REDUCED COMPLEXITY CDNA PROBES


Year of Award:
1999
Award Type:
R33
Project Number:
CA081667
RFA Number:
PAR-98-067
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
MCCLELLAND, MICHAEL
Other PI or Project Leader:
N/A
Institution:
SIDNEY KIMMEL CANCER CENTER
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.