Size Of DNA Fragments Research Articles

Evaluation of an Adapted Semi-Automated DNA Extraction for Human Salivary Shotgun Metagenomics.

Recent attention has highlighted the importance of oral microbiota in human health and disease, e.g., in Parkinson's disease, notably using shotgun metagenomics. One key aspect for efficient shotgun metagenomic analysis relies on optimal microbial sampling and DNA extraction, generally implementing commercial solutions developed to improve sample collection and preservation, and provide high DNA quality and quantity for downstream analysis. As metagenomic studies are today performed on a large number of samples, the next evolution to increase study throughput is with DNA extraction automation. In this study, we proposed a semi-automated DNA extraction protocol for human salivary samples collected with a commercial kit, and compared the outcomes with the DNA extraction recommended by the manufacturer. While similar DNA yields were observed between the protocols, our semi-automated DNA protocol generated significantly higher DNA fragment sizes. Moreover, we showed that the oral microbiome composition was equivalent between DNA extraction methods, even at the species level. This study demonstrates that our semi-automated protocol is suitable for shotgun metagenomic analysis, while allowing for improved sample treatment logistics with reduced technical variability and without compromising the structure of the oral microbiome.

Amplicon size and non-encapsidated DNA fragments define plasma cytomegalovirus DNA loads by automated nucleic acid testing platforms: A marker of viral cytopathology?

Management of cytomegalovirus (CMV) in transplant patients relies on measuring plasma CMV-loads using quantitative nucleic acid testing (QNAT). We prospectively compared the automated Roche-cobas®6800-CMV and Roche-CAP/CTM-CMV with laboratory-developed Basel-CMV-UL54-95bp, and Basel-CMV-UL111a-77bp. Roche-cobas®6800-CMV and Roche-CAP/CTM-CMV were qualitatively concordant in 142/150 cases (95%). In-depth comparison revealed higher CMV-loads of the laboratory-developed assay and correlated with smaller amplicon size. After calibration to the 1.WHO-approved CMV international standard, differences were reduced but remained significant. DNase-I pretreatment significantly reduced CMV-loads for both automated Roche-CAP/CTM-CMV and Roche-cobas®6800-CMV assays, whereby 90% and 95% of samples became undetectable. DNase-I pretreatment also reduced CMV-loads quantified by Basel-CMV-UL54-95bp and Basel-CMV-UL111a-77bp, but remaining detectable in 20% and 35%, respectively. Differences were largest for 110 samples with low-level CMV-DNAemia being detectable but not-quantifiable by Roche-cobas®6800-CMV, whereby the smaller amplicon sizes yielded higher viral loads for concordant positives. We conclude that non-encapsidated fragmented CMV-DNA is the major form of plasma CMV-loads also measured by fully-automated platforms. Amplicons of <150 bp and calibrators are needed for reliable and commutable QNAT-results. We hypothesize that non-encapsidated fragmented CMV-DNA results from lysis of CMV-replicating cells and represent a direct marker of viral cell damage, which contribute to delayed viral load responses despite effective antivirals.

Processing DNA Storage through Programmable Assembly in a Droplet-Based Fluidics System.

DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot be actively processed in DNA pools. An active DNA data editing process is developed using splint ligation in a droplet-controlled fluidics (DCF) system. DNA fragments of discrete sizes (100-500bps) are synthesized for droplet assembly, and programmed sequence information exchange occurred. The encoded DNA sequences are processed in series and parallel to synthesize the determined DNA pools, enabling random access using polymerase chain reaction amplification. The sequencing results of the assembled DNA data pools can be orderly aligned for decoding and have high fidelity through address primer scanning. Furthermore, eight 90bps DNA pools with pixel information (png: 0.27-0.28kB), encoded by codons, are synthesized to create eight 270bps DNA pools with an animation movie chip file (mp4: 12kB) in the DCF system.

Blood and saliva-derived ctDNA is a marker of residual disease after treatment and correlates with recurrence in human papillomavirus-associated head and neck cancer.

There is an alarming increase in human papillomavirus-associated head and neck cancer (HNC), reaching epidemic levels. While patient prognosis is generally good, off-target treatment effects are associated with decreased quality of life. Thus, non-invasive strategies to predict treatment response and risk of recurrence could help de-escalate treatment. In this study, we tested circulating tumor (ct)DNA in liquid biopsies (blood/saliva) of HPV-positive HNC patients to assess treatment response and disease progression. A total of 235 blood and saliva samples were collected from 60 HPV-positive and 17 HPV-negative HNC patients (control group) before and/or after treatment. Samples were analyzed using ddPCR for HPV16/18/31/33/35/45 and correlated with imaging and pathological examination. HPV-ctDNA detection was significantly higher prior to treatment (91%) than after treatment (8.0%) (χ2 p < 0.00001), with high concordance between saliva and blood (93%). In matched samples, all patients positive for ctDNA before treatment showed significant reductions in ctDNA levels post treatment (p < 0.0001). All but one patient with persistent ctDNA after treatment showed residual tumor and subsequent recurrence. Finally, fragmentomic analysis revealed shifts in cell-free DNA fragment size after treatment, suggesting a complementary biomarker for treatment response. Blood and saliva were found to be good sources of HPV-ctDNA. The presence of ctDNA strongly correlated with treatment response, demonstrating clinical utility as a non-invasive biomarker to monitor tumor progression in HPV-positive HNC. Liquid biopsy based ctDNA testing could be an effective approach to predict recurrence and stratify patients for de-escalation of treatment, thereby improving quality of life.

Nanopore sequencing of DNA barcodes succeeds in unveilling the diversity of fungal mock communities

Background: The fungal component of the gut microbiome has been underrepresented in most gut microbiome studies. While metabarcoding approaches have been used to assess the diversity and role of the gut mycobiome, differences in experimental design and downstream analyses may induce bias and differential outcomes. This study assessed the capacity of nanopore sequencing to retrieve the microbial profile and relative abundance of a mock fungal community comprised of Candida glabrata, Meyerozyma guilliermondii, Pichia kudriavzevii, Clavispora lusitaniae and Candida parapsilosis. Methods: The approach was implemented using the MinION system and involved the analysis of sequencing libraries made from equimolar mixtures of the PCR-amplified nuclear ribosomal internal transcribed spacer (ITS) of the target species. Results: Nanopore sequencing successfully retrieved the composition of the fungal mock community in terms of the different taxa present. However, the approach was unable to correctly assess the expected relative abundances of each species in the same community, showing some yet undetermined bias that may be related to the size of the respective target DNA fragments. Clavispora lusitaniae was consistently overrepresented in the mixtures, while C. glabrata was underrepresented. The remaining three species showed relative abundances more aligned with the expected values of an equimolar mixture. Conclusions: Although not yielding the expected results for the relative abundances, the values obtained from independent sequencing runs were similar for all species, suggesting a good reliability but questionable accuracy in this sequencing approach.

O-287 Preimplantation genetic testing for monogenic disease (PGT-M) where results are only based upon analysis of linked polymorphisms/haplotypes risks serious diagnostic errors

Abstract Study question PGT-M often involves diagnoses based upon the analysis of polymorphisms linked to the mutant gene. Are such methods sufficiently reliable to be used alone? Summary answer Several problems can lead to misdiagnosis when linkage analysis is used in isolation. Therefore, PGT-M should also include direct mutation testing (DMT) whenever possible. What is known already Many PGT-M strategies involve analysis of DNA sequence polymorphisms in close proximity to the mutant gene, which have specific alleles that are inherited along with the disease. Unlike diagnostics that focus on detection of specific mutations, which can be unique to individual families, PGT-M protocols using linked polymorphisms can usually be reused for multiple families. Indeed, strategies such as karyomapping, which assess thousands of polymorphisms across the genome, provide a single method applicable to numerous diseases. Such methods are attractive since the work-up required for individual cases is minimal, reducing costs and patient waiting times. However, are such methods safe? Study design, size, duration Over a period of three years, we carried out 261 PGT-M cases covering 312 different mutations in 116 genes. Each couple requesting PGT-M provided blood samples from which DNA was extracted. The patients underwent IVF and embryos that reached the blastocyst stage were subjected to trophectoderm biopsy. All PGT-M cases involved the use of DMT to interrogate the mutation site(s). The DMT result was supplemented by analysis of multiple informative linked polymorphisms, as described below. Participants/materials, setting, methods Embryo biopsy samples were subjected to multiple displacement amplification (MDA). Mutation site(s) were amplified from MDA products using PCR and mutations were revealed using minisequencing, Sanger sequencing, or DNA fragment size analysis. Parental and embryo samples were also analysed using karyomapping, involving the genotyping ∼300,000 polymorphisms scattered across the genome with a microarray. Where possible, samples from additional family members were also tested, allowing determination of which alleles of linked polymorphisms accompanied mutant gene copies. Main results and the role of chance Multiple PGT-M cases were identified where DMT prevented potentially serious errors. In five cases, diagnostic reports provided to the PGT laboratory were incorrect. These reports are vital for defining the genetic status of an individual, allowing specific alleles of linked polymorphisms to be correctly associated with mutant or normal gene copies. In three cases, DMT carried out on patient samples during the initial work-up revealed that, contrary to the report, the patient did not carry a mutation. Therefore, PGT-M was not indicated, saving patients from the stress and expense of an unnecessary PGT-M cycle and avoiding discard of healthy embryos. In the other two cases, errors in the reports would have inverted all results based upon linkage analysis, leading to transfer of affected embryos and discard of unaffected embryos, potentially causing a serious misdiagnosis. Thirteen more cases had recombination events extremely close to the mutation site, preventing determination of the status of the embryos based on analysis of nearby polymorphisms. Two further cases displayed consanguinity (undisclosed by the patients), leading to large areas of homozygosity in the genome, precluding use of linked polymorphisms for diagnosis. In all these cases DMT unequivocally confirmed the true status of patients and their embryos. Limitations, reasons for caution In ∼8% of PGT-M cases at least one embryo could not be accurately diagnosed without DMT, while in ∼1% of cases our routine use of DMT averted a serious misdiagnosis. Nonetheless, even when DMT and linkage analysis are combined, it must be acknowledged that misdiagnoses remain possible (although extremely rare). Wider implications of the findings PGT-M is a valuable reproductive strategy for patients at high-risk of transmitting a single gene disorder. Linkage analysis is a valid strategy for PGT-M, but misdiagnoses are possible when testing relies entirely on such methods. These errors can be virtually eliminated by including direct testing of the mutation site. Trial registration number Not applicable

Isolation of salivary cell-free DNA for cancer detection.

Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA- a bona fide cancer biomarker in a subset of head and neck cancers- from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.

Nanopore sequencing of DNA barcodes to unveil the diversity of fungal mock communities

Background: The fungal component of the gut microbiome has been underrepresented in most gut microbiome studies. While next-generation sequencing (NGS) approaches have been used to assess the diversity and role of the gut mycobiome, differences in experimental design and downstream analyses may induce bias and differential outcomes. This study assessed the capacity of nanopore sequencing to retrieve the microbial profile and relative abundance of a mock fungal community comprised of Candida glabrata, Meyerozyma guilliermondii, Pichia kudriavzevii, Clavispora lusitaniae and Candida parapsilosis. Methods: The approach was implemented using the MinION system and involved the analysis of sequencing libraries made from equimolar mixtures of the PCR-amplified internal transcriber spacer genomic regions of the target species. Results: Nanopore sequencing successfully retrieved the composition of the fungal mock community in terms of the different taxa present. However, the approach was unable to correctly assess the expected relative abundances of each species in the same community, showing some yet undetermined bias that may be related to the size of the respective target DNA fragments. Clavispora lusitaniae was consistently overrepresented in the mixtures, while C. glabrata was underrepresented. The remaining three species showed relative abundances more aligned with the expected values of an equimolar mixture. Conclusions: Although not yielding the expected results for the relative abundances, the values obtained from independent sequencing runs were similar for all species, suggesting a good reliability but questionable accuracy in this sequencing approach.

NanoNIPT: Short-fragment nanopore sequencing of cell-free DNA for non-invasive prenatal testing of fetal aneuploidies and sex chromosome aberrations.

N/A This article is protected by copyright. All rights reserved.

Development and validation of blood tumor mutational burden reference standards.

Tumor mutational burden (TMB), measured by exome or panel sequencing of tumor tissue or blood (bTMB), is a potential predictive biomarker for treatment benefit in patients with various cancer types receiving immunotherapy targeting checkpoint pathways. However, significant variability in TMB measurement has been observed. We developed contrived bTMB reference materials using DNA from tumor cell lines and donor-matched lymphoblastoid cell lines to support calibration and alignment across laboratories and platforms. Contrived bTMB reference materials were developed using genomic DNA from lung tumor cell lines blended into donor-matched lymphoblastoid cell lines at 0.5% and 2% tumor content, fragmented and size-selected to mirror the size profile of circulating cell-free tumor DNA with TMB scores of 7, 9, 20, and 26 mut/Mb. Variant allele frequency (VAF) and bTMB scores were assessed using PredicineATLAS and GuardantOMNI next-generation sequencing assays. DNA fragment sizes in the contrived reference samples were similar to those found within patient plasma-derived cell-free DNA, and mutational patterns aligned with those in the parental tumor lines. For the 7, 20, and 26 mut/Mb contrived reference samples with 2% tumor content, bTMB scores estimated using either assay aligned with expected scores from the parental tumor cell lines and showed good reproducibility. A bioinformatic filtration step was required to account for low-VAF artifact variants. We demonstrate the feasibility and challenges of producing and using bTMB reference standards across a range of bTMB levels, and how such standards could support the calibration and validation of bTMB platforms and help harmonization between panels and laboratories.

Comparative Assessment of DNA Extraction Techniques From Formalin-Fixed, Paraffin-Embedded Tumor Specimens and Their Impact on Downstream Analysis.

Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits. Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing. In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results. The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance.

Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer

Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (95% CI [87, 93]). Sensitivity was 55% at 95% specificity. Performance was similar for early stage 1 (AUC 89%; 95% CI [83, 94]) and late stage 4 CRC (AUC 94%; 95% CI [89, 98]). The classifier could detect CRC even when the proportion of tumor DNA in blood was undetectable by other methods. Expanding the classifier to include information about cell-free DNA fragment size and abundance across the genome led to gains in sensitivity (63% at 95% specificity), with similar overall performance (AUC 91%; 95% CI [89, 94]). We confirm that 5-hydroxymethylcytosine can be used to detect CRC, even in early-stage disease. Therefore, the inclusion of 5-hydroxymethylcytosine in multianalyte testing could improve sensitivity for the detection of early-stage cancer.

EV-ADD, a database for EV-associated DNA in human liquid biopsy samples.

Extracellular vesicles (EVs) play a key role in cellular communication both in physiological conditions and in pathologies such as cancer. Emerging evidence has shown that EVs are active carriers of molecular cargo (e.g. protein and nucleic acids) and a powerful source of biomarkers and targets. While recent studies on EV‐associated DNA (EV‐DNA) in human biofluids have generated a large amount of data, there is currently no database that catalogues information on EV‐DNA. To fill this gap, we have manually curated a database of EV‐DNA data derived from human biofluids (liquid biopsy) and in‐vitro studies, called the Extracellular Vesicle‐Associated DNA Database (EV‐ADD). This database contains validated experimental details and data extracted from peer‐reviewed published literature. It can be easily queried to search for EV isolation methods and characterization, EV‐DNA isolation techniques, quality validation, DNA fragment size, volume of starting material, gene names and disease context. Currently, our database contains samples representing 23 diseases, with 13 different types of EV isolation techniques applied on eight different human biofluids (e.g. blood, saliva). In addition, EV‐ADD encompasses EV‐DNA data both representing the whole genome and specifically including oncogenes, such as KRAS, EGFR, BRAF, MYC, and mitochondrial DNA (mtDNA). An EV‐ADD data metric system was also integrated to assign a compliancy score to the MISEV guidelines based on experimental parameters reported in each study. While currently available databases document the presence of proteins, lipids, RNA and metabolites in EVs (e.g. Vesiclepedia, ExoCarta, ExoBCD, EVpedia, and EV‐TRACK), to the best of our knowledge, EV‐ADD is the first of its kind to compile all available EV‐DNA datasets derived from human biofluid samples. We believe that this database provides an important reference resource on EV‐DNA‐based liquid biopsy research, serving as a learning tool and to showcase the latest developments in the EV‐DNA field. EV‐ADD will be updated yearly as newly published EV‐DNA data becomes available and it is freely available at www.evdnadatabase.com.

First large‐scale quantification study of DNA preservation in insects from natural history collections using genome‐wide sequencing

Abstract Insect declines are a global issue with significant ecological and economic ramifications. Yet, we have a poor understanding of the genomic impact these losses can have. Genome‐wide data from historical specimens have the potential to provide baselines of population genetic measures to study population change, with natural history collections representing large repositories of such specimens. However, an initial challenge in conducting historical DNA data analyses is to understand how molecular preservation varies between specimens. Here, we highlight how Next‐Generation Sequencing methods developed for studying archaeological samples can be applied to determine DNA preservation from only a single leg taken from entomological museum specimens, some of which are more than a century old. An analysis of genome‐wide data from a set of 113 red‐tailed bumblebee Bombus lapidarius specimens, from five British museum collections, was used to quantify DNA preservation over time. Additionally, to improve our analysis and further enable future research, we generated a novel assembly of the red‐tailed bumblebee genome. Our approach shows that museum entomological specimens are comprised of short DNA fragments with mean lengths below 100 base pairs (BP), suggesting a rapid and large‐scale post‐mortem reduction in DNA fragment size. After this initial decline, however, we find a relatively consistent rate of DNA decay in our dataset, and estimate a mean reduction in fragment length of 1.9 bp per decade. The proportion of quality filtered reads mapping to our assembled reference genome was around 50%, and decreased by 1.1% per decade. We demonstrate that historical insects have significant potential to act as sources of DNA to create valuable genetic baselines. The relatively consistent rate of DNA degradation, both across collections and through time, mean that population‐level analyses—for example for conservation or evolutionary studies—are entirely feasible, as long as the degraded nature of DNA is accounted for.

Isogenic Japonica Rice Koshihikari Integrated with Late Flowering Gene Hd16 and Semidwarfing Gene sd1 to Prevent High Temperature Maturation and Lodging by Typhoon.

We developed semidwarf and late-maturing isogenics of Koshihikari to stabilize high yield and avoid high temperature maturation. Whole-genome analysis (WGS) was conducted to examine the transitional changes in the entire genome, the size of DNA fragments integrated with the target gene, and genes accompanying the target gene owing to the progress of backcrossing. In both Koshihikari Hd16 (BC7F4) and Koshihikari sd1Hd16 (BC8F2), an SNP from adenine to guanine was detected in Hd16 at 32,996,608 bp on chromosome 3, which is known to be a causative mutation of Hd16 in Nipponbare. In Koshihikari sd1Hd16 (BC8F2), an SNP from thymine to guanine was detected in sd1 at 38,267,510 bp on chromosome 1. From BC7 to BC8, the size of the DNA fragment integrated with Hd16 decreased by 5871 bp. Koshihikari sd1Hd16 flowered 12.1 days later than Koshishikari or Koshihikari sd1 did and was 14.2 cm (15%) shorter than Koshihikari. The yield in Koshishikari sd1Hd16 (63.2 kg/a) was 7.0% higher than that of Koshihikari. This is a new germplasm designed to avoid heat damage at ripening during high-temperature summer periods by late maturation owing to Hd16 as well as to avoid lodging by autumn typhoons by semidwarfness owing to sd1.

Fragment size and dynamics of EGFR-mutated tumor-derived DNA provide prognostic information regarding EGFR-TKI efficacy in patients with EGFR-mutated NSCLC

Circulating tumor DNA (ctDNA)-based next-generation sequencing (NGS) is a complementary and alternative test to tissue-based NGS. We performed NGS analysis of ctDNA samples collected from patients with EGFR-mutated non-small cell lung cancer (NSCLC) who received osimertinib; the samples were collected after second-line treatment, before osimertinib treatment, one week and one month after osimertinib treatment, and at the time of resistance formation. We examinedthe correlation with osimertinib efficacy. From January to December 2018, 34 patients with EGFR-mutated NSCLC harboring EGFR T790M mutations were enrolled, and a total of 132 peripheral blood samples were collected. The fragment sizes of EGFR-mutated ctDNAs were significantly shorter than that of their corresponding normal fragments. Osimertinib treatment of patients with shorter EGFR-mutated ctDNA fragments resulted in shorter progression-free survival (PFS). The disappearance time of EGFR-mutated fragment fractions and clonal evolution patterns (new driver mutation group, additional mutation group vs. attenuation group) were each associated with the PFS achieved with osimertinib treatment; however,multivariate analysis revealed that only shorter EGFR-mutated ctDNA fragments were associated with the PFS resulting from osimertinib treatment. EGFR-mutated ctDNA fragment size, time of disappearance of these fragments, and clonal evolution pattern were related to the effects of osimertinib. In particular, short EGFR-mutated ctDNA fragmentation may be closely related to osimertinib efficacy prediction.

Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination.

SummaryFunctional genomics, synthetic biology and metabolic engineering require efficient tools to deliver long DNA fragments or multiple gene constructs. Although numerous DNA assembly methods exist, most are complicated, time‐consuming and expensive. Here, we developed a simple and flexible strategy, unique nucleotide sequence‐guided nicking endonuclease (UNiE)‐mediated DNA assembly (UNiEDA), for efficient cloning of long DNAs and multigene stacking. In this system, a set of unique 15‐nt 3′ single‐strand overhangs were designed and produced by nicking endonucleases (nickases) in vectors and insert sequences. We introduced UNiEDA into our modified Cre/loxP recombination‐mediated TransGene Stacking II (TGSII) system to generate an improved multigene stacking system we call TGSII‐UNiE. Using TGSII‐UNiE, we achieved efficient cloning of long DNA fragments of different sizes and assembly of multiple gene cassettes. Finally, we engineered and validated the biosynthesis of betanin in wild tobacco (Nicotiana benthamiana) leaves and transgenic rice (Oryza sativa) using multigene stacking constructs based on TGSII‐UNiE. In conclusion, UNiEDA is an efficient, convenient and low‐cost method for DNA cloning and multigene stacking, and the TGSII‐UNiE system has important application prospects for plant functional genomics, genetic engineering and synthetic biology research.

Rapid, inexpensive fabrication of electrophoretic microdevices for fluorescence detection.

The laser print, cut, and laminate (PCL) method for microfluidic device fabrication can be leveraged for rapid and inexpensive prototyping of electrophoretic microchips useful for optimizing separation conditions. The rapid prototyping capability allows the evaluation of fluidic architecture, applied fields, reagent concentrations, and sieving matrix, all within the context of using fluorescence‐compatible substrates. Cyclic olefin copolymer and toner‐coated polyethylene terephthalate (tPeT) were utilized with the PCL technique and bonding methods optimized to improve device durability during electrophoresis. A series of separation channel designs and centrifugation conditions that provided successful loading of sieving polymer in less than 3 min was described. Separation of a 400‐base DNA sizing ladder provided calculated base resolution between 3 and 4 bases, a greater than 18‐fold improvement over separations on similar substrates. Finally, the accuracy and precision capabilities of these devices were demonstrated by separating and sizing DNA fragments of 147 and 167 bases as 148.62 ± 2 and 166.48 ± 3 bases, respectively.

Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes.

Inflammation is central to the mechanisms of parturition, but the lack of understanding of how it is controlled in normal parturition hampers our ability to understand how it may diverge resulting in preterm birth. Cell-free fetal DNA is found in the amniotic fluid, and it is thought to be able to activate inflammation as a danger-associated molecular pattern. Although its levels increases with gestational age, its effect has not been studied on the human fetal membranes. Thus, the aim of this study was to determine if the fetal DNA can trigger inflammation in the human fetal membranes and, thus, potentially contribute to the inflammatory load. Isolated human amniotic epithelial cells and fetal membrane explants were treated apically with fetal DNA causing the translocation of NF-KB into the nucleus of cells and throughout the cells of the explant layers with time. Fetal membrane explants were treated apically with either small or larger fragments of fetal DNA. IL-6, TNFα, and GM-CSF secretion was measured by ELISA, and pro-MMP2 and pro-MMP9 activity was measured by zymography from apical and basal media. Increased apical IL-6 secretion and basal pro-MMP2 activity was seen with small fragments of fetal DNA. When the data were disaggregated based on fetal sex, males had significant increases in IL-6 secretion and basal increased activity in pro-MMP2 and 9, whereas females had significantly increased basal secretion of TNFα. This was caused by the smaller fragments of fetal DNA, whereas the larger fragments did not cause any significant increases. Male fetal DNA had significantly lower percentages of methylation than females. Thus, when the cytokine and pro-MMP activity data were correlated with methylation percentage, IL-6 secretion significantly correlated negatively, whereas GM-CSF secretion positively correlated. These data support the role of fetal DNA as an inflammatory stimulus in the FM, as measured by increased NF-κB translocation, cytokine secretion, and increased pro-MMP activity. However, the data also suggested that the responses are different from FM tissues of male and female fetuses, and both the fragment size and methylation status of the fetal DNA can influence the magnitude and type of molecule secreted.

Abstract 1694: Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells

Abstract Introduction: Detection of circulating free DNA (cfDNA) is a promising tool to monitor and predict tumor progression and treatment efficacy. Our group has shown the clinical relevance of cfDNA as a biomarker of tumor development and progression in several cancer models, including colorectal carcinoma and melanoma. cfDNA may stem from a combination of apoptosis, necrosis, and cellular secretions. However, the source and mechanism of cfDNA release in cancer cells are still unclear. Indeed, while some data state that cfDNA is derived from apoptosis, other studies report it is mainly a product of cellular secretions. To better understand cfDNA release and its correlation with tumor biology, this study aimed to assess cfDNA kinetics under cell death, cell cycle arrest, and senescence by using an in vitro cancer model. Methods: Colorectal (HTC116 and HT29), lung (A549), and melanoma (MP41 and OMM2.5) cancer cells underwent cytotoxic conditions by drugs: TRIAL/APO2L, Roscovitine, and valproic acid (VAP), as well as environmental stress by heat and irradiation. Cell death and cell cycle were evaluated by FACS. β-galactosidase staining assessed senescence. cfDNA was extracted from 3 mL supernatant using QIAamp nucleic acid kit and evaluated by digital droplet PCR through hot spot mutations (Mut), and a mitochondrial gene (mt-cfDNA). Isolated cfDNA was visualized by fragment size using Bioanalyzer 2100. Results: Mut and mt-cfDNA increased during cytotoxic conditions in a dose-dependent manner. Cells under apoptosis released greater mut and mt-cfDNA compared to necrotic and untreated cells. Similarly, cell cycle disturbances by VAP were associated with an increase in Mut and mt-cfDNA. Moreover, β-galactosidase positive cells (5D post-irradiation) showed lower Mut and mt-cfDNA levels than control conditions. Electropherogram images showed that cytotoxic conditions alter fragment size distribution. While fragments about 100-200 bp were observed in apoptosis, necrosis showed more abundant fragments about &amp;gt;1000 bp. Conclusions: The release of cfDNA is influenced by cytotoxic and stress conditions. Knowing the biology of cfDNA can help understand its role in cancer development and how best to utilize this marker clinically, especially post cytotoxic agents that induce different cell death mechanisms. These findings have major implications in the interpretation of ctDNA in liquid biopsy as a biomarker of treatment response and tumor progression. Citation Format: Prisca Bustamante, Thupten Tsering, Julia Valdemarin Burnier. Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1694.