Use of elemental analysis to determine comparative performance of established DNA quantification methods.

Quantification of genomic DNA is critical for many analyses in molecular biology. Current methods include optical density (OD) measurements or fluorescent enhancement but both approaches have limitations on achievable accuracy. In this study we performed an elemental analysis to quantify genomic DNA to provide an independent value for comparing the performance of four quantification methods. Specifically ICP-OES (inductively coupled plasma-optical emission spectroscopy) was used to assign a concentration value to a DNA stock solution, based on the stoichiometry of phosphorus within the molecule. Two absorbance- and two fluorescence-based methods were then used to quantify the same DNA solution using replicate analyses. The precision of each method was assessed by measurement of replicate spread (coefficient of variation) and trueness by t-test. Results showed that performance of the methods was variable, both in terms of concordance with the independent ICP-OES value and repeatability of data. While need for expensive equipment and technical expertise may preclude widespread replacement of more traditional methods for DNA quantification, use of primary methods such as ICP-OES analysis for production of accurate calibrants may increase quantitative accuracy and give greater appreciation of the true performance of current methods.

Routes to improving the reliability of low level DNA analysis using real-time PCR.

BACKGROUND: Accurate quantification of DNA using quantitative real-time PCR at low levels is increasingly important for clinical, environmental and forensic applications. At low concentration levels (here referring to under 100 target copies) DNA quantification is sensitive to losses during preparation, and suffers from appreciable valid non-detection rates for sampling reasons. This paper reports studies on a real-time quantitative PCR assay targeting a region of the human SRY gene over a concentration range of 0.5 to 1000 target copies. The effects of different sample preparation and calibration methods on quantitative accuracy were investigated. RESULTS: At very low target concentrations of 0.5-10 genome equivalents (g.e.) eliminating any replicates within each DNA standard concentration with no measurable signal (non-detects) compromised calibration. Improved calibration could be achieved by eliminating all calibration replicates for any calibration standard concentration with non-detects ('elimination by sample'). Test samples also showed positive bias if non-detects were removed prior to averaging; less biased results were obtained by converting to concentration, including non-detects as zero concentration, and averaging all values. Tube plastic proved to have a strongly significant effect on DNA quantitation at low levels (p = 1.8 x 10(-4)). At low concentrations (under 10 g.e.), results for assays prepared in standard plastic were reduced by about 50% compared to the low-retention plastic. Preparation solution (carrier DNA or stabiliser) was not found to have a significant effect in this study.Detection probabilities were calculated using logistic regression. Logistic regression over large concentration ranges proved sensitive to non-detected replicate reactions due to amplification failure at high concentrations; the effect could be reduced by regression against log (concentration) or, better, by eliminating invalid responses. CONCLUSION: Use of low-retention plastic tubes is advised for quantification of DNA solutions at levels below 100 g.e. For low-level calibration using linear least squares, it is better to eliminate the entire replicate group for any standard that shows non-detects reasonably attributable to sampling effects than to either eliminate non-detects or to assign arbitrary high Ct values. In calculating concentrations for low-level test samples with non-detects, concentrations should be calculated for each replicate, zero concentration assigned to non-detects, and all resulting concentration values averaged. Logistic regression is a useful method of estimating detection probability at low DNA concentrations.

Multiple and simultaneous fluorophore detection using fluorescence spectrometry and partial least-squares regression with sample-specific confidence intervals.

Fluorescent labeling is widely used in biological and chemical analysis, and the drive for increased throughput is stretching multiplexing capabilities to the limit. The limiting factor in multiplexed analyses is the ability to subsequently deconvolute the signals. Consequently, alternative approaches for interpreting complex data sets are required to allow individual components to be identified. Here we have investigated the application of a novel approach to multiplexed analysis that does not rely on multivariate curve resolution to achieve signal deconvolution. The approach calculates a sample-specific confidence interval for a multivariate (partial least-squares regression (PLSR)) prediction, thereby enabling the estimation of the presence or absence of each fluorophore based on the total spectral signal. This approach could potentially be applied to any multiplexed measurement system and has the advantage over the current algorithm-based methods that the requirement for resolution of spectral peaks is not central to the method. Here, PLSR was used to obtain the concentrations for up to eight dye-labeled oligonucleotides at levels of (0.6-5.3) x 10(-6) M. The sample-specific prediction intervals show good discrimination for the presence/absence of seven of the eight labeled oligonucleotides with efficiencies ranging from approximately 91 to 100%.

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures.

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr(128) protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationary-phase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the rpsL gene promoter (which heads the Mycobacterium smegmatis operon encoding the tuf gene encoding EF-Tu) or the rrnA promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so rpsL expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing rpsL at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.

Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation.

BACKGROUND: Detection of fetal DNA in maternal plasma is achievable at 5 weeks of gestation, but few large-scale studies have reported circulating fetal and maternal DNA across all trimesters. METHODS: Blood samples were collected from 201 women between 5 and 41 weeks of pregnancy. Quantitative PCR was used to assess total and fetal DNA concentrations, and allelic discrimination analysis was investigated as a route to detecting specifically fetal DNA. RESULTS: Male fetuses were detectable from 5 weeks amenorrhea with increasing fetal DNA concentrations across gestation. The sensitivity of fetal male gender determination in pregnancies with live birth confirmation was 99%, with 100% specificity. Total DNA concentrations did not correlate with gestational age, but appeared slightly higher in the first and third trimesters than in mid-pregnancy. Analysis of short tandem repeats demonstrated that significant improvements in the detection limit are required for specific detection of fetal DNA. CONCLUSIONS: The high sensitivity of PCR-based detection, together with quantification provided by real-time DNA analysis, has clear potential for clinical application in noninvasive prenatal diagnosis. However, accurate quantification using best-fit data analysis, standardization of methods, and performance control indicators are necessary for robust routine noninvasive diagnostics.

S-Nitrosoglutathione cytotoxicity to Mycobacterium smegmatis and its use to isolate stationary phase survival mutants.

We report that stationary phase Mycobacterium smegmatis is more sensitive than exponential phase cells to the nitric oxide donor S-Nitrosoglutathione (GSNO). This finding was used to select for both spontaneous and transposon mutants of M. smegmatis with increased resistance to GSNO in stationary phase. Some of these mutants were also defective in stationary phase survival, demonstrating a link between sensitivity to GSNO and stationary phase survival. Transduction of the disrupted region from seven selected mutants indicated that the transposon insertion was linked to the GSNO-resistance and stationary phase survival phenotypes. For five mutants, the disrupted genes were identified. Three were homologous to genes with possible roles in nutrient scavenging, including: (i) a putative amino acid efflux pump, (ii) a putative thioesterase and (iii) an enoyl-CoA-hydratase. One mutant was disrupted in the atpD gene, encoding the beta chain of F1 F0 ATP synthase. We independently isolated a stationary phase survival mutant disrupted in the atpA gene (encoding the alpha chain) of the F1 F0 ATP synthase of the same operon, suggesting an important role for efficient ATP synthesis in stationary phase survival.

Generic scheme for independent performance assessment in the molecular biology laboratory.

BACKGROUND: A variety of proficiency testing schemes are available for specific molecular analyses, but there is an acute need for more widely accessible schemes to assess and demonstrate general competence in DNA analysis. METHODS: Fifteen laboratories, including academic, clinical, and commercial organizations, were recruited into the prototype assessment exercise. A range of test samples were provided, and participants were required to extract DNA from simple matrices, perform PCR amplification, and score the samples as positive or negative by electrophoretic analysis of the amplification products. Results were requested as both gel images and a completed results table, and the performance of each laboratory was then scored on the submitted analytical results. RESULTS: Overall, laboratories performed the analysis successfully, with participants scoring a high proportion of the samples correctly in the two rounds of the scheme. However, not all of the laboratories were able to achieve amplification for all samples, and the performance of some laboratories was not consistent in the two rounds. In addition, several analytical problems were encountered at all stages of the process, including DNA extraction, PCR amplification, and correct recording of results. CONCLUSIONS: The generic approach described here has enabled effective cross-sectoral benchmarking of laboratories from a variety of analytical sectors. The problems encountered by some participating laboratories highlight the need for quality control and checks at all stages of the process to ensure accuracy of results. A statistical analysis of the results (ANOVA) allowed meaningful comparison of the consistency and sensitivity achieved by laboratories, demonstrating that an effective balance was achieved between the level of data obtained from laboratories and the time expenditure required from participants.

SERRS as a more sensitive technique for the detection of labelled oligonucleotides compared to fluorescence.

This communication contains data from a comparison between the detection limits obtained using surface enhanced resonance Raman scattering (SERRS) and fluorescence detection of dye labelled oligonucleotides. The results show that the detection limits for SERRS are generally at least three orders of magnitude lower than those obtained for fluorescence.

A purF mutant of Mycobacterium smegmatis has impaired survival during oxygen-starved stationary phase.

In this study it was demonstrated that a range of transposon mutants of Mycobacterium smegmatis, previously described as having impaired survival in carbon-starved stationary phase, were not markedly affected in O(2)-starved stationary-phase survival. One exception was 329B, a purine auxotroph, which showed a precipitous reduction in viability from approximately 10(8) to approximately 10(3) c.f.u. ml(-1) during the first 5-10 d in O(2)-starved stationary phase. This was followed by an equally rapid recovery in culturability to a level within 10-100-fold of wild-type levels by 10-20 d into stationary phase. Transduction of the mutation into a clean genetic background demonstrated that the phenotype was due to the transposon insertion, which was shown to be in the purF gene. purF encodes phosphoribosylpyrophosphate amidotransferase, which catalyses the first committed step in purine biosynthesis. The M. smegmatis purF gene, which encodes a protein with a very high degree of similarity to the PurF homologues of Mycobacterium tuberculosis and Mycobacterium leprae, was cloned and shown to substantially complement the O(2)-starvation phenotype. The recovery in culturabilty of the purF mutant in O(2)-starved stationary phase did not involve movement of the transposon. In addition, when cells that had recovered culturability were retested, their survival kinetics in stationary phase were identical to the original culture, indicating that their recovery was not explained by the accumulation of suppressor mutations. It is concluded that the survival curve in O(2)-starved stationary phase for the purF mutant represents its true phenotype and is not a result of subsequent genetic changes in the culture. It is argued that the purF cells lose culturability for a finite period of time in stationary phase. Whether this is due to a fraction of the population dying and then regrowing using a previously undiscovered fermentation pathway, or becoming transiently dormant, or entering an active nonculturable state and subsequently undergoing resuscitation cannot be distinguished at this stage.

Mutants of Mycobacterium smegmatis impaired in stationary-phase survival.

A bank of 600 insertional mutants of Mycobacterium smegmatis was screened for mutants defective in stationary-phase survival. Of 74 mutants picked by the initial screen, 21 had stationary-phase survival defects and 7 of these were studied in more detail. In general, mutants survived stationary phase significantly less well in rich medium than under carbon-starvation conditions. In all cases the loss of viability in stationary phase was not complete even after prolonged incubation. All mutants showed an initial decrease in viability, during the first 40 d in stationary phase, followed by an increase in viable counts that returned viability close to the levels of the wild-type. Southern hybridization experiments showed that recovery of viability was not a consequence of precise excision or movement of the transposon. Two of the survival mutants differed from the wild-type in their colony morphology, and recovery of their viability in stationary phase was coincident with the return of wild-type colony morphology. It is possible that second-site suppressor mutations accumulate that alleviate the effects of the original mutation. For five of the mutants the DNA flanking the site of transposition was amplified by ligation-mediated PCR and sequenced to identify the disrupted locus. In each case, homologous genes were identified in the Mycobacterium tuberculosis genome, three of which have clearly predicted functions in M. tuberculosis as a penicillin-binding protein, in biotin biosynthesis and as a polyketide synthase. This is the first identification of genes implicated in the stationary-phase survival of mycobacteria.