Cesar E. Guerra

Expression profiling using a hexamer-based universal microarray

We describe a transcriptional analysis platform consisting of a universal micro-array system (UMAS) combined with an enzymatic manipulation step that is capable of generating expression profiles from any organism without requiring a priori species-specific knowledge of transcript sequences. The transcriptome is converted to cDNA and processed with restriction endonucleases to generate low-complexity pools (∼80–120) of equal length DNA fragments. The resulting material is amplified and detected with the UMAS system, comprising all possible 4,096 (46) DNA hexamers. Ligation to the arrays yields thousands of 14-mer sequence tags. The compendium of signals from all pools in the array-of-universal arrays comprises a full-transcriptome expression profile. The technology was validated by analysis of the galactose response of Saccharomyces cerevisiae, and the resulting profiles showed excellent agreement with the literature and real-time PCR assays. The technology was also used to demonstrate expression profiling from a hybrid organism in a proof-of-concept experiment where a T-cell receptor gene was expressed in yeast.

Q-Beta Amplification Assays

Many diseases are characterized by a long incubation period during which the patient is asymptomatic and has a low titer of the infectious agent, yet throughout this period the patient is infectious. It is desirable to detect these agents when they are present in a low titer in order to treat infected individuals and to prevent the spread of the disease to other people. Assays that utilize nucleic acid hybridization probes (Gillespie and Spiegelman 1965) offer the promise of sufficient sensitivity to detect these agents at an early stage. Single-stranded oligonucleotide probes can seek out and bind to unique complementary nucleotide sequences from the infectious agent. The resulting probe-target hybrids are the strongest and most specific intermolecular complexes known. Moreover, these complexes form rapidly in solution. The problem with using oligonucleotide probes is that when the number of targets is small, it is very difficult to detect the correspondingly small number of probes that are bound to the targets. The traditional solution to this problem is to tag the probes with radioactive groups or fluorescent moieties, or to link the probes to enzymes and then incubate them with colorless substrates that are converted to colored products. However, the limit of detection with these schemes is 200 000 target molecules. It is desirable to improve the sensitivity so that as few as 2000 target molecules can be detected. This would, for example, enable the detection of a single, infected cell in a blood sample from a patient exposed to human immunodeficiency virus (Pelligrino et al. 1987).