REDUCED COMPLEXITY CDNA PROBES

RNA任意引物PCR （RAP-PCR）具有较低的复杂性和非化学计量扩增的特点，可用于cDNA序列的差异筛选。与整个mRNA群体相比，RAP-PCR指纹图谱具有较低的复杂性，因为它们只采样混合物中某些mRNA的一小部分。与全复杂性探针相比，降低的复杂性应该导致与非同源克隆的低背景杂交。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，一个罕见的mRNA可能产生丰富的PCR产物。这种非化学计量允许每个不同的RAP-PCR指纹扩增一组不同的速率mrna，然后可以很容易地在廉价的阵列上检测到。我们将尝试各种参数来优化探针的性能。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采样特定感兴趣序列的概率，(3)探索化学计量方法以降低探针的复杂性。这些方法将应用于简单的细胞培养系统和一小部分来自癌症患者的样本。我们对这些系统中差异表达基因的研究将有助于比较各种策略的表现。

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.