Non-Covalent Interactions between dUTP C5-Substituents and DNA Polymerase Decrease PCR Efficiency.

The approach based on molecular modeling was developed to study dNTP derivatives characterized by new polymerase-specific properties. For this purpose, the relative efficiency of PCR amplification with modified dUTPs was studied using Taq, Tth, Pfu, Vent, Deep Vent, Vent (exo-), and Deep Vent (exo-) DNA polymerases. The efficiency of PCR amplification with modified dUTPs was compared with the results of molecular modeling using the known 3D structures of KlenTaq polymerase-DNA-dNTP complexes. The dUTPs were C5-modified with bulky functional groups (the Cy5 dye analogs) or lighter aromatic groups. Comparing the experimental data and the results of molecular modeling revealed the decrease in PCR efficiency in the presence of modified dUTPs with an increase in the number of non-covalent bonds between the substituents and the DNA polymerase (about 15% decrease per one extra non-covalent bond). Generalization of the revealed patterns to all the studied polymerases of the A and B families is discussed herein. The number of non-covalent bonds between the substituents and polymerase amino acid residues is proposed to be a potentially variable parameter for regulating enzyme activity.

Microarray analyzer based on wide field fluorescent microscopy with laser illumination and a device for speckle suppression.

A microarray analyzer was developed to obtain images and measure the fluorescence intensity of microarrays at three wavelengths from 380 nm to 850 nm. The analyzer contains lasers to excite fluorescence, barrier filters, optics to project images on an image detector, and a device for suppressing laser speckles on the microarray support. The speckle suppression device contains a fibre-optic bundle and a rotating mirror positioned in a way to change the distance between the bundle butt and mirror surface during each mirror revolution. The analyzer provides for measurements with accuracy within ± 5%. Obtaining images at several exposure times allowed a significant expansion in the range of measured fluorescence intensities. The analyzer is useful for high throughput analysis of the same type of microarrays.

UV fluorescence of tryptophan residues effectively measures protein binding to nucleic acid fragments immobilized in gel elements of microarrays.

Microarrays allow for the simultaneous monitoring of protein interactions with different nucleic acid (NA) sequences immobilized in microarray elements. Either fluorescently labeled proteins or specific fluorescently labeled antibodies are used to study protein-NA complexes. We suggest that protein-NA interactions on microarrays can be analyzed by ultraviolet (UV) fluorescence of tryptophan residues in the studied proteins, and this approach may eliminate the protein-labeling step. A specialized UV microscope was developed to obtain fluorescent images of microarrays in the UV wavelengths and to measure the fluorescence intensity of individual microarray elements. UV fluorescence intensity of BSA immobilized in microarray gel elements increased linearly with increased BSA amount with sensitivity of 0.6 ng. Real-time interaction curves between the DNA-binding domain of the NFATc1 transcription factor (NFATc1-DBD) and synthetic hairpin-forming oligodeoxyribonucleotides immobilized within 0.2 nL microarray gel elements at a concentration 5 × 10(-5) M and higher were obtained. The UV fluorescence intensities of microarray gel elements containing NFATc1-DBD-DNA complexes at equilibrium allowed the estimation of the equilibrium binding constant for complex formation. The developed method allows the protein-NA binding to be monitored in real time and can be applied to assess the sequence-specific affinity of NA-binding proteins in parallel studies involving many NA sequences.

Rapid genotyping of common deficient thiopurine S-methyltransferase alleles using the DNA-microchip technique.

Thiopurine drugs are metabolized, in part, by S-methylation catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low or undetectable TPMT activity are at high risk of severe, potentially fatal hematopoietic toxicity when they are treated with standard doses of thiopurines. As human TPMT activity is controlled by a common genetic polymorphism, it is an excellent candidate for the clinical application of pharmacogenetics. Here, we report a new molecular approach developed to detect point mutations in the TPMT gene that cause the loss of TPMT activity. A fluorescently labeled amplified DNA is hybridized with oligonucleotide DNA probes immobilized in gel pads on a biochip. The specially designed TPMT biochip can recognize six point mutations in the TPMT gene and seven corresponding alleles associated with TPMT deficiency: TPMT*2; TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8. The effectiveness of the protocol was tested by genotyping 58 samples of known genotype. The results showed 100% concordance between the biochip-based approach and the established PCR protocol. The genotyping procedure is fast, reliable and can be used for rapid screening of inactivating mutations in the TPMT gene. The study also provides the first data on the frequency of common TPMT variant alleles in the Russian population, based on a biochip analysis of 700 samples. TPMT gene mutations were identified in 44 subjects; genotype *1/*3A was most frequent.

Fluorescence data analysis on gel-based biochips.

A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This feature is valuable for monitoring the kinetics of sample interaction with immobilized probes. Depending on the model and the label used, the sensitivity of these readers approaches 0.3 amol of a labeled sample per gel element. In the selective scanner, both the spot size of the excitation laser beam and the detector field of view match the size of the biochip array elements so that the whole row of the array can be read in a single scan. The portable version reads 50-mm long, 150-element, one-dimensional arrays in 5 s. With a dynamic range of 4000:1, a sensitivity of 1-5 amol of a labeled sample per gel element, and a data format facilitating online processing, the scanner is an attractive, inexpensive solution for biomedical diagnostics. Fluorophores for sample labeling were compared experimentally in terms of detection sensitivity, influence on duplex stability, and suitability for multilabel analysis and thermodynamic studies. Texas Red and tetracarboxyphenylporphyn proved to be the best choice for two-wavelength analysis using the imaging readers.