[Ru(bpy)2dppz]2+ electrochemiluminescence switch and its applications for DNA interaction study and label-free ATP aptasensor

[Ru(bpy)2dppz]2+ electrochemiluminescence (ECL) was studied, and it was used to investigate DNA interaction and develop a label-free ATP aptasensor for the first time. ECL of [Ru(bpy)2dppz]2+ is negligible in aqueous solution, and increases approximately 1000 times when [Ru(bpy)2dppz]2+ intercalates into the nucleic acid structure. The ECL switch behavior of [Ru(bpy)2dppz]2+ is ascribed to the intercalation that shields the phenazine nitrogens from the solvent and results in a luminescent excited state. The ECL switch by DNA was applied to investigate the interaction of [Ru(bpy)2dppz]2+ with herring sperm DNA. The calculated equilibrium constant (K) is 1.35 x 10(6) M(-1), and the calculated binding-site size (s) is 0.88 base pair, which is consistent with the reported values.

Effects of 17-α-ethynylestradiol on Hybrid Striped Bass Sperm

Gemstone Team FISH

Reduced sperm yield from testicular biopsies of vasectomized men is due to increased apoptosis

Objective: To compare sperm yields, apoptotic indices, and sperm DNA fragmentation from vasectomized men and fertile men undergoing vasectomy.

Design: Testicular biopsies from vasectomized (n 26) and fertile men (n 46), were milked to calculate sperm/gram and also formalin-?xed to determine the numbers of developing sperm and incidence and intensities of testicular FasL, Fas, Bax, and Bcl-2. Testicular sperm DNA fragmentation was assessed using the alkaline Comet assay.

Setting: An ART unit.

Patient(s): Twenty-six men attending for intracytoplasmic sperm injection (ICSI) and 46 men attending for vasectomies.

Main Outcome Measure(s): Spermatocyte, spermatid and sperm yields, Fas, FasL, and Bax staining.

Result(s): Sperm yields from men vasectomized 5 years previously were markedly reduced compared to fertile men. Increased intensities of FasL and Bax staining were observed in the seminiferous tubules of vasectomy men. FasL positivity (percentage) also increased in Sertoli cells, and both FasL and Fas positivity (percentage) increased in primary spermatocytes and round spermatids of vasectomized men. Sperm DNA fragmentation, an end point marker of apoptosis, increased signi?cantly in vasectomized men compared to fertile men.

Conclusion(s): Reduced sperm yields after vasectomy are associated with increased apoptosis through the Fas–FasL and Bax pathways. Sperm after vasectomy displayed increased DNA fragmentation. (Fertil Steril 2007;87:834–41. ©2007 by American Society for Reproductive Medicine.)

Increased concentrations of the oxidative DNA adduct 7, 8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 105 molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 105 molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.

Comparison of serum and whole-blood specimens for the detection of Candida DNA in critically ill, non-neutropenic patients

In contrast to the multitude of studies on fungal PCR assay methods, little work has been reported evaluating Candida PCR performance when using whole blood compared with serum in candidaemic patients. Here, a comparison of the performance of whole-blood and serum specimens using a set of real-time PCR Candida species assays is described. Specimens were collected prospectively from non-neutropenic adults who were recruited to a diagnostic clinical trial, the primary purpose of which was to verify the performance of the assays using serum; in all, 104 participants also had whole-blood specimens submitted for analysis in addition to the serum specimen. Of these participants, 10 had laboratory-confirmed candidaemia and 94 were categorized as being 'unlikely' to have invasive Candida infection. PCR results from the whole-blood specimens are presented here and compared with the results from serum specimens in this subgroup among whom both specimen types were obtained contemporaneously. All participants with candidaemia were PCR-positive from serum samples; however, only seven were PCR-positive from whole blood. All specimens from patients in the 'unlikely' category were PCR-negative in both types of specimen. Moreover, DNA extraction from serum required 1 h; extraction from whole blood required approximately 3 h. These data tentatively suggest that, overall, serum is an appropriate specimen for Candida PCR for detection of candidaemia in non-neutropenic adults.

Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus , is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the Msp I satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus , up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

DGGE with genomic DNA: Suitable for detection of numerically important organisms but not for identification of the most abundant organisms

Identification of all important community members as well as of the numerically dominant members of a community are key aspects of microbial community analysis of bioreactor samples. A systematic study was conducted with artificial consortia to test whether denaturing gradient gel electrophoresis (DGCE) is a reliable technique to obtain such community data under conditions where results would not be affected by differences in DNA extraction efficiency from cells. A total of 27 consortia were established by mixing DNA extracted from Escherichia coli K12, Burkholderia cepacia and Stenotrophomonas maltophilia in different proportions. Concentrations of DNA of single organisms in the consortia were either 0.04, 0.4 or 4 ng/mu l. DGGE-PCR of genomic DNA with primer sets targeted at the V3 and V6-V8 regions of the 16S rDNA failed to detect the three community members in only 7% of consortia, but provided incorrect information about dominance or co-dominance for 85% and 89% of consortia with the primer sets for the V6-V8 and V3 regions, respectively. The high failure rate in detection of dominant B. cepacia with the primers for the V6-V8 region was attributable to a single nucleoticle primer mismatch in the target sequences of both, the forward and reverse primer. Amplification bias in PCR of E. coli and S. maltophilia for the V6-V8 region and for all three organisms for the V3 region occurred due to interference of genomic DNA in PCR-DGGE, since a nested PCR approach, where PCR-DGGE was started from mixtures of 16S rRNA genes of the organisms, provided correct information about the relative abundance of original DNA in the sample. Multiple bands were not observed in pure culture amplicons produced with the V6-V8 primer pair, but pure culture V3 DGGE profiles of E. coli, S. maltophilia and B. cepacia contained 5, 3 and 3 bands, respectively. These results demonstrate DGGE was suitable for identification of all important community members in the three-membered artificial consortium, but not for identification of the dominant organisms in this small community. Multiple DGGE bands obtained for single organisms with the V3 primer pair could greatly confound interpretation of DGGE profiles. (C) 2008 Elsevier Ltd. All rights reserved.

Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent

The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at -20 degrees C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human P-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully. (c) 2007 Elsevier B.V. All rights reserved.

Assessment of the quality of dna extracted by two techniques from Mycobacterium tuberculosis for fast molecular identification and genotyping

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Extração de DNA de materiais de arquivo e fontes escassas para utilização em reação de polimerização em cadeia (PCR)

Este trabalho visou a comparação de cinco métodos diferentes de extração de DNA de materiais de arquivo (tecidos incluídos em parafina, esfregaços de sangue periférico - corados e não corados com Leishman, lâminas com mielogramas, gotas de sangue em Guthrie Card) e de fontes escassas (células bucais, um e três bulbos capilares e 2 mL de urina), para que fossem avaliadas a facilidade de aplicação e a facilidade de amplificação deste DNA pela técnica da reação de polimerização em cadeia (PCR). Os métodos incluíram digestão por proteinase K, seguida ou não por purificação com fenol/clorofórmio; Chelex 100® (BioRad); Insta Gene® (BioRad) e fervura em água estéril. O DNA obtido foi testado para amplificação de três fragmentos gênicos: Brain-derived neutrophic factor (764 pb), Factor V Leiden (220 pb) e Abelson (106 pb). de acordo com o comprimento do fragmento gênico estudado, da fonte potencial de DNA e do método de extração utilizado, os resultados caracterizaram o melhor caminho para padronização de procedimentos técnicos a serem incluídos no manual de Procedimentos Operacionais Padrão do Laboratório de Biologia Molecular do Hemocentro - HC - Unesp - Botucatu.

Sperm Organelle Morphologic Abnormalities: Contributing Factors and Effects on Intracytoplasmic Sperm Injection Cycles Outcomes

OBJECTIVE To (1) analyze possible relationships between motile sperm organelle morphology examination (MSOME) and sperm chromatin status, aneuploidy incidence, and patient's age; (2) determine the effects of sperm morphologic abnormalities on intracytoplasmic sperm injection (ICSI) outcomes; and (3) identify the benefits of intracytoplasmic morphologically selected sperm injection (IMSI) in patients with high DNA fragmentation rate.METHODS The study was performed in 50 patients undergoing ICSI cycles. The MSOME, sperm DNA fragmentation, and sperm aneuploidy incidence were performed in 200 sperm cells of each patient. Regression models were used to assess the relationships among sperm morphology and sperm aneuploidy, sperm DNA fragmentation, patient's age, and ICSI outcomes. In cycles with patients showing a high incidence of DNA fragmentation, oocytes were split into 2 groups according to the sperm selection method: Standard-ICSI (n = 82) and IMSI (n = 79). Fertilization and high-quality embryo rates were compared between the groups.RESULTS A close relationship between sperm DNA fragmentation and the presence of vacuoles in the MSOME was noted. The patient's age was correlated to the presence of vacuoles. No correlation between sperm aneuploidy and IMSI was observed. Vacuolated cells were negatively correlated with fertilization, pregnancy, and implantation. In patients with a high incidence of sperm DNA fragmentation, fertilization and high-quality embryo rates were similar when comparing IMSI and Standard-ICSI.CONCLUSIONS Our data demonstrate a correlation between paternal age and the incidence of nuclear vacuoles, as well as an effect of large and small vacuoles on late embryo development. UROLOGY 78: 786-791, 2011. (C) 2011 Elsevier B.V.

Avaliação de dois métodos de extração de DNA de material parafinado para amplificação em PCR

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Coagglutination for viral DNA preparation of canine parvovirus for molecular diagnosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Suitability of DNA extracted from archival specimens of fruit-eating bats of the genus Artibeus (Chiroptera, Phyllostomidae) for polymerase chain reaction and sequencing analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)