Andrei Mirzabekov

Sequence analysis by hybridization with oligonucleotide microchip: identification of β-thalassemia mutations

Diagnostics for genetic diseases were run and sequence analysis of DNA was carried out by hybridization of RNA transcripts with oligonucleotide array microchips. Polyacrylamide gel pads (100 x 100 x 20 microm) were fixed on a glass slide of the microchip and contained allele-specific immobilized oligonucleotides (10-mers). The RNA transcripts of PCR-amplified genomic DNA were fluorescently labeled by enzymatic or chemical methods and hybridized with the microchips. The simultaneous measurement in real time of the hybridization and melting on the entire oligonucleotide array was carried out with a fluorescence microscope equipped with CCD camera. The monitoring of the hybridization specificity for duplexes with different stabilities and AT content was enhanced by its measurement at optimal, discrimination temperatures on melting curves. Microchip diagnostics were optimized by choosing the proper allele-specific oligonucleotides from among the set of overlapping oligomers. The accuracy of mutation detection can be increased by simultaneous hybridization of the microchip with two differently labeled samples and by parallel monitoring their hybridization with a multi-wavelength fluorescence microscope. The efficiency and reliability of the sequence analysis were demonstrated with diagnostics for beta-thalassemia mutations.

Detection of single nucleotide polymorphisms of the human mu opioid receptor gene by hybridization or single nucleotide extension on custom oligonucleotide gelpad microchips: Potential in studies of addiction

The human mu opioid receptor (MOR) plays a central role in mediating the effects of opioids, both endogenous and exogenous. Epidemiological studies have shown that addiction in general, and especially opiate addiction, has a heritable component. Clinical and laboratory studies suggest that the MOR gene may contribute to the heritable component of vulnerability to develop opiate addiction. Naturally occurring single nucleotide polymorphisms (SNPs) have been identified in the MOR gene by conventional methods. Two coding region SNPs, the A118G and C17T substitutions, occur at high allelic frequencies (10.5% and 6.6%, respectively, in our previous studies). These common SNPs cause amino acid changes in the receptor, and may have implications for differences in individual responses to opioids, as well as decreased or increased vulnerability to opiate addiction. The A118G substitution encodes a variant receptor with binding and signal transduction differences in response to beta-endorphin in cellular assays. Recent innovations in microchip technology offer new potential methods for SNP detection. We report here on the development of two separate approaches using custom oligonucleotide gelpad microarrays for detection of these two common SNPs of the MOR gene in human DNA samples. First, PCR-amplified genomic DNA samples were used to produce target sequences, which were labeled with fluorescent dye and hybridized to custom microchips. Oligonucleotides on these reusable microchips were designed to query nucleotide substitutions at positions 17 and 118 of the MOR gene. Thirty-six human DNA samples were assayed both on these custom microchips and by conventional automated gel sequencing, with highly concordant identification of both heterozygous and homozygous substitutions. A second approach was developed for the C17T SNP utilizing single nucleotide extension on custom microchips. These custom gelpad microchips have potential for the rapid and inexpensive detection of specific SNPs for genetic and genomic studies.

The method of crosslinking histones to DNA partly depurinated at neutral pH

Abstract The procedure of either reversible or stable “zero-length” crosslinking of histones through their lysine residues directly to DNA partly depurinated under mild conditions at neutral pH is described. DNA in chromatin was methylated with dimethyl sulfate. Methylated purine bases in DNA can be readily excised under mild conditions at neutral pH. Aldehyde groups formed in the depurinated residues of DNA react with the ϵ-amino groups of lysines in histones and crosslink histones to DNA through the Schiff bases. The Schiff bases catalyze the β-elimination reactions causing quantitative single-stranded scissions of DNA at the points of crosslinking in such a way that only the 5′-terminal fragments of DNA formed upon the breakage become attached to histones. These covalent DNA-protein crosslinks are reversible and can be split at pH 3 or stabilized by reduction of the Schiff bases with NaBH 4 .

Discrimination of Bacillus anthracis and Closely Related Microorganisms by Analysis of 16S and 23S rRNA with Oligonucleotide Microarray

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.

Methidium intercalator inserted into synthetic oligonucleotides.

Abstract A new methidium intercalator phosphoramidite has been synthesized. Methidium incorporation into an oligonucleotide during the synthesis was confirmed by UV and MALDI TOF MS data. UV melting experiments showed enhanced stability of a duplex, containing internal methidium.