplified cDNA samples. Current cDNA amplification methods are based on RT-PCR technology and suffer from the same defects of general PCR: e.g. successive high temperature denaturing steps leading to template damage; use of low fidelity, low processivity Taq DNA polymerase; primer-dimer amplification. Molecular Staging Inc. (MSI) is a life science company developing a portfolio of advanced nucleic acid amplification technologies. MSI's DNA amplification technology, Multiple Displacement Amplification (MDA), being marketed as Repli-g, amplifies whole genomic DNA under isothermal conditions. Our proposed Isothermal Total Transcript Amplification (ITTA) technology will also utilizes key components of MDA. 1st strand cDNA is performed using a 5'-phosphorylated primer. Single-stranded (ss) cDNA molecules are then ligated using a universal bridge oligonucleotide. The 3'-end of the bridge oligonucleotide contains six random nucleotides and hybridizes to the 3'-end of the cDNAs whereas the 5'-end is complementary to the cDNA primer sequences. Ligated cDNA molecules are amplified using 1229 DNA polymerase and exonuclease-resistant hexamer primers to give long multimeric double-stranded (ds) DNA products. Multimer amplified products are converted to unit length amplified cDNA molecules upon digestion with Not I or other rare cutter restriction endonuclease, whose site is incorporated in 1st strand cDNA primer, for expression profiling or other applications. The objective of the Phase-1 segment of this project is to establish scientific and technical feasibility of this cDNA/mRNA amplification technology, referred to as ITTA (patent pending). Specifically, we propose to: 1. Establish and optimize protocols for various steps of ITTA procedure to obtain efficient amplification of all transcripts. We will also determine the sensitivity of ITTA procedure regarding minimum RNA input, maximum fold amplification, and size-dependency on transcript amplification. 2. Demonstrate non-biased amplification of at least 40 different transcripts from more than two RNA samples using Taqman quantitative-PCR technology. 3. Document all assay protocols as standard operating procedures (SOP) for future use. The successful development of ITTA technology will transform global amplification of mRNAs/cDNAs. Isothermal amplification will provide a cost effective and simple technology that will not require expensive equipment or reagents. Given the fact that whole genome amplification using our isothermal amplification technology generates insignificant amplification bias compare to PCR based technologies (as determined by allele representation studies using amplified gDNA), we anticipate that isothermal cDNA amplification technology will also produce little amplification bias of the RNA population.

描述(申请人提供)：高通量信使核糖核酸表达谱研究有助于评估不同生理环境下全球基因表达的变化，例如疾病组织与正常组织、药物治疗组织与未治疗组织、致癌物/毒素暴露组织与未暴露组织。通常有少量的总RNA样本可用于这些研究，例如从通过针吸活组织检查获得的细胞中获得的样本。因此，需要使用扩增的cdna样本进行高通量表达谱研究。目前的基因扩增方法都是基于RT-PCR技术，存在着与普通PCR相同的缺陷：如连续的高温变性步骤导致模板损伤；使用低保真度、低加工能力的Taq DNA聚合酶； 分子分期公司(MSI)是一家生命科学公司，正在开发一系列先进的核酸扩增技术。MSI的DNA扩增技术，即多重置换扩增(MDA)，被称为Repli-g，在等温条件下扩增整个基因组DNA。我们提出的等温总转录扩增(ITTA)技术也将利用丙二醛的关键成分。 用5‘-磷酸化的引物扩增第一链cDNA.然后使用通用桥联寡核苷酸连接单链(Ss)cdna分子。桥联寡核苷酸的3‘端含有6个随机核苷酸，与cDNAs的3’端杂交，而5‘端与cDNA3’端互补。用1229DNA聚合酶和抗外切核酸酶的六聚体引物扩增连接的cDNA分子，得到长的多聚体双链(DS)DNA产物。多聚体扩增产物经Not I或其他罕见的切酶限制性内切酶消化后转化为单位长度扩增的cDNAs分子，其位点被结合在第一链cdna引物中，用于表达谱分析或其他应用。 该项目第一阶段的目标是建立这种被称为ITTA(正在申请专利)的cdna/mrna扩增技术的科学和技术可行性。具体来说，我们建议： 1.建立和优化ITTA程序各个步骤的操作规程，以获得所有转录本的有效扩增。我们还将确定ITTA程序关于最小RNA输入、最大折叠扩增和转录本扩增的大小依赖关系的敏感性。 2.利用Taqman定量聚合酶链式反应技术对两个以上的RNA样本中至少40个不同的转录本进行无偏扩增。 3.将所有化验方案记录为标准操作程序(SOP)，以备将来使用。 ITTA技术的成功开发将改变mRNAs/cDNA的全球扩增。等温扩增将提供一种成本效益高且简单的技术，不需要昂贵的设备或试剂。考虑到使用我们的等温扩增技术的全基因组扩增与基于PCR的技术(通过使用扩增的gDNA进行的等位基因表示研究确定)相比，产生的扩增偏差不显著，我们预计等温扩增技术也将产生很小的RNA群体的扩增偏差。