Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstractPCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA Electronic supplementary materialThe online version of this article (10.1007/s00216-018-0931-z) contains supplementary material, which is available to authorized users.

We present a novel method for monitoring isothermal lipid hydration using a sorption microcalorimeter. A measuring cell of the double-twin calorimeter consists of two vessels connected by a stainless steel tube. The upper vessel contains pure water, and the bottom vessel is loaded with the lipid sample. This calorimeter allows for simultaneous measurement of the partial molar enthalpy and the chemical potential (or the partial molar free energy) of the water. The versatility of the method is demonstrated by studies of the hydration of the phospholipids dipalmitoyl phosphatyl choline (DPPC), dimyristoyl phosphatidyl choline (DMPC), and dilauroyl phosphatidyle choline (DLPC) at 25 and 27 °C. The measurements provide a relation between water content and water chemical potential, which, in these lamellar systems, is often recast as a force−distance relation and has been called the hydration force. Through the simultaneously monitored calorimetric values, the partial molar enthalpy of water is also obtained. The method consequently provides a rather unique combination of information on both partial molar enthalpy and partial molar free energy and thus also the partial molar entropy of the process. We find that the incorporation of the first three to four water molecules per lipid is exothermic. These water molecules presumably interact directly with oxygen atoms on the phosphate of the lipid headgroup. When the first waters have been added, the remaining ones are incorporated endothermically. This applies to the water molecules taken up both in the gel phase and in the liquid crystalline state. We also observe that the sorption process triggers a first-order phase change from a gel (Lβ ‘) to a liquid crystalline (Lα) phase. For DLPC, this occurs at 25 °C at a relative humidity of 0.79 with an endothermic transition enthalpy of 42 ± 2 kJ/mol (DLPC) and, for DMPC, at 27 °C at 0.93 relative humidity with ΔH = 56 ± 5 kJ/mol (DMPC). We use a previously established model to quantitatively interpret these phase transitions. Furthermore, the observed endothermic nature of the sorption process above three to four waters per lipid is fully consistent with the suggestion that the negative free energy of the sorption (swelling) is due to increased thermal excitations and thus a positive entropy. It is more problematic to reconcile the data with models proposing structuring effects in the water as the main cause of the swelling.

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Glycerol and urea are examples of small, water-soluble molecules with low vapor pressure that can protect lipid membranes upon dehydration. Both are a part of the Natural Moisturizing Factor in human skin, and are also present in other organisms, where they prevent drying due to osmotic stress. This study was conducted in order to understand the mechanism of such protection. We have selected two ternary systems: dimyristoylphosphatidylcholine (DMPC)-glycerol-water and DMPC-ureawater, as models to investigate the molecular mechanisms behind this protective effect with a focus on factors that control the solid to liquid phase transition in the phospholipid bilayers. By combining a number of experimental techniques, including solid-state NMR, sorption microbalance and DSC, the structure and the phase transitions have been characterized at low water content and in excess solution. It was discovered that both glycerol and urea stabilize the liquid crystalline bilayers at low relative humidities (down to 75% RH at 27 C), whereas for the pure DMPC-water system, a solid gel phase is induced at 93% RH. This demonstrates the protective effect of glycerol and urea against osmotic stress. It is further concluded that for lipid systems with limited access to solvent, the phase behavior is determined by solvent volume, irrespective of the composition. The observation that glycerol and urea have a similar effect on the lipid phase behavior under dry conditions, together with the lack of evidence of specific interactions between the lipids and glycerol or urea, implies a general mechanism, which might also be applicable to other, similar solutes.

We present measurements of isothermal DNA-hexadecyltrimethylammonium (DNACTA) complex and pure DNA hydration at 25°C using a sorption microcalorimeter. This calorimeter provides simultaneous measurement of (i) water activity (sorption isotherms) and (ii) the partial molar enthalpy of water as a function of water uptake. For pure DNA, hydration is exothermic over the studied concentration range and we find an approximately linear relation between the partial molar enthalpy and the partial molar free energy. A kink in the isotherm appears at 20.0 ( 0.3 water molecules per base pair for a water activity of 0.80, consistent with A-B transition of the DNA. There is no detectable heat effect associated with this transition. At low water contents, the hydration of the DNACTA (1:1) complex is exothermic as for the pure DNA, but after incorporation of the first 7.0 ( 0.1 water molecules, the enthalpy changes sign. At 22 water molecules per base pair, the enthalpy levels off to 2.7 ( 0.2 kJ/mol. In a separate experiment, the swelling limit for the DNACTA complex was found to be 27 ( 1 waters per base pair. The DNACTA complex is arranged in a hexagonal structure. We propose a model for the DNACTA complex based on the packing of the components in an electroneutral way consisting of six DNA helices, presumably in an A configuration, placed around a central CTA + cylinder. The hydration of the complex is seen as a balance between the attractive electrostatic interaction causing the formation of the complex and a repulsive component arising from a hexagonal deformation of CTA + cylinders. An important contribution to the partial molar enthalpy of water comes, in this interpretation, from the release of conformational constraints of the CTA ion alkyl chains.

We investigate how a small polar molecule, urea, can act to protect a phospholipid bilayer system against osmotic stress. Osmotic stress can be caused by a dry environment, by freezing, or by exposure to aqueous systems with high osmotic pressure due to solutes like in saline water. A large number of organisms regularly experience osmotic stress, and it is a common response to produce small polar molecules intracellularly. We have selected a ternary system of urea-water-dimyristoyl phosphatidylcholine (DMPC) as a model to investigate the molecular mechanism behind this protective effect, in this case, of urea, and we put special emphasis on the applications of urea in skin care products. Using differential scanning calorimetry, X-ray diffraction, and sorption microbalance measurements, we studied the phase behavior of lipid systems exposed to an excess of solvent of varying compositions, as well as lipid systems exposed to water at reduced relative humidities. From this, we have arrived at a rather detailed thermodynamic characterization. The basic findings are as follows: (i) In excess solvent, the thermally induced lipid phase transitions are only marginally dependent on the urea content, with the exception being that the P(beta) phase is not observed in the presence of urea. (ii) For lipid systems with limited access to solvent, the phase behavior is basically determined by the amount (volume) of solvent irrespective of the urea content. (iii) The presence of urea has the effect of retaining the liquid crystalline phase at relative humidities down to 64% (at 27 degrees C), whereas, in the absence of urea, the transition to the gel phase occurs already at a relative humidity of 94%. This demonstrates the protective effect of urea against osmotic stress. (iv) In skin care products, urea is referred to as a moisturizer, which we find slightly misleading as it replaces the water while keeping the physical properties unaltered. (v) In other systems, urea is known to weaken the hydrophobic interactions, while for the lipid system we find few signs of this loosening of the strong segregation into polar and apolar regions on addition of urea.

In this work, we describe the application of a sorption calorimeter to study the binary system n-octyl β-Dglucoside/water at 25, 40, and 60 °C. At 25 °C we also carried out experiments with heavy water which showed similar results to those with normal water. The method used allows one to simultaneously obtain information about the activity of water and the enthalpy of mixing in binary systems. The obtained results show that the hydration in the system is endothermic and hence driven by entropy. We have also specified the concentration ranges and thermodynamic properties of the different phases in the system. The first step of isothermal hydration in the system when the lamellar phase is formed involves high endothermic effect and is similar to the transition observed during the heating of the pure surfactant. Forces between the layers of the lamellar phase in the next step of hydration have also been studied at three temperatures. Using the calorimetric method, we have determined concentration ranges of the narrow two-phase regions of the transitions from lamellar to cubic and isotropic phases. We have used van der Waals differential equation to calculate temperature-composition slopes of the phase boundaries using the isothermal properties of the system.

Milk coagulation is an important processing trait, being the basis for production of both cheese and fermented products. There is interest in including technological properties of these products in the breeding goal for dairy cattle. The aim of the present study was therefore to estimate genetic parameters for milk coagulation properties, including both rennet- and acid-induced coagulation, in Swedish Red dairy cattle using genomic relationships. Morning milk samples and blood samples were collected from 395 Swedish Red cows that were selected to be as genetically unrelated as possible. Using a rheometer, milk samples were analyzed for rennet- and acid-induced coagulation properties, including gel strength (G'), coagulation time, and yield stress (YS). In addition to the technological traits, milk composition was analyzed. A binary trait was created to reflect that milk samples that had not coagulated 40min after rennet addition were considered noncoagulating milk. The cows were genotyped by using the Illumina BovineHD BeadChip (Illumina Inc., San Diego, CA). Almost 600,000 markers remained after quality control and were used to construct a matrix of genomic relationships among the cows. Multivariate models including fixed effects of herd, lactation stage, and parity were fitted using the ASReml software to obtain estimates of heritabilities and genetic and phenotypic correlations. Heritability estimates (h(2)) for G' and YS in rennet and acid gels were found to be high (h(2)=0.38-0.62) and the genetic correlations between rennet-induced and acid-induced coagulation properties were weak but favorable, with the exception of YSrennet with G'acid and YSacid, both of which were strong. The high heritability (h(2)=0.45) for milk coagulating ability expressed as a binary trait suggests that noncoagulation could be eliminated through breeding. Additionally, the results indicated that the current breeding objective could increase the frequency of noncoagulating milk and lead to deterioration of acid-induced coagulation through unfavorable genetic associations with protein content (0.38) and milk yield (-0.61 to -0.71), respectively. The outcome of this study suggests that by including more detailed compositional traits genetically associated with milk coagulation or by including milk coagulation properties directly within the breeding goal, it appears possible to breed cows that produce milk better suited for production of cheese and fermented products.