DNA Hyperstructure

This study presents a new procedure to condense DNA molecules and precipitate them onto a glass slide. The resulting DNA molecules undergo autonomous self-assembly, creating closed superstructures on the micrometer scale, which are called DNA hyperstructures. These structures can be observed using low-magnification (4×) light microscopy. Precisely controlling the alcohol/glacial acetic acid ratio and DNA concentration during precipitation enabled the regulation of structure compaction on the slide. The alcohol/glacial acetic acid ratio is inversely proportional to the DNA concentration to achieve optimal compaction on the slide. Confocal microscopy fluorescence analysis of DNA extracts stained with DAPI shows that nucleic acids self-assemble to form structures during precipitation on the slide. This methodology is relevant since it facilitates the precipitation and visualization of DNA, regardless of its origin or molecular weight. To confirm its versatility, results with DNA extracted from human peripheral blood, the Lambda virus, and plasmid pBR322 are presented. The study examined the morphological features of DNA hyperstructures in both healthy individuals and those diagnosed with different medical conditions or illnesses, revealing distinct patterns specific to each case. This innovative technology has potential for disease detection in peripheral blood samples, ranging from cancer and Alzheimer’s disease to determining the gender of the gestational product at an early stage.


■ INTRODUCTION
Diagnostics are essential in determining medical strategies.The COVID-19 pandemic highlighted the need for methodologies that allow for rapid and reliable diagnoses.The DNA molecule is composed of a deoxyribose alternating with phosphate groups, where each sugar is linked to one of four nitrogen-containing bases.In recent years, it has been shown that several diseases are related to changes experienced by the chemical environment of the DNA, which in turn translates into conformational modifications of the molecule. 1,2Various methods have been utilized for DNA research aimed at diagnostic purposes, among which is the examination of peripheral blood for the acquisition of circulating free DNA (cfDNA).−5 Cell-free DNA has been detected in blood through a quantitative polymerase chain reaction (qPCR).cfDNA originates from various tissues and possesses specific characteristics, including its size in base pairs.The condensed phase of DNA occurs naturally within cells and serves to shield molecules from external agents, preventing damage or mutations. 6DNA is a negatively charged molecule.Electrostatic shielding happens in environments with enough cations to cause condensed molecules. 7Condensed DNA is formed by reaching the isoelectric point, which happens when the molecule's surface charge reaches zero and molecules associate to form larger structures.Pincus et al. conducted a study on this topic, finding that using spermine (a tetravalent cation) leads to structures of approximately 100 nm or even micrometers in size. 8In the laboratory, the condensation process is achieved through DNA oligomerization; 9−11 the use of salts with mono-, di-, and tetravalent cations in varying concentrations has been studied; 12,13 cationic surfactants 14 and ethanol are also required to precipitate the DNA and create the conditions necessary for its self-assembly. 15In 1991, DNA was first observed under a light microscope (patent application filed in 1991 with the Mexican Institute of Industrial Property, granted in 1996) 16−18 with the goal of finding an alternative method to replace electrophoresis.Methylation of the DNA molecule generates local structural changes in the double helix.Roll and propeller twist were the DNA shape features most sensitive to the methylation process. 19,20Modifications have also been detected in its mechanical properties, 21,22 and changes in electronic properties ■ EXPERIMENTAL SECTION Materials.All chemicals and biological samples were purchased from Sigma and used as received without further purification.Trizma hydrochloride, molecular biology grade (BioUltra, ≥99%); sodium hydroxide, reagent grade (98%); ethylenediaminetetraacetic acid disodium salt dihydrate, molecular biology grade (BioUltra, ≥99.0%); phenol, molecular biology grade (≥99%); chloroform/isoamyl alcohol 24:1, molecular biology grade (BioUltra, ≥99.5%); absolute ethyl alcohol, molecular biology grade (≥99.45%);glacial acetic acid, ACS reagent grade (≥99.7%).Lambda Phage DNA and pBR322 Phage DNA were purchased as lyophilized powder.
Methods.The BFC buffer was obtained by following a specific procedure for DNA extraction and precipitation.First, a 100 mL solution of 10 N NaOH in distilled water was prepared.Then, a solution containing 7.9 g of Trizma HCl, 7.5 g of EDTA, and 0.6 g of NaCl in 80 mL of distilled and sterile water was prepared to obtain the BFC buffer.After a pH range of 9.5−10 was achieved using the 10 N NaOH solution, it was added drop by drop, and the volume was adjusted with distilled and sterile water.Phenol was heated to 40 °C, and 15 mL of liquid phenol was obtained, which was then mixed with 10 mL of BFC through vortexing for 1 min at 2000 rpm.The mixture was refrigerated at 4 °C until use and separated into two immiscible phases.Phenol (P) was located at the top.
Protocol DNA Extraction.The study obtained informed consent from all participants prior to sample collection.Samples were collected from volunteer patients who had predetermined diagnoses, and the study followed the Helsinki Declaration.To collect the samples, 3−4 mL of peripheral blood was drawn into an EDTA-containing tube, incubated at room temperature for 24 h, and centrifuged at 3000 rpm for 5 min, and the resulting plasma was discarded.(1) Following the removal of plasma, the remaining material is identified as R and stored at a temperature of −20 °C until usage.(2) In a 1.5 mL tube, 4 μL of BFC and 40 For confocal microscopy analysis, LSM800 equipment (Carl Zeiss, Jena Germany) mounted on an Axio Observer.Z1 inverted microscope (Carl Zeiss, Jena Germany) was used and with the objective of lower amplification mounted on the equipment (EC Plan-Neofluar 10x/0.3).A 405 nm laser with a maximum power of 5 mW was used as the excitation source, without exceeding 3.5% of the maximum value.For fluorescence detection, a high-sensitivity GaAsP detector was used and the bright-field image was obtained with laser light transmitted to a photomultiplier tube (PMT).For large work areas, the Tiles module of the Zen2 Blue Edition software (Carl Zeiss) allowed the acquisition of individual images and their merge into a mosaic of several photos, according to the required extension.
DNA Extraction of Bloodstream: Condensation and Precipitation for Different Conditions.DNA hyperstructure of a healthy subject.DNA ext was prepared as follows: 2 mL of peripheral blood was collected in a tube containing EDTA, incubated for 24 h at room temperature, then centrifuged at 3000 rpm for 5 min, the plasma was discarded, and the R residue was stored at −20 °C until use.For DNA ext , 40 μL of R was taken and transferred to a 1.5 mL tube containing 4 μL of BFC, vortexed, 10 μL of P was added, vortexed, 2 μL of P and then 2 μL of C were added, vortexed, and centrifuged at 1000 rpm for 5 min; finally 5 μL of P was added, vortexed, and centrifuged at 1000 rpm for 30 min.DNA ext was then precipitated directly onto the slide by precipitating 1 μL of DNA ext with 10 μL of AA ga containing 5% A ga (see Table S1).
DNA hyperstructure of patients with breast cancer and patients with uterine cancer were analyzed, DNA ext was obtained with 0 μL of BFC in a 1.5 mL tube capacity, 44 μL of R was added, and it was homogenized in vortex, 2 μL of P was added, homogenized in vortex, 2 μL of P was added, and 2 μL of C was homogenized in vortex and centrifuged at 1000 rpm for 5 min.Then, 2 μL of P was added, homogenized by vortexing, and centrifuged at 1000 rpm for 30 min.Then, 0.4 μL of DNA ext was taken and precipitated directly on the slide with 20 μL A containing 0.1% A ga (see Table S2).
DNA of a subject with Alzheimer's disease was extracted from 8 mL of peripheral blood, in an EDTA tube, and centrifuged at 3000 rpm, and 3.2 mL of plasma was taken, centrifuged at 4000 rpm, and the supernatant was discarded, the residue was resuspended with 20 μL of BFC and homogenized in a vortex, 80 μL of P and 160 μL of C were added and homogenized in a vortex and centrifuged at 10 000 rpm for 15 min.It was precipitated directly on the slide, and 1 μL of DNA ext was taken and precipitated with 75 μL of AA ga with 20% A ga .
DNA hyperstructure from an adolescent with Down Syndrome was analyzed in this study.DNA extraction utilized 25 μL of R, with the addition of 2.5 μL of BFC.The solution was homogenized in a vortex, followed by the addition of 25 μL of P and 25 μL of C.After further homogenization, the solution was centrifuged at 10 000 rpm for 15 min.3 μL of DNA ext and 25 μL of AA ga with 30% A ga were used for precipitation.The sample was observed under a microscope and photographed.
DNA hyperstructure of a pregnant woman results in the birth of a girl child.The DNA was extracted by combining 22 μL of P and 44 μL of R, followed by homogenization by using a vortex.Additional homogenization was performed using 1 μL of F and 1 μL of C, which were also homogenized in a vortex.The resulting extract was then centrifuged at 4000 rpm for 5 min, followed by a second centrifugation at 2000 rpm.After 60 min, 0.4 μL of DNA extract was precipitated with 8 μL of A and 0.1% A ga .The sample was observed under a microscope, and a photograph was taken (Table S3).
DNA hyperstructure of a pregnant woman from which a male child was born was extracted from peripheral blood using 22 μL of P and 44 μL of R, homogenized in a vortex, and then further homogenized using 1 μL of F and 1 μL of C, followed by centrifugation at 4000 rpm for 5 min and another round of centrifugation at 2000 rpm.For 60 min, 0.4 μL of DNA ext was precipitated with 8 μL of A containing 0.1% A ga .Subsequently, it was observed under a microscope and photographed for further analysis (see Table S4).The DAPI stain associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. 33Once the DAPI−DNA coupling is formed, the emission of the dye is amplified with respect to its free coupling emission 34 facilitating its detection by fluorescent techniques.In the present study, we used DAPI staining of DNA ext obtained from circulating peripheral blood to demonstrate that the structures formed by precipitating the extracts on slides correspond to the self-organization of genetic material down to millimeter sizes.Figure 2 shows a sample of DNA ext precipitated on slides with the ratio 1−10−5.Conditions of precipitation are defined: DNA ext volume (μL), AA ga volume (μL), percentage of A ga , and then (1−10−5) means 1 μL of DNA ext , 10 μL of AA ga and AA ga contains a 5% in volume of A ga .The sample corresponds to a 75-year-old woman diagnosed with breast cancer.In the bright-field image (Figure 2a), a closed structure stands out with a length of 1.96 mm of semimajor axis.Going around the perimeter, it is observed that the structure is made up of several strands or threads that in some regions join to form a compact and uniform strand that is darker than the rest with diameters between 4.5 and 10 μm.Interestingly, these compact strands can be fully stretched as at the top or forming "random coil" 35 -type structures as seen on the left side (see Figure S1).On the right side of the structure, we observe that the compact strand loses its homogeneity, and the constituent strands "open" or disperse to form ovoid structures that close at the other end to continue with the compact strand.This behavior is repeated every certain distance, breaking the continuity of the strand but completely closing the precipitated structure.The area bounded by the structure is 2.377 mm 2 .Figure 2b corresponds to the emission of the DAPI dye from the precipitated DNA sample.An intense emission is observed along the perimeter, indicating that the genetic material is found mainly in that region; therefore, the compact and scattered strands that are observed in the bright field are formed by the precipitated DNA and that it self-assembles forming these arrangements.To a lesser intensity, an emission is also observed inside the region delimited by the structure.Apparently, in the self-assembly process, not all of the molecules were integrated into the DNA hyperstructure formed.indicates the perfect splicing (merge) between the DAPI− DNA ext emission and the structure formed in the bright field.To our knowledge, there are previous works with DAPI staining where DNA condensation by charge shielding is studied, 36,37 and there are currently reports showing the formation of selfassembled DNA structures at millimeter scales. 38,39In this sense, in our work, we refer to structures with a characteristic pattern after precipitating the DNA on slides that can be systematically reproduced by applying an experimental protocol.Therefore, the term "DNA hyperstructure" is used to refer to the structures that are formed during the precipitation of DNA on a slide.Although it is not clear what is the precise mechanism that originates the precipitated structures, we maintain that some fundamental factors are the appropriate electrostatic shielding of the DNA strands by the presence of the salts in the concentrated buffer (BFC), the adequate value of the dielectric constant ε r of the solvent during the precipitation on the slide, and the adequate relation between the concentration of DNA and the volume of AA ga used in the precipitation.
From the same DNA ext analyzed above, another precipitation was performed for reproducibility purposes under the same conditions (1−10−5).Figure 3 shows the DNA hyperstructure obtained by the precipitation of the extract on slide.In the bright-field image (Figure 3a), a closed structure with a semimajor axis of 2.19 mm is observed.The elements in Figure 3 are similar to those in Figure 2.However, in the upper region of the image, there is an extension of the condensed DNA strand that measures 1.36 mm and extends beyond the enclosed area, which is the most significant and notable difference.The area bounded by this DNA hyperstructure is 1857 mm 2 .As will be seen later, the delimited area is the ideal parameter to compare the compaction of the DNA hyperstructure formed in the precipitation.For example, the value obtained for this second case is 80% of that of the previous area.Shown in red is the region where an image capture was performed at higher magnification (40×) as shown in the bright-field image (Figure 3b) and its corresponding DAPI fluorescence emission capture by confocal microscopy (Figure 3c).Both images are merged into Figure 3d.From these figures, the strands are formed by the nucleic acids of the DAPI-stained DNA precipitate, as evidenced by their intense and well-localized emission along the chain, as also shown in Figure S2.
Figure 4 shows the DNA hyperstructure obtained with the precipitation condition of 1.5−15−1.In general, the structure is formed by an intense emission in the periphery that is mostly straight with little curvature (except in the upper part) and whose continuity is lost in the left part (Figures 4a and S3).A relevant aspect in the figure is indicated in the red box, which is amplified in Figure 4b.The image indicates that after the DNA molecules self-associate to form a compact strand or chain, this can fold or twist on itself without losing its integrity.In the upper part of Figure 4b, a well-defined strand of 3.5 μm in average diameter presents great flexibility, folding on itself twice.On the right side of the figure, the chain divides into other strands that emit with less intensity and with smaller diameters (2.5 μm).
Condensed DNA Phase.Buffer (BFC) used in this work is a buffer solution where the monovalent salt (NaCl) is 100 mM, and pH is around 10; these conditions are essential for the condensed DNA phase, reported by diverse authors. 40Similar results have been obtained by Shupeng He et al. using ethanol to precipitate DNA and salts for condensation; these salts can be monovalent as Na + , K + , divalent as Ca 2+ , Mg 2+ , and trivalent as Co 3+ , La 3+ , Al 3+ . 15Further, Carrivain et al. studied the condensation mechanism that generates a supercoiling molecule of DNA when monovalent ions have been used.Melnikov et al. demonstrated that the reduction of the dielectric permittivity of the solvent by the addition of primary alcohols to a dilute DNA solution promotes the compaction of individual DNA molecules.This effect is due to the increased electrostatic forces resulting from the decreased (dielectric permittivity of the solvent) ε r , which in turn increases the attraction between similarly charged monomers due to the increased ion−ion correlations. 41A study of the monovalent ion and interaction with DNA has determined the length value for which the interaction energies between two ions and thermal energy are equal; 42 this depends on a dielectric constant relative to NaCl in water, Boltzmann constant (K B ), and temperature (T) all in the where e is the proton charge and has a Bjerrum length of 7.06 Å at room temperature.Given that b represents the distance between the DNA molecule's phosphates along the DNA axial axis 44−46 and has a value of 1.7 Å, we calculate the ratio l B /b is 4.15, where this number is known as Manning fraction (ξ) and predicts an electrostatic screening when ξ > 1.When using water and AA ga , ε r is obtained using the Onsager theory (2) (2) where ε 1 and ε 2 are the dielectric constants for the solutions, and φ 1 and φ 2 are the fraction volumes.We used three solutions, A ga , A, and water + NaCl. Figure 5 shows the charge coefficient of Manning (ξ) as a function of the value of ε r that changes as a function of Osanger eq 3, Θ is the number of contra-ions condensed for each phosphate group where N is the valence of the salt solution; in our case, the values of ε r vary between 15 and 20, and this increases the value of Θ to around 93−94%, which means that neutralization of charges is effective using the protocol proposed.Determination of AA ga Content for the Precipitation of Condensed DNA ext .The amount of AA ga necessary for the precipitation of condensed DNA ext was performed using a calibration curve.First, Lambda Virus lyophilized DNA was used to build a calibration curve by UV−vis spectroscopy and then determine the DNA concentration in the extracts obtained from peripheral blood.Condensed DNA ext from a healthy female was used as a control, and their estimated concentration was 28 μg/μL.We obtained the linear fit equation from the calibration curve with the Lambda virus by UV−vis spectroscopy: A 260 nm = 0.01687 × C DNA .Subsequently, to measure the absorbance of the DNA ext used as a control, 10 μL of DNA ext was mixed with 2990 μL of BFC (total volume in the quartz cell was 3000 μL).The absorbance obtained under these conditions was A 260 nm = 1.586.Thus, multiplying by the dilution factor gives DNA ext = 28.2μg/μL.Gong and Li mention that the conventional phenol-chloroform extraction method allows the recovery of an average of 4.5 μg of genomic DNA from 200 μL whole blood samples. 47Our method uses 4 mL of whole blood samples for DNA extraction.Theoretically, we would have 90 μg of DNA available in the extract obtained, so we consider the reported concentration range for the DNA ext to be affordable.Subsequently, a new calibration curve relating the volume of AA ga needed to precipitate DNA ext on the slide adequately was constructed.For this, dilutions of DNA ext were made, and for each concentration, the AA ga volume that formed the optimal DNA hyperstructure in the precipitate was sought.
In the precipitation tests, the volume of DNA ext was kept constant (1 μL).The optimal DNA hyperstructure was considered to cover most of the visual field allowed by a 4× objective in a conventional optical microscope, as illustrated in Figure S4.The graph shown in Figure 6 was constructed with the optimal volumes for each concentration.The behavior of the straight line that fits the experimental data shows a negative slope.This indicates that the higher the DNA concentration, the smaller the volume of AA ga required for adequate precipitation on slides.Diluted samples will require larger volumes of AA ga to be properly observed when precipitating.The explicit dependence of the AA ga volume required according to the concentration of DNA ext is summarized in the following linear relationship, eq 4 Furthermore, He et al. 15 found that in DNA solutions with concentrations near 1 μg/μL and monovalent salts (100 mM), ethanol at a concentration of 60 vol % led to almost complete precipitation of DNA.In our study, we utilized AA ga proportions for DNA precipitation ranging from 80 to 95% by volume, guaranteeing optimal conditions for efficient precipitation.Controlling the DNA Hyperstruture Compaction.We have found that a fixed concentration and volume of DNA ext and the amount of AA ga used during precipitation are the variables that regulate the compaction of the DNA hyperstructure obtained on the slide.To study the effect of AA ga on compaction, Lambda virus lyophilized was acquired and used as a control, 50 μg of Lambda virus DNA was dissolved in 100 μL of BFC.Then, 1 μL of Lambda virus DNA was precipitated with 1000, 200, and 100 μL of AA ga (95−5% v/v).Figure 7 shows (a) extended DNA hyperstructure, (b) circular DNA, and (c) compacted DNA of Lambda virus.The higher AA ga content causes a more extended precipitate and compaction is achieved with the lower AA ga content.Relevantly, these results indicate that the methodology proposed in this work also allows obtaining DNA hyperstructure from sources other than human DNA. Figure S5 shows the DNA hyperstructure obtained from plasmid pBR322.This cloning vector was purchased freeze-dried from a commercial company (Sigma) and dissolved in BFC under the same conditions as those for the Lambda virus.Surprisingly, the circular morphology of pBR322 DNA hyperstructure coincides with that reported by TEM. 48he study of DNA compaction was extended to peripheral blood samples.The images in Figure 8 correspond to the same DNA ext sample at 1.8 μg/μL precipitated on slides with different proportions.In tests, the DNA ext volume (1 μL) and A ga content in the alcohol (1% v/v) were kept constant.The AA ga volumes were 20, 15, 10, and 7.5 μL generating the structures shown in (a−d), respectively.Figure S6 shows the bright-field images merged with the fluorescent image associated with Figure 8.The perimeter-bounded area of the DNA hyperstructures was measured by using ImageJ software.We define the DNA concentration in the precipitation as an appropriate parameter to compare the effect of AA ga on the DNA hyperstructure compaction.The volume of the solution is given by the DNA ext volume + AA ga volume, in each case.The DNA ext content did not vary.Thus, Figure 8e shows a graph of the area about the DNA concentration.It is observed that there is a monotonous decreasing behavior of the area as the concentration increases.Therefore, the decrease in the AA ga content during the precipitation of the sample on the slide generates more compact DNA hyperstructures.
DNA Extraction of Bloodstream: Condensation and Precipitation for Different Conditions.The formation of DNA hyperstructures is closely tied to the solvent-drying process on the slide.Sufficient AA ga is crucial for enabling DNA molecules self-assembly during slide precipitation.Without AA ga , DNA hyperstructure formation is unattainable.Figure 9 illustrates 1 μL of DNA ext deposited on AA ga free slides.In this scenario, the formation of DNA hyperstructures does not occur, resulting in the anticipated ring-like structure that is characteristic of a drying process involving a particle or macromoleculefilled solution drop, commonly known as the "coffee ring pattern".The solvent in this case is solely composed of BFC, within which DNA ext is dispersed.
When adding AA ga during DNA ext precipitation on the slide, we should expect a ring-shaped formation as the solvent evaporates.Surprisingly, the self-assembled structures present geometries far from the circular shape, and long straight extensions and other curved regions are observed on their perimeter (Figure S7).This suggests that within the DNA hyperstructure, there exist domains with varying mechanical properties.Specifically, the straight regions exhibit higher rigidity, while the remaining regions display greater flexibility.
Figure 10 depicts the DNA hyperstructure of healthy individuals of different genders and ages.The extraction and precipitation conditions are the same for all cases.The precipitated structures exhibit a uniform morphology characterized by a single closed chain with a well-defined and uninterrupted perimeter featuring extended straight segments.Notably, the patterns generated exhibit strikingly similar morphologies, despite originating from different subjects, who   When applying the extraction protocol to both healthy individuals and patients with various conditions, we acquired images of DNA hyperstructures that exhibit noticeable modifications in morphology compared with those from healthy patients.Figure 11c,d illustrates images from uterine cancer patients lacking treatment, which differ from breast cancer  images but bear similar features to one another.Cancer creates unique physical conditions that necessitate modifications to the protocol to identify the precise conditions necessary for the condensed and self-assembled DNA to form a DNA hyperstructure.It has been widely cited that cancer cell DNA has been detected in peripheral blood, 5,49−52 quantifying the increase in cfDNA concentration has been utilized as a diagnostic method for breast cancer. 53Tumoral cells release two types of DNA, the first with information about the tumor, circulating tumor DNA (ctDNA), and healthy DNA. 54,55Atomic force microscopy images of methylated DNA from a cancer patient and healthy patients have been reported in several works. 56,57Similarly, a study of the structural changes of DNA due to the effect of methylation, which generates a process called fragmentation of cell-free cfDNA in circulation, shows important differences between healthy and sick individuals.Several methods have been reported for the isolation of cfDNA to analyze the sizes of each molecule.Based on these data, the sizes of molecules (in terms of base pairs) found in a characteristic size in healthy individuals are compared.These molecule sizes are found to be distinct and unique to individuals with different types of cancer and are obtained through PCR sequencing.Furthermore, a statistical study is conducted to determine the size of the DNA fragments. 29,58n our case, the extraction and precipitation of the DNA molecule includes all of the molecules present in the peripheral blood, so we observe a mixture of different types of DNA in a large structure.In our methodology for DNA hyperstructure, unlike in healthy patients where we observe an organized structure, here we see structures that lose the ability to organize the self-assembled structure, giving rise to a disorganized structure; we speculate that this may be due to methylated sites that prevent self-assembly.
In Figure 12, we can see the DNA of a patient with Alzheimer's disease.The image illustrates an incomplete selfassembly process of DNA chains in which several strands combine in a disorganized manner to form a diffuse perimeter.The ability to form self-organized DNA structures during the slide precipitation process seems to be impacted in patients with chronic illnesses.−66 Other researchers observed alterations in the structure of DNA in Alzheimer's patients by examining modifications in the DNA of mouth cells, which were analyzed using super-resolution microscopy (Figure 12). 67gure 13 shows the DNA hyperstructure from an adolescent with Down syndrome.
In Figure 14, the DNA hyperstructure was analyzed in four different women who were pregnant at 5, 16, 17, and 19 weeks.In all cases, a small adjacent strand is observed on the outside of the main strand.−70 Figure 15a,b shows two pregnant women in the 20th week of pregnancy, and Figure 15c,d shows the same pregnant woman in the 13th and 20th weeks of pregnancy.In the case of processed samples from pregnant women (Figure 15), structures have been systematically obtained that self-assemble separately when the DNA ext precipitates on the slide.We assume that the larger chain or structure is associated with the DNA of the pregnant woman, while the smaller structure (blue circle) corresponds to the cfDNA of the pregnant product.Diverse researchers have reported the cfDNA of mother and fetus found in the bloodstream, and fetal cfDNA is shorter than maternal cfDNA. 71,72n both situations, the DNA hyperstructure is formed by a major strand and a minor strand.It is systematically observed that in pregnancies where the product is a boy, the secondary strand is confined within the region formed by the main strand.On the contrary, if the product of pregnancy is a girl, the secondary strand is attached to the main strand on the outside of the latter.

■ CONCLUSIONS
The methodology proposed for extracting DNA from peripheral blood allows for the generation of extracts with high DNA concentrations (20−30 μg/μL).The use of BFC allows for the screening of repulsive electrostatic interactions between DNA strands, thus facilitating their self-assembly in precipitation with acid alcohol on a slide.The formation of structures upon precipitation of DNA ext is marked by intense and localized DAPI emission, thereby ensuring nucleic acid participation in the   precipitates.The millimeter-scale DNA structures, known as DNA hyperstructures, can be compressed or elongated through the regulation of the acid alcohol content during precipitation.The correlation between DNA concentration and the necessary AA ga volume for precipitation is a linear, negative slope.As the DNA molecule is identical across all living organisms, this methodology permits the fabrication of DNA hyperstructures from any source of DNA and not exclusively from humans.We postulate that a comprehensive database could enable the detection of distinctive morphological patterns in DNA hyperstructures associated with individual diseases or pregnancies.Such patterns would enable the early identification of both diseases and pregnancies via a peripheral blood sample.For all diagnoses of pregnancy and disease, it is important to perform systematic studies that consider different stages of the clinical condition, which will lead us to understand the behavior of DNA hyperstructure in order to establish diagnoses at early stages and in a reliable manner.There is strong interest in understanding how methylation affects the geometric and mechanical properties associated with DNA folding and condensation.We believe that our methodology can contribute in this direction.

Figure 3 .
Figure 3. DAPI-stained DNA ext images using confocal microscopy.(a) Bright-field mosaic image formed with 15 individual photographs obtained with a 10× objective.(b−d) High-resolution images of the red square delimited region in (a) that correspond to transmitted light or bright-field mode, DAPI fluorescence, and merged images, respectively.A 40× objective was used for zoomed images.The sample was precipitated on the slide with the 1−10−5 relation.

Figure 4 .
Figure 4. Confocal fluorescence images of DAPI-stained DNA ext .The sample was precipitated with the relation 1.5−15−1.(a) Mosaic image formed with 48 individual photographs obtained with a Plan-Apochromatic 40×/0.95dry objective.(b) Individual image acquired at 40× of the selected region.For excitation, a 405 nm laser at 1.5% of the maximum power was used.

Figure 5 .
Figure 5. Charge coefficient of Manning as a function of ε r .

Figure 6 .
Figure 6.Calibration curve for AA ga needed for optimal DNA hyperstructure formation as a function of DNA concentration in the precipitation process.Origin software.
share the common characteristic of not having chronic degenerative diseases.

Figure 11
shows the DNA hyperstructure of patient with (a, b) breast and (c, d) uterine cancer.The same protocol was used for DNA extraction and precipitation; in Figure 11(a), patient with preview chemotherapy and radiation therapy treatment, and in Figure 11(b), patient surgery and radiation therapy show a DNA hyperstructure with changes with respect to healthy subjects.

Figure 8 .
Figure 8. Photographs of DAPI-stained DNA hyperstructures on slides from the same sample and different precipitation conditions.The volume of DNA ext was constant (1 μL), and the volumes of AA ga were 20, 15, 10, and 7.5 μL in (a−d), respectively.(e) Behavior of the area delimited by DNA hyperstructure when varying the AA ga content in the DNA ext precipitation.

Figure 9 .
Figure 9. Dried DNA ext (1 μL) without AA ga at room temperature.Images (a) and (c) correspond to healthy subjects and image (b) corresponds to a pregnant woman.Scale corresponds to 500 μm.

Figure 10 .
Figure10.Hyperstructure of (a−f) DNA in healthy subjects under precipitation condition 1−10−5.The image was obtained by light microscopy using a 4× objective.DNA ext and the precipitation protocol were the same for all samples.The scale bar in all images is 500 μm.

Figure 11 .
Figure 11.(a, b) DNA hyperstructure in breast cancer subjects with treatment and (c, d) uterine cancer subjects without treatment.All samples underwent the same precipitation conditions of 0.4, 20, and 0.1.Images were acquired by using optical microscopy and a 10× objective.The scale corresponds to 200 μm for all images.

Figure 15 .
Figure 15.DNA was extracted from pregnant women carrying male fetuses, including (a) Subject 1 and (b) Subject 2 at 20 weeks of gestation, (c) Subject 3 at 13 weeks of gestation, and (d) Subject 3 at 40 weeks of gestation.(e) Amplified image from (d) corresponding to secondary strand.The precipitation condition used was 0.4−8−0.1.Image obtained through optical microscopy using a 4× objective.