Biomedical subjects

M J Fiandaca

Publications and source records attributed to M J Fiandaca.

4 recordsLinked to original sources

PubMed2001

PNA beacons for duplex DNA.

We report here on the hybridization of peptide nucleic acid (PNA)-based molecular beacons (MB) directly to duplex DNA sites locally exposed by PNA openers. Two stemless PNA beacons were tested, both featuring the same recognition sequence and fluorophore-quencher pair (Fluorescein and DABCYL, respectively) but differing in arrangement of these groups and net electrostatic charge. It was found that one PNA beacon rapidly hybridized, with the aid of openers, to its complementary target within duplex DNA at ambient conditions via formation of a PD-like loop. In contrast, the other PNA beacon bound more slowly to preopened duplex DNA target and only at elevated temperatures, although it readily hybridized to single-stranded (ss) DNA target. Besides a higher selectivity of hybridization provided by site-specific PNA openers, we expect this approach to be very useful in those MB applications when denaturation of the duplex DNA analytes is unfavorable or undesirable. Furthermore, we show that PNA beacons are advantageous over DNA beacons for analyzing unpurified/nondeproteinized DNA samples. This feature of PNA beacons and our innovative hybridization strategy may find applications in emerging fluorescent DNA diagnostics.

Base Pairing↗

PubMed2001

Self-reporting PNA/DNA primers for PCR analysis.

We report a new fluorogenic method for sealed-tube PCR analysis using a quencher-labeled peptide nucleic acid (Q-PNA) probe. The Q-PNA hybridizes to a complementary tag sequence located at the 5' end of a 5' fluorophore-labeled oligonucleotide primer, quenching the primer's fluorescence. Incorporation of the primer into a doublestranded amplicon causes displacement of the Q-PNA such that the fluorescence of the sample is a direct indication of the amplicon concentration. The Q-PNA is able to quench multiple primers bearing distinct 5' fluorophores in a single reaction. We show realtime quantitative detection of a single-copy gene, K-ras, from human genomic DNA, as well as an endpoint multiplex assay for Chlamydia trachomatis and Neisseria gonorrhoeae targets. Because the Q-PNA may be used to quench any primer that contains the 5' tag sequence, it is possible to inexpensively adapt an existing primer set for use in a self-reporting fluorescent assay by including the tag sequence in one of the primers.

Chlamydia trachomatis↗

PubMed1996

Detection of rRNA from four respiratory pathogens using an automated Q beta replicase assay.

Ribosomal RNA targets from Mycobacterium avium complex (23S), Mycoplasma pneumoniae (16S), Pneumocystis carinii (18S) and Legionella pneumophila (16S) were detected in four separate assays on a model automated Q-beta amplification instrument. Sandwich hybridization, reversible target capture, detector probe amplification and fluorescent signal detection occurred in closed, disposable packs at 38 degrees C. Packs were injected with 0.5 ml samples in 3.06 M guanidine thiocyanate. Ten samples per run were read after 7 h, requiring only 4 min loading time. Synthetic RNA transcripts and purified, natural RNAs from up to four different strains per assay were diluted to 10(6) or fewer molecules per sample (approximately 100 cells for prokaryotes, 10 cells for Pneumocystis). All analytes were detected at 10(6) targets. The limits of detection were found at 10(5) to 10(4). Discrimination against competitor RNA was tested using up to 10(9) molecules (1000 X excess) of appropriate test strains. Samples containing either zero targets or 10(7) competitors produced negative results in 95 to 100% of the samples, depending on the assay. Closely related Legionella and Mycoplasma species cross-reacted at high challenge levels of 10(9) molecules as a result of sequence similarities in the target regions. These results demonstrate the utility and versatility of an automated, high sensitivity, closed system for amplified analysis of direct-from-sample testing of respiratory pathogens.

Bacteria↗

PubMed1992

Protein kinase-C activation increases the quantity and poly(A) tail length of corticotropin-releasing hormone messenger RNA in NPLC cells.

We have studied the effect of protein kinase-C activation on the regulation of CRH gene expression in the human hepatoma cell line NPLC/PRF/5 (NPLC), the only cell line known to express the endogenous CRH gene. Incubation of NPLC cells with 100 nM 12-O-tetradecanoyl phorbol 13-acetate (TPA), a phorbol ester that activates protein kinase-C, resulted in a rapid (1-h) and prolonged (72-h) increase in CRH mRNA levels, with the maximum increase of 16-fold observed at 24 h. In addition, TPA treatment increased the size of CRH mRNA by approximately 100 nucleotides. This size increase, which was blocked by protein synthesis inhibitors, occurred within 1 h of TPA addition and lasted at least 8 h, with a return toward the baseline size by 24 h. Structural analysis of CRH mRNA revealed two poly(A) addition sites and, as found in human placenta, multiple transcription start sites. The increase in CRH mRNA size was not due to changes in the sites of either transcription initiation or poly(A) addition, but, rather, to a 3-fold increase in the length of the poly(A) tail itself. The ability of TPA to increase CRH mRNA levels in NPLC cells suggests that the protein kinase-C second messenger pathway may be involved in the physiological regulation of CRH gene expression. Increases in CRH mRNA poly(A) tail length potentially may influence CRH mRNA stability or translatability and, thus, may represent a general mechanism by which the protein kinase-C pathway can influence gene expression.

Anisomycin↗