V. E. Barsky

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.

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.

Biological Microchips with Hydrogel-Immobilized Nucleic Acids, Proteins, and Other Compounds: Properties and Applications in Genomics

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single-nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, protein microchips have been created, containing immobilized antibodies, antigens, enzymes, and many other substances, as well as microchips with gel-immobilized live cells.

Gel-Based Microchips: History and Prospects

The review describes the history of formation and development of the microchip technology and its role in the human genome project in Russia. The main accent was done on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous models. The microchips are manufactured by photoinitiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical properties and stability; this allowed one to work with the DNA fragments of up to 500 nt in length, as well as with quite large protein molecules. At present, the gel-based microchips are widely applied to solve different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA–protein interactions. The oligonucleotide microchips are a cheap and reliable diagnostic tool designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; of orthopoxviruses, including the smallpox virus; of the anthrax pathogen; and chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteo-mics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which opens a wide potential for creating biosensors on the basis of microchips.

[Optical properties of fluorochromes, promising for use in biological microchips].

Fourteen fluorochromes (free and oligonucleotide-bound) and five ligands commonly used to quantitatively assess DNA duplexes or complexes with proteins in microchips were studied by measuring their fluorescence excitation and emission spectra. The spectral changes are described that were caused by oligonucleotide binding, solution hybridization, or varying the temperature. The data are discussed from the standpoint of applicability and limitations of the fluorochromes and the ligands in qualitative and quantitative assays for DNA duplexes in microchips.

Effects of various fluorochromes and competition between labeled oligonucleotides on their hybridization to oligonucleotides immobilized on biological microchips

Some technical tips are described on how to improve the discrimination between perfect and imperfect duplexes formed by hybridization of fluorescently labeled oligonucleotides to biological microchips. Model experiments were performed to assess the precision of the method. Effects of labeling on the efficiency of hybridization and some properties of competitive hybridization were studied using short synthetic oligonucleotides and three most popular fluorochromes as examples.

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.

Biochip development for determining Y-haplogroups that occur in Russian populations

A biochip has been developd that enables one to determine Y-chromosome haplogroups C, DE, G, H, I, J, L, N, O, and R in the DNA sample. SNPs M130, M145, P257, M69, U179, M304, M185, M231, M175, P224 were selected as haplogroup markers, correspondingly. The genotyping included two-round PCR with fluorescent labeling of the product followed by hybridization with immobilized probes on the biochip. An analysis of the ratios of fluorescent signals for immobilized wild-type probe-group specific probe pairs for each of the chosen polymorphic markers showed a high accuracy Y-haplogroup genotyping using the biochip. The reliability of genotyping was confirmed by direct sequencing.

Microchips in the laboratory of A.D. Mirzabekov: 1988–2007

The paper reviews the last period in the research work of A.D. Mirzabekov, when gel-based biochips were invented, studied, and introduced in practice. This work, starting from the early stages of the Human Genome Project up to the recent development of diagnostic and protein biochips, is considered in the context of the worldwide development of microarray technologies.

Analysis of NAT2 Point Mutations with Biological Microchips

The NAT2 product, N-acetyltransferase 2, is involved in biotransformation and detoxification of several aromatic amines (in particular, 2-aminofluorene, 4-aminobiphenyl, and 4-naphthylamine), which are strongly mutagenic and carcinogenic, and acetylates some drugs, affecting their metabolism. A biological microchip was developed to detect 16 point mutations, which determine 36 alleles and 660 genotypes of NAT2. The genotypes can be divided into four groups according to the acetylator phenotype: groups with rapid (R/R), intermediate (R/S), or slow (S/S) acetylation and a group combining intermediate and slow alleles (“R/S or S/S”). The last group includes the alleles determined by combinations of seven mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, and 857G/A), whose cis or trans position is detectable by restriction enzyme analysis. The NAT2 genotype was unequivocally established for 37 out of 71 DNA specimens, while the other 34 specimens were characterized by more than two genotypes. By the acetylator phenotype, 16 out of the 34 genotypes were assigned to the group “R/S or S/S,” combining mutations 282C/T, 341T/C, 481C/T, 590G/A, and 803A/G. Thus, the biochip allows primary analysis of most NAT2 polymorphic substitutions, the acetylator genotype being important to know in predictive medicine and individualized therapy.