Dynamic DNA hybridization is presented as an approach to perform gene expression analysis. The method is advantageous because of its dynamic supplies of both DNA samples and probes. The approach was demonstrated on a microfluidic platform by incorporating paramagnetic beads as a transportable solid support. A glass chip was fabricated to allow simultaneous interrogation of eight DNA target samples by DNA probes. DNA targets were immobilized on beads via streptavidin-biotin conjugation or base pairing between oligonucleotide residues. The DNA/bead complex was introduced into the device in which hybridization took place with a complementary probe. The hybridized probe was then removed by heat denaturation to allow the DNA sample to be interrogated again by another probe with a different sequence of interest. A pneumatic pumping apparatus was constructed to transport DNA probes and other reagents into the microfluidic device while hydrostatic pumping was used for the introduction of paramagnetic beads with samples. After investigating three types of paramagnetic beads, we found Dynabeads Oligo(dT)25 best suited this application. Targets on the beads could be sequentially interrogated by probes for 12 times, and the hybridization signal was maintained within experimental variation. Demonstration of specific hybridization reactions in an array format was achieved using four synthesized DNA targets in duplicate and five probes in sequence, indicating the potential application of this approach to gene expression analysis.

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The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.

Myc binds to a 6 bp 2‐fold symmetric DNA site: 5′‐C‐3A‐2C‐1G+1T+2G+3‐3′. Using site‐specific 5‐bromouracil mediated photocrosslinking, we show that His336 of Myc contacts, or is close to, the thymine 5‐methyl group at 2‐fold symmetry‐related positions ‐2 and +2 of the DNA site in the Myc‐DNA complex. Our results strongly suggest that homologous amino acids of Myc and Max make equivalent contacts in the respective protein‐DNA complexes.

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