Establishment and Application of Ligation Reaction-Based Method for Quantifying MicroR-156b

Microribonucleic acids (miRNAs) play significant roles in the regulation of biological processes and in responses to biotic or abiotic environmental stresses. Therefore, it is necessary to quantitatively detect miRNAs to understand these complicated biological regulation mechanisms. This study established an ultrasensitive and highly specific method for the quantitative detection of miRNAs using simple operations on the ground of the ligation reaction of ribonucleotide-modified deoxyribonucleic acid (DNA) probes. This method avoids the complex design of conventional reverse transcription. In the developed assay, the target miRNA miR156b was able to directly hybridize the two ribonucleotide-modified DNA probes, and amplification with universal primers was achieved following the ligation reaction. As a result, the target miRNA could be sensitively measured even at a detection limit as low as 0.0001 amol, and differences of only a single base could be detected between miR156 family members. Moreover, the proposed quantitative method demonstrated satisfactory results for overexpression-based genetically modified (GM) soybean. Ligation-based quantitative polymerase chain reaction (PCR) therefore has potential in investigating the biological functions of miRNAs, as well as in supervising activities regarding GM products or organisms.

Nanofluorescence Probes to Detect miR-192/Integrin Alpha 1 and Their Correlations with Retinoblastoma

We developed a novel nanostructure DNA probe for the in situ detection of ITGA1 and miR-192 in retinoblastoma (RB) and to study the correlation between ITGA1 and miR-192 expression and RB development. ITGA1 and miR-192 nanostructure DNA probes were carried by silica particles and coated by dioleoyl-trimethy-lammonium-propane, which enhances their organizational compatibility and infiltration capacity. This probe has stable physicochemical properties and high specificity and sensitivity to detect ITGA1 and miR-192 in situ both in RB cell lines and RB tissues. Using ITGA1 and miR-192 nanostructure DNA probes in RB tissue and cell lines, we found that the expression of ITAG1 drastically increased, but to the contrary, miR-192 was not expressed. After transfection, ITGA1 and miR-192 were overexpressed or silenced in RB116 cells, and we found that ITGA1 could effectively increase the activity and invasion of this RB cell line and reduce its apoptosis level, while miR-192 antagonized this tumor-promoting effect. Therefore, miR-192 can be used as an early biomarker of RB, and ITGA1 may be a new prognostic marker and therapeutic target for the treatment of RB.

3C Based DNA Hybridization Method for Chromosomal Translocation Screening

Background: DNA probes have been widely used as diagnostic tools for chromosomal translocations in malignancies. PCR-based methods often fail to detect translocations such as MYC/TRD in chronic lymphocytic leukemia. In addition, microscopic techniques cannot be helpful due to size detection limitations. This study sought to design a screening tool using immobilized ssDNA probes on a nitrocellulose membrane followed by 3C library fragments hybridization. Results: Hence, we focused on developing a suitable 27 bp specific probe for the juxtaposed region of MYC and TRD. Colloidal gold nanoparticles (AuNP) functionalized translocation fragments of the MYC gene with a thiol group (MYC-AuNP-probe). Then TRD-probes were immobilized on nitrocellulose surface to detect TRD/MYC translocation in the SKW3 cells. Hybridization between DNA probes and 3C-library fragments of SKW3 cells was determined by color intensity. Optimal hybridization of the 3C library sample of the cell line to TRD-probe and MYC-AuNP-probe showed higher color intensity due to their convenient proximity to the juxtaposed region compared with normal cells. Conclusions: Our results demonstrated that DNA hybridization colorimetric assay could be a helpful technique in chromosomal rearrangements screening. Accordingly, the combination of 3C based techniques and DNA-DNA hybridization can identify cancer cells with high specificity and sensitivity.

Biolayer Interferometry for DNA-Protein Interactions v1

Biolayer interferometry (BLI) is a widely utilized technique for determining the interaction dynamics between macromolecules. Most BLI instruments, such as the Octet RED96e used throughout this protocol, are completely automated and detect changes in the interference pattern of white light reflected off a biosensor tip. Biosensors are initially loaded with a stationary macromolecule, then introduced into a solution containing macromolecules of interest. Binding to the stationary molecules creates a change in optical wavelength that is recorded by the instrument in real-time. The majority of published BLI experiments assess protein-protein (such as antibody-substrate kinetics) or protein-small molecule (such as drug discovery) interactions. However, a less-appreciated assay for BLI analysis is DNA-protein interactions. In our laboratory, we have shown the practicality of using biotinylated-DNA probes to determine the binding kinetics of transcription factors to specific DNA sequences. The following protocol describes these steps, including the generation of biotinylated-DNA probes, the execution of the BLI experiment, and data analysis through GraphPad Prism.

3C based DNA hybridization method for chromosomal translocation screening

Background DNA probes have been widely used as diagnostic tools for chromosomal translocations in malignancies. PCR-based methods often fail to detect translocations such as MYC/TRD in chronic lymphocytic leukemia. In addition, microscopic techniques cannot be helpful due to size detection limitations. This study sought to design a screening tool using immobilized ssDNA probes on a nitrocellulose membrane followed by 3C library fragments hybridization. Results Hence, we focused on developing a suitable 27 bp specific probe for the juxtaposed region of MYC and TRD. Colloidal gold nanoparticles (AuNP) functionalized translocation fragments of the MYC gene with a thiol group (MYC-AuNP-probe). Then TRD-probes were immobilized on nitrocellulose surface to detect TRD/MYC translocation in the SKW3 cells. Hybridization between DNA probes and 3C-library fragments of SKW3 cells was determined by color intensity. Optimal hybridization of the 3C library sample of the cell line to TRD-probe and MYC-AuNP-probe showed higher color intensity due to their convenient proximity to the juxtaposed region compare with normal cells. Conclusions Our results demonstrated that DNA hybridization colorimetric assay could be a helpful technique in chromosomal rearrangements screening. Accordingly, the combination of 3C based techniques and DNA-DNA hybridization can identify cancer cells with high specificity and sensitivity.

Characterizing Fluorescence Emissions of Hairpin DNA-Templated Silver Nanoclusters by cDNA Hybridization

Stem-loop hairpin DNA probes have high hybridization specificity and unique selectivity to target molecules such as DNA and small molecules. DNA-templated silver nanoclusters (DNA-AgNCs) has been widely used to detect biomolecules of interest due to the photostable, bright, and efficient methods. In this study, we measured fluorescence emission of hairpin DNA upon hybridization with cDNA and mutant cDNA (cDNA-1) or mutant cDNA containing mismatched bases in the stem region (cDNA-2). Fluorescence intensity of hairpin DNA-AgNCs in the presence of cDNA was 1.80 times higher than that of hairpin DNA-AgNCs alone, but decreased to 66% in the presence of cDNA-1 containing mismatched base corresponding to the hairpin stem region. This study demonstrated that fluorescence intensities of hairpin DNA-AgNCs were dependent on hybridization with either wild-type and mutant cDNAs.

Complex approach to the study of the Derivative Chromosome 8

Введение. Дериватная хромосома (der) - структурно аномальная хромосома, формирование которой может происходить как в результате перестроек с участием двух и более негомологичных хромосом, так и вследствие аберраций внутри одной хромосомы. Дифференциальная диагностика дериватных хромосом очень важна для выяснения происхождения хромосомной аномалии и для определения тактики медико-генетического консультирования с целью оценки повторного риска рождения ребенка с хромосомным дисбалансом. В данной работе представлены семь случаев дериватной хромосомы 8, имеющих различное происхождение и механизмы формирования, а также протокол обследования пациентов с дериватной хромосомой 8 в кариотипе. Цель: изучить структуру и механизмы формирования дериватных хромосом 8. Методы: стандартное цитогенетическое исследование, M-FISH, MCB8, FISH с локус-специфичными субтеломерными ДНК-зондами, FISH с несерийными ДНК-зондами на район р23.1 хромосомы 8. Результаты. В результате проведенного стандартного цитогенетического исследования в кариотипе семи неродственных пробандов была обнаружена дериватная хромосома 8. При использовании цитогенетического и молекулярно-цитогенетического подходов было установлено, что у четырех пациентов дериватная хромосома 8 возникла в результате инвертированной дупликации/делеции 8р, а у трех - несбалансированной транслокации с участием хромосомы 8: der(8)t(8;17), der(8)t(8;12) и der(8)t(7;8). Во всех случаях был определен механизм формирования хромосомных перестроек. Дериватные хромосомы транслокационного происхождения в двух случаях были сформированы de novo, а в одном случае - как результат патологической мейотической сегрегации отцовской реципрокной транслокации. Все дериватные хромосомы с инвертированной дупликацией/делецией 8р были следствием эктопической рекомбинации. Заключение. Представленные результаты демонстрируют целесообразность комплексного лабораторного подхода в изучении структуры и происхождения дериватной хромосомы 8. Характеристика происхождения хромосомного дисбаланса является неотъемлемой частью обследования пациентов со структурно аномальной хромосомой 8 в кариотипе. Background. Derivative chromosome (der) is a structurally abnormal chromosome, the formation of which can occur as a result of rearrangements with the participation of two or more non-homologous chromosomes, or be the result of aberrations within one chromosome. Differential diagnosis of derivative chromosomes is very important for clarifying the origin of the chromosomal abnormality and for determining the tactics of medical genetic counseling in order to assess the repeated risk of chromosomal imbalance. This work presents seven cases of a derivative chromosome with different origins and mechanisms of formation, as well as a protocol for examining patients with derivative chromosome 8 in the karyotype. Aim: to study the structure and mechanisms of formation of the derivative chromosome 8. Methods. GTG-banded chromosomal analysis, M-FISH, MCB8, FISH with subtelomeric DNA probes, FISH with home-made DNA probes for 8p23.1. Results. As a result of a conventional cytogenetic study of seven unrelated probands a derivative chromosome 8 was found. In all cases, the mechanism of the formation of chromosomal rearrangements was determined. Derivative chromosomes of translocation origin were formed de novo in two cases- der(8)t(8;12) and der(8)t(7;8), and in one case -der(8)t(8;17) - as a result of malsegregation of the paternal reciprocal translocation. In the remaining four cases, the derivative chromosomes were identified as an inverted duplication/deletion 8p due to ectopic recombination. Conclusion. The presented results demonstrate the feasibility of an integrated laboratory approach in the diagnosis of derivative chromosome 8. Characterization of the origin of chromosomal imbalance is an integral part of the examination of patients with structurally abnormal chromosome 8 in the karyotype.

New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.