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

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.

The developing fetus is likely to be exposed to the same environmental chemicals as the mother during critical periods of growth and development. The degree of maternal-fetal transfer of chemical compounds will be affected by chemical and physical properties such as lipophilicity, protein binding, and active transport mechanisms that influence absorption and distribution in maternal tissues. However, these transfer processes are not fully understood for most environmental chemicals. This review summarizes reported data from more than 100 studies on the ratios of cord:maternal blood concentrations for a range of chemicals including brominated flame-retardant compounds, polychlorinated biphenyls (PCB), polychlorinated dibenzodioxins and dibenzofurans, organochlorine pesticides, perfluorinated compounds, polyaromatic hydrocarbons, metals, and tobacco smoke components. The studies for the chemical classes represented suggest that chemicals frequently detected in maternal blood will also be detectable in cord blood. For most chemical classes, cord blood concentrations were found to be similar to or lower than those in maternal blood, with reported cord:maternal ratios generally between 0.1 and 1. Exceptions were observed for selected brominated flame-retardant compounds, polyaromatic hydrocarbons, and some metals, for which reported ratios were consistently greater than 1. Careful interpretation of the data in a risk assessment context is required because measured concentrations of environmental chemicals in cord blood (and thus the fetus) do not necessarily imply adverse effects or risk. Guidelines and recommendations for future cord:maternal blood biomonitoring studies are discussed.

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.

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