Small Amounts Of DNA Research Articles

Heat Transfer Simulation of Various Material for Polymerase Chain Reaction Thermal Cycler

Medical diagnosis is the initial stage in identifying a person's condition, disease or injury from its signs and symptoms. The diagnostic method is carried out quantitatively by using a diagnostic kit which measures data such as blood pressure, heart rate frequency and blood cell concentration. These diagnostic kits are available in their respective capabilities and their activities require medical facilities and logistical readiness to function. Furthermore, Indonesia's geographical condition which consists of many islands and mountains causes uneven distribution of health facilities and laboratories in each region. Therefore, resulting in problems such as inadequate access and availability of these diagnostic kit in each region. Presently, one of the most widely used diagnostic methods is the Polymerase Chain Reaction (PCR) which allows the amplification of specific fragments from complex DNA. In PCR, only a small amount of DNA is needed to produce enough replication copies which were further analyzed by microscopic examination. This process begins with thermal cycling, which is the reactant's exposure to the heating cycle and repetitive repairs to produce reactions to different temperatures. This study aims to examine the material used for thermal cyclers which is an essential aspect of the heat transfer needed by the PCR process. In this study, heat transfer from several materials were simulated and analyzed by COMSOL Multiphysics 5.3 Software. The following results were obtained from the simulation: the saturation time for heating aluminum, copper and nickel were 29,37 and 51 seconds, respectively. Meanwhile, the cooling time was 26, 35 and 55 seconds, respectively. In addition, the saturation time for heating and cooling silver and Polydimethylsiloxane (PDMS) were 26 and 1480 seconds, respectively.

Development of the VISAGE enhanced tool and statistical models for epigenetic age estimation in blood, buccal cells and bones.

DNA methylation is known as a biomarker for age with applications in forensics. Here we describe the VISAGE (VISible Attributes through GEnomics) Consortium’s enhanced tool for epigenetic age estimation in somatic tissues. The tool is based on eight DNA methylation markers (44 CpGs), bisulfite multiplex PCR followed by sequencing on the MiSeq FGx platform, and three statistical prediction models for blood, buccal cells and bones. The model for blood is based on six CpGs from ELOVL2, MIR29B2CHG, KLF14, FHL2, TRIM59 and PDE4C, and predicts age with a mean absolute error (MAE) of 3.2 years, while the model for buccal cells includes five CpGs from PDE4C, MIR29B2CHG, ELOVL2, KLF14 and EDARADD and predicts age with MAE of 3.7 years, and the model for bones has six CpGs from ELOVL2, KLF14, PDE4C and ASPA and predicts age with MAE of 3.4 years. The VISAGE enhanced tool for age estimation in somatic tissues enables reliable collection of DNA methylation data from small amounts of DNA using a sensitive multiplex MPS assay that provides accurate estimation of age in blood, buccal swabs, and bones using the statistical model tailored to each tissue.

Magnetic nanoparticle mediated-gene delivery for simpler and more effective transformation of Pichia pastoris.

The introduction of exogenous DNA into a cell can be used to produce large quantities of protein. Here, we describe a novel gene delivery method for Pichia pastoris based on recombinant DNA delivery using magnetic nanoparticles (MNPs) under magnetic forces. For this purpose, a linear plasmid (pGKB-GFP) containing the Green Fluorescent Protein (GFP) gene is loaded on polyethyleneimine-coated iron oxide (Fe3O4@PEI) MNPs at doses that are non-toxic to the yeast cells. The pGKB-GFP loaded MNPs combined with enhancer PEI (Fe3O4@PEI + pGKB-GFP + PEI) are directly transferred to non-competent cells. An effective GFP expression was observed by the selection of antibiotic-resistant yeast cells and heterologous gene integration into the P. pastoris genome was provided. This method, which is very simple, effective, and advanced equipment-free compared to traditional methods, uses smaller amounts of DNA and the process can be performed in a shorter time. The suggested method might also be adapted for the transformation of other yeast species.

A novel approach for visualization and localization of small amounts of DNA on swabs to improve DNA collection and recovery process.

In this report, a simple and practical procedure is proposed for DNA localization on a solid matrix e.g., a collection swab. The approach is straightforward and employs spectrum decomposition using a model DNA intercalator Ethidium Bromide (EtBr). The proposed approach can detect picograms of DNA in solution and nanograms of DNA on solid surfaces (swabs) without the need for PCR amplification. The proposed technology offers the possibility for developing an inexpensive, sensitive, rapid, and practical method for localizing and recovering DNA deposited on collection swabs during routine DNA screening. Improved detection of low DNA concentrations is needed and, if feasible, will allow for better decision making in clinical medicine, biological and environmental research, and human identification in forensic investigations.

Amplified Fragment Length Polymorphism: Applications and Recent Developments.

AFLP or amplified fragment length polymorphism is a PCR-based molecular technique that uses selective amplification of a subset of digested DNA fragments from any source to generate and compare unique fingerprints of genomes. It is more efficient in terms of time, economy, reproducibility, informativeness, resolution, and sensitivity, compared to other popular DNA markers. Besides, it requires very small quantities of DNA and no prior genome information. This technique is widely used in plants for taxonomy, genetic diversity, phylogenetic analysis, construction of high-resolution genetic maps, and positional cloning of genes, to determine relatedness among cultivars and varietal identity, etc. The review encompasses in detail the various applications of AFLP in plants and the major advantages and disadvantages. The review also considers various modifications of this technique and novel developments in detection of polymorphism. A wet-lab protocol is also provided.

Recent advances and application in whole‐genome multiple displacement amplification

BackgroundThe extremely small amount of DNA in a cell makes it difficult to study the whole genome of single cells, so whole‐genome amplification (WGA) is necessary to increase the DNA amount and enable downstream analyses. Multiple displacement amplification (MDA) is the most widely used WGA technique.ResultsCompared with amplification methods based on PCR and other methods, MDA renders high‐quality DNA products and better genome coverage by using phi29 DNA polymerase. Moreover, recently developed advanced MDA technologies such as microreactor MDA, emulsion MDA, and micro‐channel MDA have improved amplification uniformity. Additionally, the development of other novel methods such as TruePrime WGA allows for amplification without primers.ConclusionHere, we reviewed a selection of recently developed MDA methods, their advantages over other WGA methods, and improved MDA‐based technologies, followed by a discussion of future perspectives. With the continuous development of MDA and the successive update of detection technologies, MDA will be applied in increasingly more fields and provide a solid foundation for scientific research.

Characterization of Recombinant Maintenance DNA Methyltransferase (Dnmt1) and Application to DNA Methylation Analysis

A change doesn't occur to base sequence of DNA in the process of the human development. However, one fertilized egg differentiates into cells with sixty trillion of abundant individuality. The reason is DNA is methylated. The DNA methylated correctly is necessary for correct differentiation. Aberrant methylation induce various diseases including cancer, mental disorder and lifestyle disease.Elucidating essential of diseases needs analyzing methylation of DNA.Various DNA methylation analysis methods have been developed, among which bisulfite sequencing (BS-seq) is common.BS-seq has problems such as low sensitivity, which can not analyze cell types with insufficient sample amount, and may miss specific methylation of individual cells because it is an averaged data of cell population.Conventional BS-seq, hydroxymethylation (hmC), which is an intermediate of demethylation, is detected as methylation (mC). Since hmC is not recognized as mC in vivo, conventional BS-seq can not perform accurate DNA methylation analysis.New analytical methods are needed to assess pure DNA methylation even from small numbers of cells, such as single cells. In this study, we aim to develop a novel methylation analysis method that enables accurate methylation to be performed even with a small amount of DNA by repeating amplification and methylation of DNA using PCR and DNA (cytosine-5)-methyltransferase 1 (DNMT1).

Effect of DNA on the optical properties of ZnO:SiO2:La3+ films

ZnO–SiO2 composite films doped with La3+ ions (0.1%–5.0% mass.) were obtained using the sol–gel method. The absorption and ultraviolet luminescence spectra of the films, as well as the change in spectral characteristics under the influence of DNA, were studied. Doping of ZnO:SiO2 films with La3+ ions leads to an increase in the transmittance in the UV range (λ = 357 nm) from 4.5% (for undoped films) to 32%–34%, and the transmittance is inversely proportional to the amount of dopant. In all doped films, the optical band gap increases in comparison with undoped films (3.35 eV) and amounts to 3.830–3.853 eV. The application of DNA to the surface weakly affects the value of optical band gap. The maximum of the ultraviolet luminescence (UVL) band in ZnO:SiO2:La3+ films is slightly shifted depending on the amount of dopant and is located at λem = 363–367 nm. An increase in UVL intensity was observed upon doping by a factor of 8.4–9.6 compared to ZnO:SiO2 films. A change in the amount of dopant does not lead to sharp changes in the magnitude of the intensity. Thus, the UVL intensity (λex = 260), compared with films with 0.1% La3+ content, varies by 5%–11%. The effect of small amounts of DNA was studied for films doped with La3+. In the case of ZnO:SiO2:La3+, both extinguishing (up to 12%) and ignition (up to 5%) of UVL were observed. Monotone dependencies (which are desirable for the biosensor sensing element) were obtained for samples with La3+ 0.1 and 1.0% content. SEM images show that the images for zinc oxide are white crystallites of different sizes: from 0.5 µm to conglomerates of crystallites −1 µm. In the presence of DNA, the surface structure changes due to the formation of rhomboid structures, whose sizes vary from 3 µm to 35 µm. It is known that lanthanum ions induce the compaction of DNA with the formation of nanoscale structures.

Developmental validation of the Yfiler Platinum PCR Amplification Kit for forensic genetic caseworks and databases.

Y chromosome kits are successfully applied in cases where human biological material exists. With the development of genotyping ability, more Y chromosomal markers are needed for finer identification of male individuals and lineages. In this study, a developmental validation of a newly emerged Y chromosome kit that combines two different kinds of markers: 38 Y-STRs and 3 Y-indels are conducted. The results show that this kit has high sensitivity when there is a small amount of DNA (125 pg), more than one male (minor:major = 1:7), or a mixture of males and females (male:female = 125pg:1875pg), inhibited substances (800 μM hematin and more than 1600ng/μL humic acid). The kit exhibits high precision level with a standard deviation of allele size no more than 0.14nt. Locus DYS481 shows the largest stutter rate, with three stutters per true allele. Population samples are well identified (MP of 0.001106), and mutations can be observed in father-son pairs (46 mutations in 70 pairs, 10 in locus DYS627). Out of all the population samples, 13.2% belong to haplogroup M117-O2a2b1a1, with their ethnic group being Han Chinese. The results show that this kit can improve the performance of identifying male individuals, obtaining more unique haplotypes (increasing from 894 to 918 of 1000 male samples) and higher discrimination capacity (increasing from 0.942 to 0.955) in this study compared to previous widely used Yfiler Plus kit. Besides, it gives information about their paternal lineages in forensic genetic casework and genealogical database construction.

Multiplexed Droplet Digital PCR Assays for the Simultaneous Screening of Major Genetic Alterations in Tumors of the Central Nervous System.

The increased integration of molecular alterations to define tumor type or grade in central nervous system (CNS) tumor classification brings new challenges for the pathologist to make the best use of a precious limited tissue specimen for molecular studies. Within the different methods available to identify gene alterations, the droplet digital PCR (dPCR) constitutes a rapid, cost-effective, and very sensitive tool. In this study, we describe the development and validation of five multiplexed dPCR assays to detect major CNS biomarkers by using only small amounts of DNA extracted from formalin-fixed paraffin-embedded specimens. When compared to HRM-sequencing, NGS-sequencing, RNA-sequencing, or simplex digital PCR assays used as “gold standard” methods, these multiplexed dPCR assays displayed 100% specificity and sensitivity for the simultaneous detection of: 1/BRAF V600E mutation and KIAA1549:BRAF fusion; 2/FGFR1 N546K and K656E mutations and FGFR1 duplication; 3/H3F3A K27M and G34R/V mutations; 4/IDH1 R132X and IDH2 R172X mutations; and 5/TERT promoter mutations C228T and C250T. In light of the increased integration of molecular alteration, we believe that such strategies might help laboratories to optimize their screening strategies for routine diagnosis of pediatric and adult CNS tumors.

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.

DNA recovery from unfired and fired cartridge cases: A comparison of swabbing, tape lifting, vacuum filtration, and direct PCR

The ability to recover trace DNA from fired cartridge cases can help establish important leads regarding the handler of the ammunition. Over recent years, several DNA recovery techniques for fired ammunition have been published. Three techniques of significant interest include tape lifting, direct PCR, and vacuum filtration. This study aimed to compare these to the swabbing method currently employed in our jurisdiction. Brass and nickel cartridges of five different calibres were spiked with 20ng of saliva and subject to DNA collection using all four DNA recovery methods. Unfired and fired cartridges were tested to examine the effects of firing.Swabbing recovered a greater quantity of DNA than vacuum filtration while no significant differences were found between swabbing and tape-lifting. The calibre of ammunition had no effect on DNA recovery. Firing significantly reduced DNA yield from nickel cartridges, while unfired brass cartridges returned less DNA than unfired nickel cartridges.PCR inhibition was not observed in any samples, although degradation indices suggested that most samples were slightly or moderately degraded. Analysis of profiles showed that swabbing and tape lifting resulted in greater numbers of alleles from fired nickel and brass cartridges compared to direct PCR. Samples from nickel cartridges were found to have a greater number of uploadable profiles than samples from brass cartridges. In addition, three mixed profiles were obtained from the single source spiked cartridges as well as evidence of pre-existing DNA on uncleaned cartridges and contaminating alleles on cleaned cartridges.Our results suggest that tape-lifting can be a suitable alternative to swabbing, but that caution must be taken when interpreting profiles from fired cartridge cases as small amounts of DNA not associated with the handling of the cartridges may be present.

Genotyping complex structural variation at the malaria-associated human glycophorin locus using a PCR-based strategy.

Structural variation in the human genome can affect risk of disease. An example is a complex structural variant of the human glycophorin gene cluster, called DUP4, which is associated with a clinically significant level of protection against severe malaria. The human glycophorin gene cluster harbours at least 23 distinct structural variants, and accurate genotyping of this complex structural variation remains a challenge. Here, we use a polymerase chain reaction-based strategy to genotype structural variation at the human glycophorin gene cluster, including the alleles responsible for the U- blood group. We validate our approach, based on a triplex paralogue ratio test, on publically available samples from the 1000 Genomes project. We then genotype 574 individuals from a longitudinal birth cohort (Tori-Bossito cohort) using small amounts of DNA at low cost. Our approach readily identifies known deletions and duplications, and can potentially identify novel variants for further analysis. It will allow exploration of genetic variation at the glycophorin locus, and investigation of its relationship with malaria, in large sample sets at minimal cost, using standard molecular biology equipment.

Analysis of evidence in forensic medicine. Validation of the test method as a tool to control parameters determining the reliability of the analysis results

Currently, forensic research foundation is comprised of the methods of genetic analysis. The study of DNA polymorphism became a source of highly reliable evidence, which allow the determination of the genetic profile of biological traces containing genetic material of barely a few cells, collected from all possible substrates, and their further comparison with the genetic profile of the victim or suspect. The forensic expertise of such research, supported by relevant statistical calculations is one of the few objective evidence in criminal proceedings.The process of DNA testing for forensic purposes is composed of several stages and a number of analyzes. After securing suitable test specimen, the isolation of genetic material is performed. For the quantitative and qualitative analysis of the isolate, the Real Time PCR reaction is performed. This is a crucial moment for the study because it is the assessment of the suitability of samples for further testing. The next step is the amplification of DNA fragments with Multiplex PCR technique. Then, as a result of electrophoretic separation of the sequencer we obtain a genetic profile. Thus obtained profiling results are interpreted as well as compared by specialists.For analysts in the field of forensic medicine it is necessary to obtain clear genetic profiles. Given the often small amount of DNA that can be obtained and the need for unquestionability of the evidence, it is very important to develop a method with high sensitivity, which allows to obtain completely reliable results. In order to ensure the quality of the research, forensic laboratories must apply the standards and guidelines set by international associations, periodically calibrate their testing equipment, keep complete records of the results and, above all, to validate the used methods. In the laboratory, which intends to apply the procedure in question, internal validation must be carried out. The aim of this process is to determine the reliability and limitations of the method. During the test, the results of manufacturers’ verification are validated and the level of efficiency of the said procedure within a particular lab is checked.

The mutREAD method detects mutational signatures from low quantities of cancer DNA

Mutational processes acting on cancer genomes can be traced by investigating mutational signatures. Because high sequencing costs limit current studies to small numbers of good-quality samples, we propose a robust, cost- and time-effective method, called mutREAD, to detect mutational signatures from small quantities of DNA, including degraded samples. We show that mutREAD recapitulates mutational signatures identified by whole genome sequencing, and will ultimately allow the study of mutational signatures in larger cohorts and, by compatibility with formalin-fixed paraffin-embedded samples, in clinical settings.

Validation of novel forensic DNA markers using multiplex microhaplotype sequencing

Microhaplotypes (MH) are comprised of multiple single nucleotide polymorphisms (SNPs) that are located within 300 bases of genomic sequence. Improved tools are needed to facilitate broader application of microhaplotypes in a diverse range of populations and forensic settings. We designed an assay for multiplex sequencing of 90 microhaplotypes (mMHseq) that include 46 MH loci with high Effective Number of Alleles (Ae) from previous studies [1], and 44 high Ae MH loci containing between four to fourteen SNPs that were identified from the 1000 Genomes (1KG) Project. The unique design of mMHseq integrates a novel method for multiplex amplification from small DNA amounts, and multiplex sequencing of 48 samples in a single MiSeq run to detect all relevant MH variation. Assay performance was evaluated in a cohort of 156 individuals from seven different world populations from Africa, Asia, and Europe. Three of those populations from East Africa (Chagga, Sandawe, and Zaramo) and one from Eastern Europe (Adygei) had sufficient individuals sequenced by the assay to be included in statistical analyses with the 26 1KG populations. For those 30 populations the mean global average Ae was 5.08 (range: 2.7–11.54) and mean informativeness for biogeographic variation (In) was 0.30 (range: 0.08-0.70). Eighty-five novel SNPs were detected in 58 of the 90 microhaplotypes. Open-source, web-based software was developed to visualize haplotype phase data for each microhaplotype and individual. Our approach for multiplex microhaplotype sequencing can be customized and expanded as novel loci are being discovered.

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles.

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4-5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.

Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma.

Liquid biopsies are not used in practice for hepatocellular carcinoma (HCC). Epi proColon is the first commercial blood‐based test for colorectal cancer screening based on methylated DNA testing of the septin 9 gene (SEPT9). However, Epi proColon has some disadvantages, including the requirement of a large amount of blood and lack of quantitative performance. Therefore, we previously developed a novel liquid biopsy test that can quantitatively detect even a single copy of methylated SEPT9 in a small amount of DNA. In the current study, we evaluated the application potential of this assay for diagnosing HCC. Study subjects included 80 healthy volunteers, 45 patients with chronic liver disease (CLD) without HCC, and 136 patients with HCC (stage 0, 12; stage A, 50; stage B, 31; stage C, 41; and stage D, 2), according to the Barcelona Clinic Liver Cancer staging system. For the assay, DNA was treated with methylation‐sensitive restriction enzymes in two steps, followed by multiplex droplet digital polymerase chain reaction. The median copy number of methylated SEPT9 was 0.0, 2.0, and 6.4 in the healthy control, CLD, and HCC groups, respectively, with significant differences among the groups (HCC vs. healthy control, P < 0.001; HCC vs. CLD, P = 0.002; CLD vs. healthy control, P = 0.008). Assay sensitivity and specificity were 63.2% and 90.0%, respectively (cutoff value, 4.6 copies), in detecting HCC when compared with healthy subjects. The positive rate of methylated SEPT9 increased with HCC progression (stage 0, 41.7%; stage A, 58.0%; stage B, 61.3%; stage C, 75.6%; and stage D, 100%). Conclusion: We developed a sensitive methylated SEPT9 assay that might serve as a liquid biopsy test for diagnosing HCC.

Postlabeling/PAGE Method for Detection of DNA Adducts.

In DNA adduct analysis, the 32P-postlabeling technique is a powerful tool due to its high detection sensitivity. It is performed by enzymatic digestion of DNA samples, enrichment of the adduct nucleotides, and then 5'-labeling with 32P. This method is particularly useful for detection of bulky adducts. An additional advantage is that only a small amount of DNA is required for detecting DNA adducts. This chapter describes the experimental procedure for separation and detection of DNA adducts by polyacrylamide gel electrophoresis, which is an attractive method for visually assessing differences in adduct formation between samples.

DNA Yields and PCR Amplification Success Using Degraded Animal Corpses

A recurring problem with ancient specimens from wildlife animals is that their preserved tissues contain small amounts of DNA in a degraded state. The specific objectives of the present study were: (1) To determine DNA yields from different animal tissues; (2) to compare traditional (manual) DNA extraction protocols with commercial procedures and (3) to assess the success of PCR amplification of Inter-Simple Sequence Repeats (ISSR) loci in degraded animal samples. Liver, stomach and muscle samples were extracted from coyote (Canis latrans) and long-tailed weasel (Mustela frenata) for this research. Manual protocols for DNA extraction were compared to a commercial kit procedure (Qiagen DNeasy kit). Genomic DNA in different states (intact, apoptotic and degraded) were amplified using a panel of ISSR primers. No DNA was recovered from coyote stomach samples using the manual extraction protocol. DNA concentrations in stomach and liver samples from coyote were 10.31 ng/μL and 15.8 ng/μL, respectively using the Qiagen extraction kit. In general, the kit extraction method yielded more DNA than the manual extraction procedure but it is more expensive. Intact and apoptotic genomic DNA were successfully amplified by PCR resulting in a similar profile. Artificially degraded DNA showed partial amplification. Thus, the ISSR marker system is suitable for animal population genetics when only limited and/or degraded animal DNA is available.