Background DNA Research Articles

Clonal Heavy and Light Chain Immunoglobulin DNA in Plasma/Serum of AIDS Lymphoma Patients.

Background DNA tumor markers in plasma and serum such as point mutations, hypermethylation of tumor suppressor genes, and viral sequences have attracted much interest. We investigated clonal Ig DNA rearrangements in the plasma of AIDS-related lymphoma (ARL) patients (pts).Methods We evaluated specimens for Ig rearrangements from pts with ARL using fluorescently-labeled multiplex primers targeting IgH and IgK. PCR products were analyzed by capillary electrophoresis. Re-amplified PCR products from plasma and tumor were cloned and sequenced.Results Clonal IgH DNA spikes were seen in plasma at baseline in 5 of 10 pts. Clonal DNA was detected in plasma in pts with either early or advanced stage disease, with or without marrow involvement. Presence of a plasma clone was associated with high LDH. Serial specimens were available from 4 pts. In 2 pts who died with primary refractory disease, clonal peaks remained present after the administration of chemotherapy. In a pt with disease that initially responded and then relapsed, clonal DNA disappeared with chemotherapy, then reappeared presaging relapse and death. In 4 pts, matching serum specimens were available. Results were similar in both types of specimens with identical sized rearrangements amplified from matched specimens. Furthermore in 2 pts, the IgH amplification products from plasma and tumor were sequenced and confirmed to be identical. Two pts with positive IgH amplification products were also screened for light chains in the plasma and in both, clonal IgK rearrangements were detected. For purposes of comparison, plasma from healthy donors (n=20) and AIDS pts with Kaposi's sarcoma (KS) (n=10) were also surveyed. Clonal Ig DNA rearrangements were not detected in any of these.Conclusions These results are consistent with observations suggesting that free tumor DNA can often be detected in plasma or serum of cancer pts. The absence of clonal Ig DNA in plasma from healthy donors and pts with AIDS KS suggests that detection of such DNA may be highly specific. Identification of the same Ig rearrangement and sequence in plasma and tumor is consistent with this interpretation. Serial monitoring of circulating clonal DNA appears to track—and perhaps anticipate—clinical evidence of disease. These approaches may be especially valuable in ARL where FDG uptake reflective of inflammatory disease or benign lymphoproliferation detected by PET scan often renders interpretation of imaging ambiguous. Similarly, elevated LDH in this population is relatively non-specific.

Effect of the mutation rate and background size on the quality of pathogen identification

Genomic-based methods have significant potential for fast and accurate identification of organisms or even genes of interest in complex environmental samples (air, water, soil, food, etc.), especially when isolation of the target organism cannot be performed by a variety of reasons. Despite this potential, the presence of the unknown, variable and usually large quantities of background DNA can cause interference resulting in false positive outcomes. In order to estimate how the genomic diversity of the background (total length of all of the different genomes present in the background), target length and target mutation rate affect the probability of misidentifications, we introduce a mathematical definition for the quality of an individual signature in the presence of a background based on its length and number of mismatches needed to transform the signature into the closest subsequence present in the background. This definition, in conjunction with a probabilistic framework, allows one to predict the minimal signature length required to identify the target in the presence of different sizes of backgrounds and the effect of the target's mutation rate on the quality of its identification. The model assumptions and predictions were validated using both Monte Carlo simulations and real genomic data examples. The proposed model can be used to determine appropriate signature lengths for various combinations of target and background genome sizes. It also predicted that any genomic signatures will be unable to identify target if its mutation rate is >5%. Supplementary data are available at Bioinformatics online.

Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR

Unspecific background DNA in quantitative universal real-time PCR utilizing a hydrolysis probe was completely suppressed by the addition of EMA or PMA to the PCR mix via cross-linking of the dyes to DNA during 650 W visible light exposure. The proposed procedure had no effect on the sensitivity of the real-time PCR reaction.

Risk assessment of renal cell carcinoma using alkaline comet assay

DNA damage induced by mutagens has been associated with an individual's susceptibility to cancer. In the current study, which involved 193 renal cell carcinoma (RCC) patients and 193 controls, DNA damage before mutagen induction (baseline), after benzo(alpha)pyrene dio epoxide (BPDE) treatment, and after gamma-radiation induction were assayed by comet assay in peripheral blood lymphocytes. Olive tail moments were used as DNA damage parameters. The 5 variables that were analyzed for their associations with RCC risk were baseline, BPDE-induced, gamma-radiation-induced, net BPDE-induced (BPDE-induced subtract baseline), and net gamma-radiation-induced (gamma-radiation-induced subtract baseline) Olive tail moments. Significantly higher Olive tail moments were observed in cases compared with controls at baseline (1.95 vs 1.65; P = .008), after BPDE induction (3.10 vs 2.38; P < .001), and after gamma-radiation induction (4.25 vs 3.47; P < .001). The net BPDE-induced and gamma-radiation-induced DNA damage was also found to be significantly higher in cases compared with controls (P < .001 for both mutagens). Using the 75th percentile Olive tail moments in the controls as the cutoff point, the authors found that high levels of baseline DNA damage, BPDE-induced DNA damage, and gamma-radiation-induced DNA damage were associated with significantly increased risks of RCC, with odds ratios of 1.96 (95% confidence interval [95% CI], 1.26-3.06), 2.70 (95% CI, 1.72-4.23), and 3.13 (95% CI, 1.99-4.92), respectively. Similarly, net BPDE-induced and net gamma-radiation-induced DNA damages were also found to be significantly associated with elevated risks of RCC. The results of the current study suggest that both baseline and mutagen-induced DNA damages assessed by comet assay are associated with an increased risk of RCC.

Biological significance of DNA adducts investigated by simultaneous analysis of different endpoints of genotoxicity in L5178Y mouse lymphoma cells treated with methyl methanesulfonate

The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O 6-methyl-2′-deoxyguanosine ( O 6-mdGuo) by LC–MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS). The background level of 7-mG was 1440 adducts per 10 9 nucleotides while O 6-mdGuo was almost 50-fold lower (32 adducts per 10 9 nucleotides). In the comet assay and the micronucleus test, background was in the usual range seen with smaller batches of cells (2.1% Tail DNA and 12 micronuclei-containing cells per 1000 binucleated cells, respectively). For the comparison of the four endpoints for dose-related increments above background in the low-response region we assumed linearity at low dose and used the concept of the “doubling dose”, i.e., we estimated the concentration of MMS necessary to double the background measures. Doubling doses of 4.3 and 8.7 μM MMS were deduced for 7-mG and O 6-mdGuo, respectively. For doubling the background measures in the comet assay and the micronucleus test, 5 to 15-fold higher concentrations of MMS were necessary (45 and 66 μM, respectively). This means that the contribution of an increase in DNA methylation to biological endpoints of genotoxicity is overestimated. For xenobiotics that generate adducts without background, the difference is even more pronounced because the dose–response curve starts at zero and the limit of detection of an increase is not affected by background variation. Consequences for the question of thresholds in dose–response relationships and for the setting of tolerable exposure levels are discussed.

Development and evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in Acid Mine Drainages and bioleaching systems

To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. The functional genes in the microarray were involved in carbon metabolism (158), nitrogen metabolism (72), sulfur metabolism (39), iron metabolism (68), DNA replication and repair (97), metal-resistance (27), membrane-relate gene (16), transposon (13) and IST sequence (11).Based on the results of microarray hybridizations, specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was 5 ng of genomic DNA in the absence of background DNA. Strong linear relationships between the signal intensity and the target DNA were observed ( r 2 ≈ 0.98). Application of this type of the microarray to analyze the acidic environments and bioleaching systems demonstrated that the developed microarray appeared to be useful for profiling differences in microbial community structures of acidic environments and bioleaching systems. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of genes related with acidophilic microorganism and the microbial community in acidic environments and bioleaching systems, although more work is needed to improve.

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.

Analysis of the methylation status of HPV DNA in human cell lines

Background DNA methylation is an important epigenetic process that regulates gene expression mainly through its effect on chromatin conformation. It involves the stable maintenance of methylated cytosines at CpG dinucleotides (meCpGs) which in turn can regulate gene expression by two mechanisms: by direct interference with transcription factors binding to their target sequences, or by binding MeCP2 proteins, which recognize methylated DNA and recruit histone deacetilases, resulting in a highly compact chromatin structure. Human Papillomavirus (HPV) E2 protein controls various viral processes, including gene transcription and DNA replication. These activities rely upon its ability to bind as an homodimer to its target sequences (5'-ACCGN4CGGT-3') in the viral DNA. Since these sequences contain CpG dinucleotides, they are potential targets for DNA methylation, and, in fact, it has been demonstrated that E2's ability to bind to its cognate sequence is inhibited by methylation. Different studies have shown that the methylation patterns of HPV DNA, in the long control region (LCR) and the 3' region of the L1 gene, are heterogeneous in human cell lines and clinical samples. The objective of this study was to determine the methylation status of HPV type 18 and 16 DNA in different human-derived cell lines.

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga ( Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.

An investigation of relationships between hypoxia-inducible factor-1α gene polymorphisms and ovarian, cervical and endometrial cancers

Background DNA sequence variations in HIF-1α gene might yield changes both in the production outcomes and in the activities of the gene. Overexpression of the HIF-1α subunit, resulting from intratumoral hypoxia and genetic alterations, has been demonstrated in common human cancers and is correlated with tumor angiogenesis and patient mortality. In this study, we aimed to determine how the three single nucleotide polymorphisms (SNPs, C1772T and G1790A exon 12, C111A exon 2) in the HIF-1α gene coding regions affect the ovarian, cervical and endometrial cancer patients in the Turkish population. A study on this relationship has not been conducted to date. Method 102 gynecologic cancer patients and 107 healthy controls were studied. Genotypes of the three polymorphisms were analyzed by PCR-RFLP. Results There was no significant difference between ovarian cancer patients and controls in terms of the distribution of C1772T genotypes and alleles ( P > 0.05). However, there was a highly significant increase in the frequency of both CT 1772 and TT 1772 genotypes in patients with cervical and endometrial cancers compared with healthy controls. In fact, 1772T allele-carriers (CT + TT genotypes) showed an association with the risk of cervical and endometrial cancers compared to the wild type (OR = 3.84, 95% CI: 1.65–8.93; OR = 7.41, 95% CI: 2.33–23.59, respectively). C1772T polymorphism was not associated with family history concerning gynecologic and/or other cancer types, stages (I–IV) and grades of tumor, smoking habits and existence of other diseases that generate a hypoxic microenvironment even after multivariable logistic regression analysis. As for HIF-1α G1790A genotypes, the frequencies of G alleles were 98% in ovarian patients and 100% in the control group. We found no significant difference in the genotype distribution and allele frequencies between the ovarian patients and healthy control subjects. There were no GA and AA genotypes among the cervical and endometrial cancer patients. As for HIF-1α C111A polymorphism, we did not find CA and AA variants of the gene in controls or in any of the three types of patients. Conclusion Our results suggest that the C1772T polymorphism of the HIF-1α may be associated with cervical and endometrial cancers.

A Robust Procedure for Removing Background Damage in Assays of Radiation-Induced DNA Fragment Distributions

The non-random distribution of DNA breakage in PFGE (pulsed-field gel electrophoresis) experiments poses a problem of proper subtraction of the background DNA damage to obtain a fragment-size distribution due to radiation only. A naive bin-to-bin subtraction of the background signal will not result in the right DNA mass distribution histogram. This problem could become more pronounced for high-LET (linear energy transfer) radiation, because the fragment-size distribution manifests a higher frequency of smaller fragments. Previous systematic subtraction methods have been based on random breakage, appropriate for low-LET radiation. Moreover, an investigation is needed to determine whether the background breakage is itself random or non-random. We consider two limiting cases: (1) the background damage is present in all cells, and (2) it is present in only a small subset of cells, while other cells are not contributing to the background DNA fragmentation. We give a generalized formalism based on stochastic processes for the subtraction of the background damage in PFGE experiments for any LET and apply it to two sets of PFGE data for iron ions.

Equal Performance of TaqMan, MGB, Molecular Beacon, and SYBR Green-Based Detection Assays in Detection and Quantification of Roundup Ready Soybean

We have tested and compared the performance of 12 different assays representing four different real-time polymerase chain reaction (PCR) chemistries in the context of genetically modified organism detection. Several different molecular beacon, SYBR Green, TaqMan, and MGB assays were designed for the event specific detection and quantification of the 3' integration junction of GTS 40-3-2 (Roundup Ready) soybean. Sensitivity as well as robustness in the presence of background DNA were tested. None of the PCR-based approaches appeared to be significantly better than any of the other, but the molecular beacon assays had the lowest efficiency and also seemed more sensitive to changes in experimental setup.

Influence of Dangling Ends and Surface-Proximal Tails of Targets on Probe-Target Duplex Formation in 16S rRNA Gene-Based Diagnostic Arrays

Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.

Multiple Displacement Amplification Enables Large Scale Clonal Analysis after Retroviral Gene Therapy.

Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole genome amplification named multiple displacement amplification (MDA) with respect to the even and accurate representation of retrovirally transduced genomic DNA. We were able to show that MDA is a suitable method to subsequently specify engraftment efficiencies by quantitative real-time PCR as the retroviral integrations are amplified the same way and by the same probability as all other parts of the genome. We validated the method by analyzing a dilution series containing retrovirally transduced DNA and untransduced background DNA and retroviral integrations found in primary material from a retroviral transplantation model by quantitative real-time PCR. The representation of the portion of retroviral DNA in the amplified samples was 0.9-fold (range 0.2 – 2.1-fold) of the portion determined in the original genomic DNA. Furthermore, the succession of the combination of MDA and integration site analysis by ligation-mediated PCR showed an increase in the sensitivity of the method as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show that MDA enables large scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow up analysis in gene therapy studies even from smallest amounts of starting material.

Real-Time Quantitative Broad-Range PCR Assay for Detection of the 16S rRNA Gene Followed by Sequencing for Species Identification

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.

Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique for the amplification of DNA under isothermal conditions. For the first time, we applied this method to develop a simple and quantitative monitoring method for environmental microorganisms targeting amoA gene in ammonia-oxidizing bacteria. Quantitative analysis was performed first by measuring fluorescence derived from an intercalation dye using a real-time thermal cycler, and then by measuring the turbidity of the reaction solution using a real-time turbidimeter. As a result, it was possible to quantify the initial amoA DNA concentration from an environment with a sensitivity down to 10(2) DNA copies of target DNA and a dynamic range of 7-9 orders in magnitude. Background DNA from nontargeted bacteria (Pseudomonas denitrificans) that does not encode amoA gene did not affect the quantitative capability of LAMP. Over results suggested that the real-time LAMP is effective for monitoring microorganisms and their gene expression in environments.

The relationship between HVRII mutation of mitochondrial DNA and squamous cell carcinoma of the lung

Mitochondrial DNA (mtDNA) is the only hereditary substance besides nucleus, which is composed of a code region and a non-code D-loop region. The aim of this study is to investigate the hypervariable region II (HVRII) mutation of mtDNA in peripheral blood leucocyte, pericancerous tissues and cancer tissues of lung squamous cell carcinoma patients, and to explore its significance. White blood cells, pericancerous tissues and cancer tissues were obtained from 15 cases of lung squamous cell carcinoma patients and mtDNA were extracted by one step method. HVRII fragments were amplified by PCR. Mutations were determined by DNA sequencing and the mutations of HVRII were analysed. In 15 lung squamous cell carcinoma patients, 14 patients showed mutation in HVRII(93.33%), 88 mutations were found totally. Eighty-seven mutations located in H-strand origin region, especially in the conserved sequence blocks and the mtTF1, 2 binding site (TFX and TFY). The results suggest that the mutation frequency of HVRII in cancer tissues of lung squamous cell carcinoma patients is very high and it might play an important role in carcinogenesis of the lung.

Single DNA molecule stretching in sudden mixed shear and elongational microflows

High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.

Comparison of a LightCycler-based real-time PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR

Measurement of viral load is important in predicting and monitoring of Epstein-Barr virus (EBV)-associated diseases especially in immunocompromised patients. The objectives of this study were the development of a LightCycler-based real-time PCR assay using primers and probes which recognize the virus capsid antigen p23-encoding region and its comparison to the semiquantitative PCR. The LightCycler protocol shows a high degree of specificity and inter- and intra-assay reproducibility. Concerning sensitivity, a good correlation between both methods was demonstrated for standard plasmid DNA, reference DNA isolated from the EBV-genome containing Namalwa cell line, and DNA extracted from plasma/cerebrospinal fluid (CSF). The detection limit was determined with 1 copy/microl eluate for the standard plasmid DNA and with 500 copies/ml plasma or CSF. For DNA derived from peripheral blood mononuclear cells (PBMCs), a decrease of sensitivity by factor 10-100 was found when larger amounts of background DNA (500 and 100 ng) were used presuming an inhibitory effect of cellular DNA. This was supported by running dilutions of the plasmid standard carried out with EBV-negative Ramos cell DNA. Thus, the cut-off level was estimated with 100-500 copies/10(5) PBMCs, when 50 or 10 ng total DNA were tested. The results indicate that the real-time PCR described here is a first line tool for the determination of viral load in plasma and CSF. Semiquantitative nested PCR is used for screening of PBMCs viral load. Positive specimens containing more than 500 copies/10(5) cells are measured for exact values by real-time PCR. To circumvent inhibitory effects of cellular DNA, measurements should be carried out generally with 50-10 ng DNA.

Simultaneous determination of O6-methyl-2′-deoxyguanosine, 8-oxo-7,8-dihydro-2′-deoxyguanosine, and 1,N6-etheno-2′-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

O(6)-Methyl-2'-deoxyguanosine (O(6)-mdGuo), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), and 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O(6)-[(2)H(3)]mdGuo was synthesized and used as internal standard. The limits of quantification for O(6)-mdGuo, 8-oxodGuo, and epsilondAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24-125 pmol/ml, 0.98-125 pmol/ml, and 0.49-62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O(6)-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for epsilondAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O(6)-mdGuo was above the limit of detection (37 adducts per 10(9) normal nucleosides) but could not be quantified. 8-oxodGuo and epsilondAdo showed background levels of 500 and 130 adducts per 10(9) normal nucleosides, respectively. DNA analyzed 1h after treatment of rats with dimethylnitrosamine by oral gavage of 50 microg/kg b.wt. did not affect the levels of 8-oxodGuo and epsilondAdo but resulted in 200 O(6)-mdGuo adducts per 10(9) normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.