Specimen Preparation for In Situ PCR

Our objective is to increase the sensitivity of in situ PCR , by improving methods of specimenpreparation. We have devised a LYOPHILM technology, which comprises lyophilization of specimen onspecialized microporous nitrocellulose films, to allow reactants to thoroughly penetrate specimen tomaximize reactant access to template, under conditions which do not consequently redistribute templateand amplified product within the specimen. In Phase I, we propose to validate LYOFlLM as a single steppreservation method, by head-to-head comparison with a "conventional" preservation method whichemploys three separate chemical steps of fixation (vis. paraformaldehyde), dehydration (vis. alcoholseries) and permeation (vis. proteolysis). We will use b-actin primer pairs selected to provide multi-levelnon-specific controls to determine the most sensitive specimen preparation format for both DNA andmRNA (cDNA) PCR in thin sections of breast cancer. We will then use the most sensitive method todetermine ability to detect 1-3 copies of p53 sequences in nuclear DNA of cloned prostate cancer cells.We will use digoxygenin-conjugated nucleotide incorporation and FlTC-conjugated antibody detectionmethods to quantitatively measure sensitivity of in situ PCR. In Phase 2, we will optimize conditionsto maximize sensitivity and accuracy; and develop the technology into a standard method suitable forquantitative cell analysis in automated format.