Isothermal cDNA/mRNA Amplification Technology

描述（由申请人提供）：高通量mRNA表达谱研究可用于评价不同生理环境下整体基因表达的变化，例如患病组织与正常组织、药物处理组织与未处理组织、致癌物/毒素暴露组织与未暴露组织。通常，少量的总RNA样品，例如从通过抽吸针活检获得的细胞中获得的那些，可用于这些研究。因此，高通量表达谱研究需要使用扩增的cDNA样品进行。目前的cDNA扩增方法基于RT-PCR技术，并且具有与一般PCR相同的缺陷：例如，导致模板损伤的连续高温变性步骤;使用低保真度、低持续合成能力的Taq DNA聚合酶;引物-二聚体扩增。 Molecular Staging Inc. (MSI)是一家开发先进核酸扩增技术的生命科学公司。MSI的DNA扩增技术，多重置换扩增（MDA），以d-g销售，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MDA的关键组成部分。 使用5'-磷酸化引物进行第一链cDNA。然后使用通用桥寡核苷酸连接单链（ss）cDNA分子。桥寡核苷酸的3 ′-末端含有6个随机核苷酸并与cDNA的3 ′-末端杂交，而5 ′-末端与cDNA引物序列互补。使用1229 DNA聚合酶和核酸外切酶抗性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。多聚体扩增产物在用Not I或其它稀有切割限制性内切酶消化后转化为单位长度扩增的cDNA分子，其位点并入第一链cDNA引物中，用于表达谱分析或其它应用。 该项目第一阶段的目标是建立这种cDNA/mRNA扩增技术的科学和技术可行性，称为ITTA（专利申请中）。具体而言，我们建议： 1.建立和优化ITTA程序的各个步骤的方案，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、最大倍数扩增和转录本扩增的大小依赖性的灵敏度。 2.证明使用Taqman定量PCR技术从两个以上的RNA样品中无偏倚扩增至少40种不同的转录本。 3.将所有试验方案记录为标准操作规程（SOP），以备将来使用。 ITTA技术的成功开发将改变mRNA/cDNA的全球扩增。等温扩增将提供成本有效且简单的技术，其将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的技术相比产生不显著的扩增偏差（如通过使用扩增的gDNA的等位基因代表性研究所确定的），我们预期等温cDNA扩增技术也将产生RNA群体的很小的扩增偏差。

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.