Quantitative DNA Analysis Research Articles

A New Electrochemical Biosensor of Host-guest Recognition Mode for DNA Hybridization Detection

This paper reports on an electrochemical biosensor to detect the hybridization specificity by using host-guest recognition technique. A hairpin DNA with a dabcyl molecule which is typical guest molecule to b-Cyclodextrins (b-CD) at the 3’-terminus and a NH2 linked at the 5’-terminus as the probe DNA. The probe DNA was immobilized on the PdS nanoparticle to construct a double-labeled probe (DLP) and could selectively hybridize with its target DNA. A b-CD modified Poly(N-acetylaniline) glassy carbon electrode was used for capturing dabcyl in DLP. Without binding with target DNA, the DLP keep stem-loop structure and block dabcyl enter into the cavity of b-CD on electrode. However, a target-binding DLP is incorporated into double stranded DNA, causing loop-stem structure opened and dabcy l could be easy captured by b-CD which brought DLP on electrode surface. With electrochemical measurement, the signal come from Pd2+ be used for target DNA quantitative analysis.

Ordered silicon nanocones arrays for label-free DNA quantitative analysis by surface-enhanced Raman spectroscopy

Ordered vertical silicon nanocones arrays coated with silver nanoparticles (AgNPs@SiNCs) are developed as surface-enhanced Raman scattering (SERS)-active substrate, which features good uniformity and reliable reproducibility of SERS signals. Label-free DNA at low concentrations (10−8 M) could be quantitatively analyzed via SERS using the AgNPs@SiNCs. The Raman peak at 732 cm−1 due to adenine breathing mode was selected as an endogenous Raman marker for quantitative detection of label-free DNA. The AgNPs@SiNCs as high-performance SERS-active substrates are attractive for surface enhancement mechanism investigation and biochemical sensing applications.

Identifikasi Polimorfisme Gen GDF-9 dan BMP-15 pada Kambing Kacang

The aim of this research was to determine the polymorphisms of BMP-15 and GDF-9 gene of Kacang goat. DNA was isolated from blood samples of five female goats with salting out method. Quantitative and qualitative analysis of DNA was measured using spectrophotometer and agarose gel electrophoresis. To get DNA fragment GDF-9 gene was amplified using Forward 5'-AGTTTGTACTGAGCAGGTCT -3 ', Reverse 5'-ACTCCGCTTCGTATGTCAG-3', while to get DNA fragmen BMP-15 gene was amplified using Forward primer 5’-CAAGGAGGGGACCCCTAAAT-3’, reverse primer 5’- ACCAGAGGCTCAAGAGGAGC-3 for GDF-9. The results of amplification was a specific DNA band with the length of 1400 bp for GDF-9 gene and 400 bp for BMP-15 gene. Restriction with HaeIII enzyme of GDF-9 PCR product was resulted a haplotype with fragment size 1100 bp, 300 bp, and 100 bp. While, restriction with AluI enzymes of BMP-15 PCR product was resulted a haplotype which consist of two fragments with sizes 180 bp and 120 bp. It was concluded that there are no polymorphism of GDF-9 and BMP-15 gene of Goat Beans.

Quantitative analysis of specific target DNA oligomers using a DNA-immobilized packed-column system

Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on T(m) values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar T(m) values were separated and detected quantitatively by using a packed column carrying two different DNA probes.

Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx

Hepatitis B virus (HBV) is one of the major pathogens of human liver disease. Studies have shown that HBV X protein (HBx) plays an important role in promoting viral gene expression and replication. In this study we performed a global proteomic profiling to identify the downstream functional proteins of HBx, thereby detecting the mechanisms of action of HBx on virion replication. HBx in the HepG2.2.15 cell line was knocked down by the transfection of small interfering RNA (siRNA). The replication level of HBV was evaluated by microparticle enzyme immunoassay analysis of HBsAg and HBeAg in the culture supernatant, and real-time quantitative PCR analysis of HBV DNA. Two-dimensional electrophoresis combined with MALDI-TOF/TOF was performed to analyze the changes in protein expression profile after treatment with HBx siRNA. Knockdown of HBx disturbed HBV replication in vitro. HBx target siRNA significantly inhibited the expression of HBsAg, HBeAg and the replication of HBV DNA. Twelve significantly changed proteins (7 upregulated and 5 downregulated) were successfully identified by MALDI-TOF/TOF using proteomics differential expression analysis after the knockdown of HBx. Among these identified proteins, HSP70 was validated by Western blotting. The results of the study indicated the positive effect of HBx on HBV replication, and a group of downstream target proteins of HBx may be responsible for this effect.

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.

Quantitative variation analysis of fetal DNA in maternal plasma samples collected before and after amniocentesis

The aim of this study was to evaluate possible procedure-related variations in the levels of cell-free fetal DNA (fDNA) in maternal plasma of women undergoing genetic amniocentesis. Blood samples were collected at 16-18 weeks' gestation from 33 pregnant women attending the Fetal Medicine Unit for genetic amniocentesis. For each woman, two blood samples were obtained: the first immediately before amniocentesis and the second one 15 min after the procedure. A real-time quantitative polymerase chain reaction assay, using primers for SRY and beta-globin genes, was used to assess fDNA concentrations in maternal plasma. A Wilcoxon signed-rank test was used for statistical analysis. The karyotype on cultured amniocytes showed that 15 out of 33 women had a male fetus. Real-time quantitative polymerase chain reaction results, on maternal plasma sample pairs from known male pregnancies, showed no significant variations of fDNA correlated to amniocentesis (P=0.394). Our preliminary study suggests that amniocentesis, although invasive, could be associated with minimal, if any, disruption at the fetal-maternal interface, as revealed by the lack of substantial modifications of fDNA levels in maternal circulation.

The Quest for Accurate Measurement of Fetal DNA in Maternal Plasma

Featured Article: Lo YMD, Tein MSC, Lau TK, Haines CJ, Leung TN, Poon PMK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768–75.3 There has been a multidecade search for noninvasive methods of prenatal diagnosis. In 1997, my group reported the presence of cell-free fetal DNA in maternal plasma and serum (1), opening up exciting possibilities that this new noninvasive source of fetal genetic materials might be used for prenatal testing. Much remained unknown, however, regarding this newly discovered phenomenon. For example, it was unclear when fetal DNA first entered into the maternal circulation and whether its concentration would be sufficient for robust diagnostic assays to be developed. The 1998 report featured here represented the first attempt to address these and other issues. I decided to use the then-new real-time quantitative PCR (2) as a tool for measuring fetal and total DNA in …

Trophic Effects of Mesenchymal Stem Cells Increase Chondrocyte Proliferation and Matrix Formation

Previous studies showed that coculture of primary chondrocytes (PCs) with various sources of multipotent cells results in a higher relative amount of cartilage matrix formation than cultures containing only chondrocytes. The aim of this study was to investigate the mechanism underlying this observation. We used coculture pellet models of human mesenchymal stem cells (hMSCs) and human PCs or bovine PCs (bPCs) and studied the fate and the contribution to cartilage formation of the individual cell populations during coculture. Enhanced cartilage matrix deposition was confirmed by histology and quantification of total glycosaminoglycan deposition. Species-specific quantitative polymerase chain reaction demonstrated that cartilage matrix gene expression was mainly from bovine origin when bPCs were used. Short tandem repeat analysis and species-specific quantitative polymerase chain reaction analysis of genomic DNA demonstrated the near-complete loss of MSCs in coculture pellets after 4 weeks of culture. In coculture pellets of immortalized MSCs and bPCs, chondrocyte proliferation was increased, which was partly mimicked using conditioned medium, and simultaneously preferential apoptosis of immortalized MSCs was induced. Taken together, our data clearly demonstrate that in pellet cocultures of MSCs and PCs, the former cells disappear over time. Increased cartilage formation in these cocultures is mainly due to a trophic role of the MSCs in stimulating chondrocyte proliferation and matrix deposition by chondrocytes rather than MSCs actively undergoing chondrogenic differentiation.

CNS-targeted Viral Delivery of G-CSF in an Animal Model for ALS: Improved Efficacy and Preservation of the Neuromuscular Unit

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons. We have recently uncovered a new neurotrophic growth factor, granulocyte-colony stimulating factor (G-CSF), which protects α-motoneurons, improves functional outcome, and increases life expectancy of SOD-1 (G93A) mice when delivered subcutaneously. However, chronic systemic delivery of G-CSF is complicated by elevation of neutrophilic granulocytes. Here, we used adeno-associated virus (AAV) to directly target and confine G-CSF expression to the spinal cord. Whereas intramuscular injection of AAV failed to transduce motoneurons retrogradely, and caused a high systemic load of G-CSF, intraspinal delivery led to a highly specific enrichment of G-CSF in the spinal cord with moderate peripheral effects. Intraspinal delivery improved motor functions, delayed disease progression, and increased survival by 10%, longer than after systemic delivery. Mechanistically, we could show that G-CSF in addition to rescuing motoneurons improved neuromuscular junction (NMJ) integrity and enhanced motor axon regeneration after nerve crush injury. Collectively, our results show that intraspinal delivery improves efficacy of G-CSF treatment in an ALS mouse model while minimizing the systemic load of G-CSF, suggesting a new therapeutic option for ALS treatment.

Growth ofCoxiella burnetiiin theIxodes scapularis–Derived IDE8 Tick Cell Line

Q fever, a zoonotic disease, is caused by a gram-negative intracellular bacterium, Coxiella burnetii. Although normally transmitted during exposure to infectious aerosols, C. burnetii is also found in arthropod vectors. In the environment, ticks are thought to play a crucial role in bacterial maintenance and transmission by infecting various mammalian species. However, the nature of the pathogen-tick relationship is not well defined. To determine C. burnetii's interactions with a cultured tick cell line, we introduced purified C. burnetii NMII into Ixodes scapularis-derived IDE8 cells and assayed for bacterial presence, replication, gene expression, and subsequent infectivity for mammalian cells. Tick cells were harvested at 24 h, 72 h, 7 days, and 11 days postinfection (PI). C. burnetii uptake and subsequent replication was demonstrated by indirect immunofluorescence assay, electron microscopy, and real-time polymerase chain reaction (PCR). When a genome equivalent multiplicity of infection of 30 was used, 30%-40% of exposed cells were seen to have small, rounded, vacuoles at 72 h PI, whereas at 7 and 11 days PI, 60%-70% of cells contained enlarged vacuoles harboring large numbers of bacteria. Quantitative PCR analysis of total genomic DNA confirmed that C. burnetii genome numbers increased significantly from 24 h to 11 days PI. Expression of C. burnetii type four secretion system homologs at 7 days PI was demonstrated by reverse transcriptase PCR. Finally, indirect immunofluorescence assay demonstrated that C. burnetii propagated within IDE8 cells were infectious for mammalian cells. These studies demonstrate the utility of cultured tick cell lines as a model to investigate C. burnetii's molecular interactions with its arthropod vectors.

FRET-Based Assimilating Probe for Sequence-Specific Real-Time Monitoring of Loop-Mediated Isothermal Amplification (LAMP)

Isothermal nucleic acid amplification offers compelling opportunities for simple gene-based diagnostics, although the technologies for monitoring these reactions in real-time are still relatively undeveloped compared to PCR-specific probes. In this article, we describe the adaptation of an innovative FRET-based probe architecture for monitoring the isothermal LAMP reaction in real-time, allowing single-step, direct identification of different isolates of the agricultural pathogen Ralstonia solanacearum. The probe design is analogous to a two-component molecular zipper used for monitoring rolling circle amplification (RCA), in which a quenching oligonucleotide strand is displaced from a fluorescent strand by a strand-displacing polymerase during the reaction. Because the fluorescent strand is stably assimilated into the LAMP amplicon in our configuration, we use the nomenclature assimilating probe to describe the probe. Using these probes, we have demonstrated the ability to perform real-time, quantitative LAMP analysis of DNA, detecting fewer than ten gene copies. Probe compositions were optimized to prevent noticeable inhibition of the LAMP reaction, allowing observation of kinetics for different LAMP reactions and inference of primer design rules for accelerated detection. The sequence-specific nature of the assimilating probes offers compelling advantages over non-specific methods for real-time detection of LAMP amplification, including improved selectivity and the potential for multiplexed detection.

Development and Evaluation of a Perfusion Decellularization Porcine Heart Model - Generation of 3-Dimensional Myocardial Neoscaffolds -

Reports about the generation of 3-dimensional neoscaffolds for myocardial tissue engineering are limited. The architecture provided by perfusion decellularization of whole hearts would support the production of human-sized 3-dimensional living tissues from an acellular matrix. The aim of this study was to evaluate the potential of a perfusion decellularization model for whole heart tissue engineering. Hearts were obtained from 12 German Landrace pigs from a selected abattoir. After preparation, the hearts were mounted and perfused on a modified Langendorff decellularization model specifically constructed for this reason. Decellularization was achieved by an ionic detergent-based perfusion protocol. The quality of the decellularization process was quantified by histology and fluorescence microscopy. Data regarding the presence of residual DNA within the decellularized hearts was measured with spectrophotometric quantification and compared to controls. After histological examination, all hearts lacked intracellular components but retained various types of collagen, proteoglycan and elastin. Quantitative DNA analysis demonstrated a significant reduction of DNA in decellularized hearts compared to controls (84.32±3.99 ng DNA/mg tissue vs. 470.13±18.77 ng DNA/mg tissue (P<0.05)). The modified Langendorff perfusion decellularization model described here is applicable for whole porcine hearts by removing cellular content and DNA. The resulting 3-dimensional matrix provides an interesting tool for further studies in the field of whole heart tissue engineering.

Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver

Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here we investigated if incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3′ untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that 5 copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and β-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23–fold in the liver. However, gene expression in other tissues including the heart was not inhibited. Similarly, we inserted 4 copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3′ UTR of the AAV-luciferase vector. We wished to see if they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly due to promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that live-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in non-hematopoietic tissues resulted from promoter shut-off.

Quantitative Detection of Glyphosate by Simultaneous Analysis of UV Spectroscopy and Fluorescence Using DNA-Labeled Gold Nanoparticles

A sandwich-type immunosensor composed of antigen-double target/probe DNA-coated gold nanoparticles (NPs) was developed for the measurement of fluorescence intensity and quantitative analysis of single-stranded DNA based on the concentration of free glyphosate. The reaction between the antigen-double DNA-gold NPs and immobilized antibody on the substrate was carried out for 2 h. The results of the antigen-antibody reaction were measured on the basis of the fluorescence intensity obtained from comparison with the free antigens at concentrations of 0.01-100 μg mL(-1) for the detection of immobilized antigen-double DNA-gold NPs. For the quantitative analysis based on the concentration of glyphosate(0.01-100 μg mL(-1)), the immunosensor response also revealed the same detection range of glyphosate using DNA detection.

Direct quantification of mitochondrial DNA and its 4.9-kb common deletion without DNA purification

Quantitative analysis of mitochondrial DNA (mtDNA) and its common deletion (CD) are sensitive and early markers for mitochondrial mutations and suffering. However, the use of purified DNA can lead to quantification errors because of variable DNA extraction yields due to the significant differences in size and structure between genomic DNA (gDNA) and mtDNA. We report a real-time qPCR-based protocol directly on tissue lysate, without DNA extraction. This method, which allows both absolute and relative measure, increases the measuring accuracy of the mtDNA/gDNA ratio and leads to reliable and more reproducible results when measuring the deleted/total mtDNA ratio.

Two Novel Homozygous SACS Mutations in Unrelated Patients Including the First Reported Case of Paternal UPD as an Etiologic Cause of ARSACS

Autosomal recessive spastic ataxia of Charlevoix-Saguenay, more commonly known as ARSACS, is an early-onset cerebellar ataxia with spasticity, amyotrophy, nystagmus, dysarthria, and peripheral neuropathy. SACS is the only gene known to be associated with the ARSACS phenotype. To date, 55 mutations have been reported; of these, only five in Italian patients. We found two novel homozygous nonsense mutations in the giant exon of SACS gene in two unrelated patients with classical ARSACS phenotype. Characterization of the homozygous nature of the mutations through genotyping of the parents, quantitative DNA analysis and indirect STS studies permitted us to confirm in one of the cases that uniparental isodisomy of the paternal chromosome 13 carrying the mutated SACS gene played an etiologic role in the disease.

Tissue tropism of the Musca domestica salivary gland hypertrophy virus

The tissue tropism of Musca domestica salivary gland hypertrophy virus (MdSGHV) infecting adult house flies was examined by transmission electron microscopy (TEM) and quantitative real-time PCR. TEM demonstrated that characteristic MdSGHV-induced nuclear and cellular hypertrophy was restricted to the salivary glands. Both nucleocapsids and enveloped virions were present in salivary gland cells. In contrast, thin sections of midguts, ovaries, abdominal fat body, crops, air sacs and brains showed the presence of enveloped virions in vacuoles of tracheal cells associated with these tissues. However, no sites of viral morphogenesis were detected in the tracheal cells. Quantitative analysis of MdSGHV DNA and transcript titers revealed that viral DNA was present in all hemolymph and tissue samples collected from MdSGHV-infected flies. Average numbers of MdSGHV genome copies per 50 ng of DNA varied significantly between examined tissues and ranged from 3.83 × 10 8 (±3.75 × 10 7) in salivary gland samples to 7.98 × 10 5 (±2.91 × 10 5) in hemolymph samples. High levels of viral genome copies were detected in midgut, fat body and brain samples. Viral transcripts were present in all examined samples, and transcript abundance was also at the highest level in salivary glands and at the lowest level in hemolymph. However, over the range of different tissues that were analyzed, there was no correlation between estimated quantities of genome copies and viral transcripts. The function of viral transcripts in host tissues that do not show sites of viral morphogenesis remains to be elucidated.

Comparing On-Site to Off-Site Biomass Collection for Dehalococcoides Biomarker Gene Quantification To Predict in Situ Chlorinated Ethene Detoxification Potential

Biostimulation and bioaugmentation have emerged as constructive remedies for chlorinated ethene-contaminated aquifers, and a link between Dehalococcoides (Dhc) bacteria and chlorinated ethene detoxification has been established. To quantify Dhc biomarker genes, groundwater samples are shipped to analytical laboratories where biomass is collected on membrane filters by vacuum filtration for DNA extraction and quantitative real-time PCR analysis. This common practice was compared with a straightforward, on-site filtration approach to Sterivex cartridges. In initial laboratory studies with groundwater amended with known amounts of Dhc target cells, Sterivex cartridges yielded one-third of the total DNA and 9-18% of the Dhc biomarker gene copies compared with vacuum filtration. Upon optimization, DNA yields increased to 94 +/- 38% (+/-SD, n = 10), and quantification of Dhc biomarker genes exceeded the values obtained with the vacuum filtration procedure up to 5-fold. Both methods generated reproducible results when volumes containing >10(4) total Dhc target gene copies were collected. Analysis of on-site and off-site biomass collection procedures corroborated the applicability of the Sterivex cartridge for Dhc biomarker quantification in groundwater. Ethene formation coincided with Dhc cell titers of >2 x 10(6) L(-1) and high (i.e., >10(5)) abundance of the vinyl chloride reductive dehalogenase genes vcrA and/or bvcA; however, high Dhc cell titers alone were insufficient to predict ethene formation. Further, ethene formation occurred at sites with high Dhc cell titers but low or no detectable vcrA or bvcA genes, suggesting that other, not yet identified vinyl chloride reductive dehalogenases contribute to ethene formation. On-site biomass collection with Sterivex cartridges avoids problems associated with shipping groundwater and has broad applicability for biomarker monitoring in aqueous samples.

Heat shock memory in preimplantation mouse embryos

To investigate the consequences of possible physiologic stress to embryos caused by in vitro fertilization procedures, we used heat shock response in preimplantation mouse embryos as a model. A heat shock "memory" was discovered that renders cleavage-stage embryos more responsive at the transcriptional level to secondary perturbation with very low doses of heat, even several cell cycles after the initial stress has occurred.