REDUCED COMPLEXITY CDNA PROBES

降低复杂性的 CDNA 探针

• 批准号: 2862491
• 负责人: MICHAEL MCCLELLAND
• 金额: $ 37.64万
• 依托单位: SIDNEY KIMMEL CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-15 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2862491
• 关键词: chemical fingerprinting; complementary DNA; gene expression; human tissue; messenger RNA; nucleic acid probes; polymerase chain reaction; technology /technique development; tissue /cell culture

The reduced complexity and non-stoichiometric amplification intrinsic to RNA arbitrarily primed PCR (RAP-PCR) could be used to advantage to generate probes for differential screening of cDNA arrays. RAP-PCR fingerprints have a reduced complexity compared to the whole mRNA population because they sample only small parts of some of the mRNAs in a mixture. The reduced complexity should result in a lower background hybridization to non-homologous clones when compared to a full complexity probe. RAP-PCR fingerprints are non-stoichiometric because the probability of amplification of a particular cDNA PCR product is dependent on the match with the arbitrary primer. Thus, a rare mRNA may yield an abundant PCR product. This non-stoichiometry allows each different RAP-PCR fingerprint to amplify a different set of rate mRNAs that can then e detected easily on inexpensive arrays. We will experiment with various parameters to optimize the properties of the probe. We will (1) adjust the complexity of the non-stoichiometric probe, (2) bias sampling by the primers to increase the probability of sampling particular sequences of interest and, (3) explore stoichiometric methods to reduce probe complexity. These methods will be applied to simple cell culture systems and to a small set of samples from cancer patients. Our search for differentially expressed genes in these systems will help to compare the performance of the various strategies.

RNA任意引物PCR（RAP-PCR）具有复杂性低和非化学计量比扩增的优点，可用于cDNA芯片差异筛选的探针制备。与整个mRNA群体相比，RAP-PCR指纹具有降低的复杂性，因为它们仅对混合物中的一些mRNA的小部分进行采样。当与完全复杂性探针相比时，降低的复杂性应导致与非同源克隆的较低背景杂交。RAP-PCR指纹是非化学计量的，因为特定cDNA PCR产物的扩增概率取决于与任意引物的匹配。因此，一种罕见的mRNA可以产生丰富的PCR产物。这种非化学计量允许每个不同的RAP-PCR指纹扩增不同的速率mRNA集合，然后可以在廉价的阵列上容易地检测到。我们将试验各种参数，以优化探头的性能。我们将（1）调整非化学计量探针的复杂性，（2）通过引物进行偏置采样以增加感兴趣的特定序列的采样概率，以及（3）探索化学计量方法以降低探针复杂性。这些方法将应用于简单的细胞培养系统和癌症患者的一小部分样本。我们在这些系统中寻找差异表达的基因将有助于比较各种策略的性能。