Research Project
6555117
DESCRIPTION (Applicant's abstract): The power of polymerase chain reaction (PCR) as an analytical tool has been widely tested. Recently, many clinical diagnostic procedures used especially for screening several types of cancer, a wide range of bacterial, viral and parasitic diseases, individual identification in paternity testing, forensic analysis, and tissue typing have started employing PCR technology. Despite the invention of PCR 14 years ago, most researchers and diagnostic labs conduct PCR analyses one step at a time, with manual transfers and dedicated, single-function instruments at each step. Furthermore, both extensive manual handling and the physical heat-transfer constraints in the thermal cycling step, can contribute to 1/2 to two day long PCR tests. Although there are some automated tools available to reduce the manual work at each step, there are currently no cost effective instruments that completely automate the PCR protocol in a flexible manner. This contrasts with many of the completely automated non-PCR-based diagnostic instruments in the modern clinical labs. We propose to apply several recent advances in small sample handling, fast thermal cycling and general sample processing to the PCR protocol in order to speed analysis and reduce costs and manual labor. A technician would simply load a plate of DNA templates and a plate of the required PCR primers, then start the instrument. The instrument would precisely aspirate the required volume of each sample, then perform the thermal cycling, post-PCR cleanup, detection and results-tracking steps in a completely automated fashion, offering full automation. The instrument would reduce entire test costs (reagents, disposables and labor) by a factor of 20 or more. Considering the wide applications of PCR, we propose to make the instrument flexible (e.g., different types of detection protocols with few changes to the instrument hardware). The basis of the new instrument is to perform all functions: mixing, thermal cycling, clean up, detection and sterilization within the same set of nano-pipetter reaction vessels. In the Phase I, the target is on two PCR-based protocols: one for DNA diagnostics and one for SNP analysis. The key features of the proposed instrument, namely fast, small-volume PCR, fluorescent detection and reaction chamber cleaning will be developed and demonstrated within the Phase I project. Phase II will entail the development of a complete prototype instrument and demonstration of high-speed, low-volume processing of samples using selected protocols. PROPOSED COMMERCIAL APPLICATION: This work will result in advanced-performance commercial PCR analysis instruments. These will allow researchers to more rapidly make important discoveries using high-throughput screening assays and be the basis for an efficient and cost effective system for genetic and infectious disease diagnosis.
