RESUMO
The use of clearing agents has provided new insights in various fields of medical research (developmental biology, neurology) by enabling examination of tissue architecture in 3D. One of the challenges is that clearing agents induce tissue shrinkage and the shrinkage rates reported in the literature are incoherent. Here, we report that for a classical clearing agent, benzyl-alcohol benzyl-benzoate (BABB), the shrinkage decreases significantly with increasing sample size, and present an analytical formula describing this.
Assuntos
Benzoatos/farmacologia , Álcool Benzílico/farmacologia , Imageamento Tridimensional/métodos , Imagem Óptica/métodos , Solventes/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Masculino , Próstata/efeitos dos fármacos , Ratos , Baço/efeitos dos fármacos , Suínos , Fixação de Tecidos/métodosRESUMO
Large variation exists in mitochondrial DNA (mtDNA) not only between but also within individuals. Also in human cancer, tumor-specific mtDNA variation exists. In this work, we describe the comparison of four methods to extract mtDNA as pure as possible from frozen tumor tissue. Also, three state-of-the-art methods for sensitive detection of mtDNA variants were evaluated. The main aim was to develop a procedure to detect low-frequent single-nucleotide mtDNA-specific variants in frozen tumor tissue. We show that of the methods evaluated, DNA extracted from cytosol fractions following exonuclease treatment results in highest mtDNA yield and purity from frozen tumor tissue (270-fold mtDNA enrichment). Next, we demonstrate the sensitivity of detection of low-frequent single-nucleotide mtDNA variants (≤1% allele frequency) in breast cancer cell lines MDA-MB-231 and MCF-7 by single-molecule real-time (SMRT) sequencing, UltraSEEK chemistry based mass spectrometry, and digital PCR. We also show de novo detection and allelic phasing of variants by SMRT sequencing. We conclude that our sensitive procedure to detect low-frequent single-nucleotide mtDNA variants from frozen tumor tissue is based on extraction of DNA from cytosol fractions followed by exonuclease treatment to obtain high mtDNA purity, and subsequent SMRT sequencing for (de novo) detection and allelic phasing of variants.
Assuntos
Neoplasias da Mama/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/isolamento & purificação , Patologia Molecular/métodos , Manejo de Espécimes/métodos , Linhagem Celular Tumoral , Feminino , Congelamento , Humanos , Espectrometria de Massas , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
We demonstrate on-chip, differential DNA and RNA extraction from a single cell using a microfluidic chip and a two-stage lysis protocol. This method enables direct use of the whole extract, without additional washing steps, reducing sample loss. Using this method, the tumor driving pathway in individual cells from a colorectal cancer cell line was determined by applying a Bayesian computational pathway model to sequences obtained from the RNA fraction of a single cell and, the mutations driving the pathway were determined by analyzing sequences obtained from the DNA fraction of the same single cell. This combined functional and mutational pathway assessment of a single cell could be of significant value for dissecting cellular heterogeneity in tumors and analyzing single circulating tumor cells.
Assuntos
DNA/isolamento & purificação , Redes Reguladoras de Genes , Microfluídica/métodos , RNA/isolamento & purificação , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Misturas Complexas/análise , Misturas Complexas/isolamento & purificação , DNA/análise , Humanos , RNA/análiseRESUMO
Hybridization of nucleic acids to microarrays is a crucial step for several biological and biomedical applications. However, the poor efficiency and resulting long incubation times are major drawbacks. In addition to diffusion limitation, back hybridization to complementary strands in solution is shown to be an important cause of the low efficiency. In this paper, repeated denaturing in an integrated device has been investigated in order to increase the efficiency of microarray hybridization. The sample solution is circulated from the microarray chamber over a denaturing zone and back in a closed loop. In addition to the improved binding rate due to flow, repeated denaturing significantly increases the total amount of molecules bound. Our results demonstrate that cyclic repeated denaturing improves the efficiency of hybridization by up to an order of magnitude over a broad range of concentrations studied (1 pM to 100 nM).
Assuntos
DNA/química , DNA/isolamento & purificação , Técnicas Analíticas Microfluídicas/instrumentação , Hibridização de Ácido Nucleico/métodos , Desnaturação de Ácido Nucleico , Soluções , Temperatura , Fatores de TempoRESUMO
The use of a priori knowledge in the alignment of targeted sequencing data is investigated using computational experiments. Adapting a Needleman-Wunsch algorithm to incorporate the genomic position information from the targeted capture, we demonstrate that alignment can be done to just the target region of interest. When in addition use is made of direct string comparison, an improvement of up to a factor of 8 in alignment speed compared to the fastest conventional aligner (Bowtie) is obtained. This results in a total alignment time in targeted sequencing of around 7 min for aligning approximately 56 million captured reads. For conventional aligners such as Bowtie, BWA or MAQ, alignment to just the target region is not feasible as experiments show that this leads to an additional 88% SNP calls, the vast majority of which are false positives (≈ 92%).
Assuntos
Algoritmos , Genômica/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de DNA , Polimorfismo de Nucleotídeo ÚnicoRESUMO
In this review, we discuss the latest targeted enrichment methods and aspects of their utilization along with second-generation sequencing for complex genome analysis. In doing so, we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a powerful tool. We explain how targeted enrichment for next-generation sequencing has made great progress in terms of methodology, ease of use and applicability, but emphasize the remaining challenges such as the lack of even coverage across targeted regions. Costs are also considered versus the alternative of whole-genome sequencing which is becoming ever more affordable. We conclude that targeted enrichment is likely to be the most economical option for many years to come in a range of settings.
Assuntos
Genoma , Genômica/métodos , Análise de Sequência de DNA/métodos , Animais , Sequência de Bases , HumanosRESUMO
Visual color changes between 300 and 510 K were observed in the photoluminescence (PL) of colloidal InP/ZnS core-shell nanocrystals. A subsequent study of PL spectra in the range 2-510 K and fitting the temperature dependent line shift and line width to theoretical models show that the dominant (dephasing) interaction is due to scattering by acoustic phonons of about 23 meV. Low temperature photoluminescence excitation measurements show that the excitonic band gap depends approximately inversely linearly on the quantum dot size d, which is distinctly weaker than the dependence predicted by current theories.
RESUMO
In studying well-characterized, exchange-biased Fe(3)O(4)/CoO superlattices, we demonstrate a causal link between the exchange bias effect and the perpendicular coupling of the ferrimagnetic and antiferromagnetic spins. Neutron diffraction studies reveal that for thin CoO layers the onset temperature for exchange biasing T(B) matches the onset of locked-in, preferential perpendicular coupling of the spins, rather than the antiferromagnetic ordering temperature T(N). The results are explained by considering the role of anisotropic exchange first proposed by Dzyaloshinsky and Moriya and developing a model based purely on information on structural defects and exchange for these oxides. The devised mechanism provides a general explanation of biasing in systems with perpendicular coupling.
RESUMO
The blocking temperature T(B) has been determined as a function of the antiferromagnetic layer thickness in the Fe3O4/CoO exchange biased system. For CoO layers thinner than 50 A, T(B) is reduced below the Néel temperature T(N) of bulk CoO (291 K), independent of crystallographic orientation or film substrate ( alpha-Al2O3, SrTiO3, and MgO). Neutron diffraction studies show that T(B) does not track the CoO ordering temperature and, hence, that this reduction in T(B) does not arise from finite-size scaling. Instead, the ordering temperature of the CoO layers is enhanced above the bulk T(N) for layer thicknesses approximately less than or equal to 100 A due to the proximity of magnetic Fe3O4 layers.