Quantitative Analysis Of Nucleic Acids Research Articles

Localization and imaging of nucleic acids on nanoporous aluminum oxide membranes.

The technologies currently available for nucleic acid analysis in the clinical laboratory rely on target amplification methods, such as PCR or transcription-mediated amplification, or signal amplification methods, such as branched-chain DNA, hybrid capture, Invader technology, and fluorescent in situ hybridization. In current nucleic acid analyses, a multistep process consisting of sample preparation and purification followed by amplification and detection is required. A comprehensive method for direct localization and visualization of individual molecules would provide an additional method for qualitative or quantitative nucleic acid analysis. We evaluated a nanoporous aluminum oxide membrane (AOM) as a support for direct localization and visualization of individual nucleic acid molecules. The process described here allows nucleic acids to be localized by filtration onto AOM with direct imaging using nucleic acid-binding stains. Additionally, we present a method for modifying the membranes to improve the optical properties. The membranes used in this study are commercially available under the trade name Anopore™ (Whatman International) and are composed of aluminum oxide with a mean pore size of 200 nm and a nominal thickness of 60 μm (1). The mean width between the pores is 50 nm, and the membranes have a pore density of ∼108 pores/cm2. The pores are evenly distributed in a honeycomb pattern across the surface, with a porosity of ∼50%. Because the length of one DNA base pair is 0.34 nm (2)(3), a linear double-stranded DNA molecule will extend ∼600 bp across a single 200 nm pore, and ∼150 bp of the molecule will cover the area between the pores. The uniform pore structure, the relatively rigid and flat surface, and the high porosity and flow rate make AOMs an attractive material for sample filtration and nucleic acid visualization. The surface properties of aluminum oxide allow for chemical modifications to the …

MALDI-TOF MS: a platform technology for genetic discovery

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been applied as a high-throughput platform technology for qualitative and quantitative nucleic acid analysis in the genetic discovery of target genes and their biological validation. Mass spectrometric methods for the elucidation of genetic variability and for subsequent large-scale genotyping of genetic markers are exemplified. The use of quantitative MALDI-TOF MS is described for large-scale validation of SNP markers and their analysis in DNA sample pools. Initial results of genome-wide association studies employing this technology are provided exemplifying a genetics-driven approach to drug discovery.

Quantitative analysis of nucleic acids--the last few years of progress.

DNA and RNA quantifications are widely used in biological and biomedical research. In the last ten years, many technologies have been developed to enable automated and high-throughput analyses. In this review, we first give a brief overview of how DNA and RNA quantifications are carried out. Then, five technologies (microarrays, SAGE, differential display, real time PCR and real competitive PCR) are introduced, with an emphasis on how these technologies can be applied and what their limitations are. The technologies are also evaluated in terms of a few key aspects of nucleic acids quantification such as accuracy, sensitivity, specificity, cost and throughput.

Relative quantitative RT-PCR protocol for TrkB expression in neuroblastoma using GAPD as an internal control.

An RT-PCR protocol for the relative quantitative measurement of TrkB transcripts using glyceraldehyde-3-phosphate-dehydrogenase (GAPD) transcripts as an internal control is described. Both TrkB and GAPD sequences were co-amplified in the exponential phase of amplification using 5'-biotinylated primers. The PCR products were subjected to PAGE, electro-transferred to nylon membrane and detected by a chemiluminescent procedure using alkaline phosphatase conjugated with avidin. Signals detected on X-ray film were analyzed by densitometry. The ratio between TrkB and GAPD expression levels was determined to normalize the expression levels of TrkB transcripts. Initially, strong signals of GAPD transcripts led to overexposure of X-ray film compared to those of TrkB, which causes difficulties in the accurate determination of the TrkB/GAPD ratio. To circumvent this problem, uniformly biotinylated GAPD primers were replaced with a mixture of biotinylated and non-biotinylated GAPD primers of the same sequence and concentration. GAPD signals detected on X-ray film were proportionally decreased as the amount of biotin-labeled primers was reduced in the total GAPD primers. This modification enabled both GAPD and TrkB signals to be analyzed within the linear range of X-ray film detection without affecting the amplification efficiency of TrkB sequence. Use of composite primers may have a wide range of applicability in quantitative analysis of nucleic acids.

Comprehensive quantification of herpes simplex virus latency at the single-cell level.

To date, characterization of latently infected tissue with respect to the number of cells in the tissue harboring the viral genome and the number of viral genomes contained within individual latently infected cells has not been possible. This level of cellular quantification is a critical step in determining (i) viral or host cell factors which function in the establishment and maintenance of latency, (ii) the relationship between latency burden and reactivation, and (iii) the effectiveness of vaccines or antivirals in reducing or preventing the establishment of latent infections. Presented here is a novel approach for the quantitative analysis of nucleic acids within the individual cells comprising complex solid tissues. One unique feature is that the analysis reflects the nucleic acids within the individual cells as they were in the context of the intact tissue-hence the name CXA, for contextual analysis. Trigeminal ganglia latently infected with herpes simplex virus (HSV) were analyzed by CXA of viral DNA. Both the type and the number of cells harboring the viral genome as well as the number of viral genomes within the individual latently infected cells were determined. Here it is demonstrated that (i) the long-term repository of HSV-1 DNA in the ganglion is the neuron, (ii) the viral-genome copy number within individual latently infected neurons is variable, ranging over 3 orders of magnitude from <10 to >1,000, (iii) there is a direct correlation between increasing viral input titer and the number of neurons in which latency is established in the ganglion, (iv) increasing viral input titer results in more neurons with greater numbers of viral-genome copies, (v) treatment with acyclovir (ACV) during acute infection reduces the number of latently infected ganglionic neurons 20-fold, and (vi) ACV treatment results in uniformly low (<10)-copy-number latency. This report represents the first comprehensive quantification of HSV latency at the level of single cells. Beyond viral latency, CXA has the potential to advance many studies in which rare cellular events occur in the background of a complex solid tissue mass, including microbial pathogenesis, tumorigenesis, and analysis of gene transfer.

Technical Considerations for the Use of Ethidium Bromide in the Quantitative Analysis of Nucleic Acids

Technical Considerations for the Use of Ethidium Bromide in the Quantitative Analysis of Nucleic Acids

The polymerase chain reaction: a new tool for the understanding and diagnosis of HIV-1 infection at the molecular level

The polymerase chain reaction (PCR) is at present the most powerful analytical tool for detection of specific nucleic acid sequences. The method is based on the in vitro amplification of DNA segments before detection with conventional hybridization techniques or visualization following electrophoresis and staining. The current diagnostic methods for HIV-1 do not allow easy identification of subgroups of infected patients including infants born to seropositive mothers, individuals with delayed serological responses to the virus, infected patients with indeterminate serology results, and patients with dual retroviral infections. Furthermore, response to antiviral therapy cannot be evaluated with serological assays. The rationale for applying PCR in those situations is elaborated here. The applications of this technique for HIV-1 as a diagnostic test and for the understanding of the pathogenesis of this retrovirus are described. Potential limitations of this technique for diagnostic purposes include mainly the possibility of false-positive results due to contamination and false-negative reactions caused by Taq polymerase inhibition. Non-isotopic means for detection of amplified products have been described and should allow for a wider application of this technology. Modifications of PCR which make use of internal standards seem promising for quantitative analysis of nucleic acids. PCR has great potential for viral diagnosis but still requires further studies and better characterization.

Quantitative analysis of nucleic acids, proteins, and viruses by Raman band deconvolution

A constrained, iterative Fourier deconvolution method is employed to enhance the resolution of Raman spectra of biological molecules for quantitative assessment of macromolecular secondary structures and hydrogen isotope exchange kinetics. In an application to the Pf1 filamentous bacterial virus, it is shown that the Raman amide I band contains no component other than that due to alpha-helix, indicating the virtual 100% helicity of coat proteins in the native virion. Comparative analysis of the amide I band of six filamentous phages (fd, If1, IKe, Pf1, Xf, and Pf3), all at the same experimental conditions, indicates that the subunit helix-percentage ranges from a high of 100% in Pf1 to a low of 71% in Xf. Deconvolution of amide I of Pf3 at elevated temperatures, for which an alpha-to-beta transition was previously reported (Thomas, G. J., Jr., and L. A. Day, 1981, Proc. Natl. Acad. Sci. USA., 78:2962-2966), allows quantitative evaluation of the contributions of both alpha-helix and beta-strand conformations to the structure of the thermally perturbed viral coat protein. Weak Raman lines of viral DNA bases and coat protein side chains, which are poorly resolved instrumentally, are also distinguished for all viruses by the deconvolution procedure. Application to the carbon-8 hydrogen isotope exchange reaction of a purine constituent of transfer RNA permits accurate determination of the exchange rate constant, which is in agreement with calculations based upon curve-fitting methods.