Circulatory cell-free DNA (cf-DNA) is increased in a variety of clinical pathologic conditions; therefore, these markers could be widely used as markers for detecting and monitoring several disorders. To better understand the biology of this molecule, we analysed the relationship between the level of circulatory cf-DNA and physiological parameters such as gender, age and frequency of blood donations. Paired plasma and serum samples were obtained from 87 blood donors and 50 healthy adults who had never donated blood. Cf-DNA was extracted from plasma and serum samples using the MagNA Pure LC Instrument. Quantity determination of circulatory cf-DNA was performed by TaqMan real-time PCR for the ubiquitous GAPDH gene. Our data showed that the concentration of cf-DNA in serum was about eightfold higher than that in plasma. Regarding the level of these circulatory species, no significant differences were observed between the age-matched men and women and gender-matched, different-age cohorts, except in women who were older than 60 years of age. Frequent blood donations did not increase the circulatory species. Circulatory cf-DNA in plasma and serum samples is not correlated with human gender and human age except in women who are older than 60 years of age. Frequent blood donation did not affect the quantity of circulatory cf-DNA. The explanation for the latter most likely is the short half-life time of free fetal DNA in maternal circulation.
AIM:A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. METHOD:We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). RESULTS:Qualitatively, both assays detected male cellfree fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT realtime qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. CONCLUSIONS:The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.The analysis of cell-free fetal DNA in maternal serum and plasma is currently the method of choice for the noninvasive determination of fetal genetic traits (1 ). Real-time quantitative PCR (qPCR)4 is used for the majority of these analyses, because this method is amenable to automation, provides data in a real-time manner, and, by being a closed system, is less prone to contamination than conventional PCR methods with longer amplicons (1, 2 ). Alternatives that are being explored and gaining in importance are mass spectrometry of primer-extended PCR products, digital PCR, and shotgun sequencing (3, 4 ).To date, clinical applications have centered largely on the rather facile detection of fetal genetic loci completely absent from the maternal genome, such as the determination of fetal sex in pregnancies at risk for X-linked disorders or the fetal Rhesus D genotype in pregnancies at risk for hemolytic disease of the fetus and newborn. This centering by clinical applications has occurred because the detection of other, more subtle genetic differences between mother and fetus is rendered more complex on account of the preponderance of maternal cell-free DNA sequences (1, 2 ).Because real-time qPCR also provides a quantitative answer, this approach has been used in a number of studies to determine the concentration of cell-free fetal DNA in maternal plasma samples. In general, these studies have indicated much higher concentrations of cell-free fetal DNA than those of rare circulating fetal cells, but they are still quite low, approximately 1%-3% early in pregnancy and progressing to approximately 5% at term. Through the use of this technology, measurements of increases i...
S U M M A R Y Current cytogenetic approaches in noninvasive prenatal diagnosis focus on fetal nucleated red blood cells in maternal blood. This practice may be too restrictive because a vast proportion of other fetal cells is ignored. Recent studies have indicated that fetal cells can be directly detected, without prior enrichment, in maternal blood samples by fluorescence in situ hybridization (FISH) analysis for chromosomes X and Y (XY-FISH). In our blinded analysis of 40 maternal blood samples, we therefore examined all fetal cells without any enrichment. Initial examinations using conventional XY-FISH indicated a low specificity of 69.4%, which could be improved to 89.5% by the use of two different Y-chromosome-specific probes (YY-FISH) with only a slight concomitant decrease in sensitivity (52.4% vs 42.9%). On average, 12-20 male fetal cells/ml of maternal blood were identified by XYand YY-FISH, respectively.
In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 2.6.1.7) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate β‐lyase, indicating that rat kidney KAT and β‐lyase represent the same molecular entity. Oligonucleotide probes derived from the β‐lyase cDNA were then used as primers for PCR of reverse‐transcribed rat brain poly(A)+ RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the β‐lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK‐293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 mM pyruvate and 2‐oxoglutarate, the Km values for l‐kynurenine were 1.2 mM and 86.3 µM, respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/β‐lyase mRNA of ∼2.1 kb.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.