We propose a novel single-cell high-sensitivity high-throughput technique for analysis of genetic heterogeneity across tissue samples. The technique involves building matrices of millions of microwells directly onto tissue slices using photolithography, performing separate but simultaneous PCR reactions in the wells, and then reading out the results by fluorescence detection. The technique would offer improved sensitivity with respect to current high-throughput methods (immunohistochemistry, in-situ hybridization) and improved throughput with respect to high-sensitivity methods (laser capture microdissection). The proposed project would produce proof of principle for the technique. The chosen model system is KRAS mutation in colorectal cancer, due to its immediate clinical usefulness in testing for drug resistance, as well as due to our local expertise and availability o tissue samples. Our preliminary results show successful amplification of tissue DNA inside the microwell matrix in- situ, but we still need to assemble mutation maps and optimize the technique. The accomplishment of these goals will prove the technique for use with colorectal cancer and serve as a basis for expansions into other applications. For example, it could revolutionize pathology by providing the best of both worlds (high sensitivity and high throughput) while preserving the morphological information. It would have very important and immediate application in clinical tests for drug resistance in cases of known mutations (KRAS in colorectal cancer and T790/MET in lung cancer), saving $100M's and improving treatment by avoiding the use of expensive targeted drugs on resistant patients. The technique would also be a wonderful tool to study the relationship between genetic heterogeneity and cancer invasiveness/lethality in prostate cancer. If extended to RT-PCR, it could also accurately stratify patients in breast cancer based on HER2 expression, thereby improving treatments and outcomes. Thus the technique would be a transformative tool of high impact and broad applicability in fundamental research of the cellular basis of disease.