Human DNA Extraction Research Articles

Evaluation of five automated and one manual method for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid

ObjectivesMolecular detection of Toxoplasma gondii plays a crucial role in the prenatal and neonatal diagnosis of congenital toxoplasmosis (CT). Sensitivity of this diagnosis is partly related to the efficiency of parasite DNA extraction and amplification. DNA extraction methods with automated platforms have been developed. Therefore, it is essential to evaluate them in combination with adequate PCR amplification assays. MethodsIn this multisite study, we investigated the suitability of two recent automated procedures for the isolation of Toxoplasma DNA from amniotic fluid (AF) (Magtration system 12GC, PSS and Freedom EVO VacS, Tecan), compared with three other automated procedures (MagNAPure Compact, Roche, BioRobot EZ1, Qiagen and modified NucliSens easyMAG, bioMérieux) and with the manual DNA extraction QIAamp DNA Mini kit (Qiagen). Two Toxoplasma PCR assays targeting the ‘529-bp’ repeat DNA element were used, based upon dual hybridization (FRET) or hydrolysis (TaqMan) probes. A total of 1296 PCRs were performed including 972 Toxoplasma PCRs. ResultsWe showed variable efficacy (4.2%–100% positive results) among the DNA extraction procedures in isolating up to five T. gondii cells/mL in AF samples. Moreover, for a given DNA extraction method, variable results were obtained among the two Toxoplasma PCR assays for detecting up to five T. gondii cells/mL: when using TaqMan PCR, all the automated systems yielded more than 60% positive results. Nevertheless, when testing the DNA extracts in triplicate, four out of six extraction methods allowed a satisfactory detection of low amounts of T. gondii DNA (≥33% of positive results) independently of the PCR assay used. ConclusionsDespite the influence of the subsequent PCR method used, this study should help microbiologists in the choice of DNA extraction methods for the detection of T. gondii in amniotic fluid. The extraction method should be checked as adequate for the PCR assay used.

A simple, rapid and economic manual method for human sperm DNA extraction in genetic and epigenetic studies

Abstract Over the recent years, isolation of high quantities of pure, intact, double-stranded, highly concentrated and not contaminated genomic DNA is prerequisite for successful and reliable large scale genetic and epigenetic analysis and also high quantities of pure DNA are required for these types of studies. The DNA extraction methods developed for human somatic cells are not effective for sperm because of the high rating of nuclear compaction and DNA integrity in sperm cells. In the present study, an economic, reliable and simple method of DNA extraction procedures, were examined to ascertain their relative effectiveness for extracting DNA from human sperm. The quality and quantity of the extracted DNA were assessed by spectrophotometric measurements, methylation-specific PCR (MSP) amplification, and gel electrophoresis. This method was applied successfully from sperm samples (N = 50, age = 33.9 ± 7.8 years) with DNAs absorbency ratios of A260/A280 (1.82 ± 0.03) and A260/230 (1.95 ± 0.16). Gel electrophoresis revealed non-degraded and suitable quality genomic DNA for epigenetic and genetic analysis. The purity and quality of this protocol was closer to the optimum value sand no contamination during manual extraction was observed. It concluded that this described method might have a sufficient quality for subsequent molecular analysis and general genetic and epigenetic methods.

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking

Genome fragment enrichment (GFE) method was applied to identify host-specific bacterial genetic markers that differ among different fecal metagenomes. To enrich for swine-specific DNA fragments, swine fecal DNA composite (n = 34) was challenged against a DNA composite consisting of cow, human, goat, sheep, chicken, duck and goose fecal DNA extracts (n = 83). Bioinformatic analyses of 384 non-redundant swine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode metabolism-associated, cellular processes and information storage and processing. After challenged against fecal DNA extracted from different animal sources, four sequences from the clone libraries targeting two Bacteroidales- (genes 1–38 and 3–53), a Clostridia- (gene 2–109) as well as a Bacilli-like sequence (gene 2–95), respectively, showed high specificity to swine feces based on PCR analysis. Host-specificity and host-sensitivity analysis confirmed that oligonucleotide primers and probes capable of annealing to select Bacteroidales-like sequences (1–38 and 3–53) exhibited high specificity (>90%) in quantitative PCR assays with 71 fecal DNAs from non-target animal sources. The two assays also demonstrated broad distributions of corresponding genetic markers (>94% positive) among 72 swine feces. After evaluation with environmental water samples from different areas, swine-targeted assays based on two Bacteroidales-like GFE sequences appear to be suitable quantitative tracing tools for swine fecal pollution.

Modifying and adapting a plant-based DNA extraction protocol for human genomic DNA extraction: a cost effective approach

This research involved modification of a standard protocol for DNA extraction in plants for its suitability as an extraction method for DNA in human whole blood in which some of the plasma was removed. Human blood samples were obtained from a 100 apparently healthy individuals residing in Calabar. The modified DNA procedure yielded good quality genomic DNA which was used in carrying out allele specific polymerase chain reaction which also yielded good quality amplicons. This method is simple and suitable for the extraction of DNA from human red cell.

A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples.

Different methods available for extraction of human genomic DNA suffer from one or more drawbacks including low yield, compromised quality, cost, time consumption, use of toxic organic solvents, and many more. Herein, we aimed to develop a method to extract DNA from 500μL of fresh or frozen human blood. Five hundred microliters of fresh and frozen human blood samples were used for standardization of the extraction procedure. Absorbance at 260 and 280nm, respectively, (A260 /A280 ) were estimated to check the quality and quantity of the extracted DNA sample. Qualitative assessment of the extracted DNA was checked by Polymerase Chain reaction and double digestion of the DNA sample. Our protocol resulted in average yield of 22±2.97μg and 20.5±3.97μg from 500μL of fresh and frozen blood, respectively, which were comparable to many reference protocols and kits. Besides yielding bulk amount of DNA, our protocol is rapid, economical, and avoids toxic organic solvents such as Phenol. Due to unaffected quality, the DNA is suitable for downstream applications. The protocol may also be useful for pursuing basic molecular researches in laboratories having limited funds.

Human autosomal DNA and X chromosome STR profiles obtained from Chrysomya albiceps (Diptera: Calliphoridae) larvae used as a biological trace.

The use of insects to answer questions in criminal investigations, as well as a combination of forensic genetic techniques to obtain human DNA from the organisms, especially necrophagous dipterians, have gained ground in recent decades among researchers and professionals in this area. The objective of our study was to evaluate and compare two methods of human DNA extraction, commonly used for forensic samples, to obtain human autosomal DNA and X chromosome short tandem repeat profiles from the digestive tract of Chrysomya albiceps (Diptera: Calliphoridae) larvae. Immature specimens were collected from corpses at the Institute of Forensic Medicine of Pernambuco and raised in bovine ground meat to allow stabilization of the colony. Groups of larvae in the third instar were provided with bovine ground meat plus human blood for 48 h, dissected, and then subjected to DNA extraction. DNA was extracted using two methods: a DNA IQ™ kit and a phenol-chloroform method. Genomic DNA was amplified using AmpFℓSTR® Identifiler® Plus PCR and Argus-X-12® kits, and samples were sequenced to determine if the two extraction techniques generated reliable profiles that were compatible with a reference sample. The existence of comparable profiles from both techniques demonstrates the usefulness of dipteran larvae for obtaining human DNA from corpses, which can be further used to correlate genetic profiles in a crime scene when other traces are not available. However, several variables still require revision; thus, the technique should be further investigated for its validity, security, and, in particular, its reproducibility.

Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains.

DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent.

The Potential of Cosmetic Applicators as a Source of DNA for Forensic Analysis.

Personal products, such as toothbrushes, have been used as both known reference and evidentiary samples for forensic DNA analysis. This study examined the viability of a broad selection of cosmetic applicators for use as targets for human DNA extraction and short tandem repeat (STR) analysis using standard polymerase chain reaction (PCR) conditions. Applicator types included eyeliner smudgers, pencils and crayons, eye shadow sponges, mascara wands, concealer wands, face makeup sponges, pads and brushes, lipsticks and balms, and lip gloss wands. The quantity and quality of DNA extracted from each type of applicator were examined by assessing the number of loci successfully amplified and the peak balance of the heterozygous alleles in each full STR profile. While degraded DNA, stochastic amplification, and PCR inhibition were observed for some items, full STR profiles were developed for 14 of 76 applicators. The face makeup sponge applicators yielded the highest proportional number of full STR profiles (4/7).

Extraction of human genomic DNA from whole blood using a magnetic microsphere method.

With the rapid development of molecular biology and the life sciences, magnetic extraction is a simple, automatic, and highly efficient method for separating biological molecules, performing immunoassays, and other applications. Human blood is an ideal source of human genomic DNA. Extracting genomic DNA by traditional methods is time‐consuming, and phenol and chloroform are toxic reagents that endanger health. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA. In this study, we developed urea–formaldehyde resin magnetic microspheres and magnetic silica microspheres for extraction of human genomic DNA. First, a magnetic microsphere suspension was prepared and used to extract genomic DNA from fresh whole blood, frozen blood, dried blood, and trace blood. Second, DNA content and purity were measured by agarose electrophoresis and ultraviolet spectrophotometry. The human genomic DNA extracted from whole blood was then subjected to polymerase chain reaction analysis to further confirm its quality. The results of this study lay a good foundation for future research and development of a high‐throughput and rapid extraction method for extracting genomic DNA from various types of blood samples.

Elimination of bioweapons agents from forensic samples during extraction of human DNA.

Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling.

Rapid extraction and preservation of genomic DNA from human samples

Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

Extraction of Human Genomic DNA from Dried Blood Spots and Hair Roots

In most studies about molecular genetics, molecular diagnostics, DNA extraction is considered as the very first step that will give a significant effective. The material used for DNA extraction is vary from fresh blood to dried blood, tissue, oral mucosa and hair root…. The dried blood and hair root are the two materials that easy to collect and can store for long term, which leads to more advantages for many time extractions to have fresh DNA and enough amount DNA for further studies. This study introduces an efficient phenol method with 2 steps of lyses which allows high yield of DNA concentration from dried blood and hair root. Comparing to the commercial QIAamp® DNA Mini Kit which applied for dried blood sample, DNA concentration extracted from this method was higher, the quality of DNA was equal and the cost is lower. Comparing to salting out method, a cheap and safe method, which an applied for hair root, the two-step lysis phenolic method also give higher efficiency. This method could be the useful tool which may apply for further molecular analysis studies using long storing time materials like dried blood spots and hair root samples. The reliability and performance of the molecular diagnostic assays which are polymerase chain reaction (PCR), enzyme digestion and recombinant selection (1) are strongly influenced by the extraction of input material, in this case, the quantity and quality of nucleic acids from clinical samples. Although blood is highly recommended for DNA extraction, for the experiments in the extremely hard working environment, blood may be difficult to be stored in long term. Furthermore the ease of collecting, costs of transporting, costs of storing and pre-processing should be taken all into consideration how to get an optimal choice of samples. Then despite of blood is the most commonly used sample since it provides enough and high quality DNA, people look for other sources which are low cost, easy to collect and convenient to store.

Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells.

We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.

PCR-ready human DNA extraction from urine samples using magnetic nanoparticles

Urine-derived human genomic DNA (gDNA) has wide application in a variety of disciplines including clinical medicine, sports, and forensic science. We describe a novel method for gDNA extraction from urine samples using carboxylated magnetic nanoparticles (CMNPs) as solid-phase adsorbents. Sedimentation associated with freezing of urine samples significantly reduces cell capture by CMNPs. However, the addition of 10 mM EDTA and subsequent pH modification (pH 6.0–7.1) can re-dissolve urine sediments. Purified gDNA ranged from around 0.1 kb to more than 23 kb. PCR using specific primers targeting K-ras, GAPDH, CYP3A4 and GDF5 amplified 100% of varying sized gene fragments, verifying the high quality of the isolated DNA. Successful PCR amplifications using DNA isolated from urine samples as small as 50 μl were demonstrated. Enrichment of urine cells and subsequent adsorption of DNA can be achieved with the same CMNPs, greatly simplifying extraction procedures. The CMNP gDNA extraction technique proved to be simple, rapid, sensitive and environmentally friendly, with application for routine laboratory use and potentially within automated urine extraction platforms.

The change in human DNA content over time in the artefacts of the blowfly Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

Adult blowflies can act as vectors of human DNA by depositing faecal or regurgitation spots (termed artefacts) after feeding on meals of human biological fluid. Given the number of backlog cases in many forensic laboratories, it is important to know the maximum time period any human DNA within a fly artefact can remain unextracted before the DNA degrades to unusable levels. The present study aimed to determine whether or not the human DNA content in fly artefacts deposited by the blowfly Lucilia cuprina changed over time, and also to provide a timeframe for forensic biologists in which the extraction of human DNA from fly artefacts should be attempted. Both the blood and semen data showed that the amount of human DNA that could be extracted increased over the first 400 days, but had decreased to one-month levels by 750 days. The saliva data showed no changes over the 60-day time period in the amount of human DNA that could be extracted.

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

BackgroundFrozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome.MethodsHigh-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene.ResultsHigh speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection.ConclusionsHigh speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.

Human DNA Extraction Methods: Patents and Applications

Since the pioneer experiments conducted by Friedrich Miescher in 1861, extraordinary advances have been achieved in the field of DNA handling. Today nucleic acids can be extracted from any type of biological material such as tissues, cells and viruses. Moreover, increasing knowledge of human genome is paving the way to an effective employment of pharmacogenomics and genetic-based predictive tests in medicine. In this context, the recovery of DNA from different sources of biological samples (e.g. archived formalin-fixed autopsy tissues, dried blood spots, frozen serum or plasma, long-term stored whole blood) is also an emerging field in genetic epidemiology studies. Thus, given the crucial role played by DNA in bio-medical research and in its related applications, here we review the main relevant issued patents and recently published advances in the field of DNA extraction and purification from human specimens.

An Efficiency Human Genomic DNA Extraction from Dried Blood Spots

Extraction of genomic DNA is the very first experiment in the study cascade of human inherited diseases. Blood is preferred to be the material for DNA extraction. In the case of limitation of the material source, for example, new born screening program, blood is saved as dried spot in filter paper. In practical, long term storing blood sample as the dried spots on filter paper leads to more advantages for DNA extraction essay which need to be performed several times. This study provides an efficient method with 2 step lyses allow high concentration of DNA is extracted from samples. Comparing to the commercial QIAamp® DNA Mini Kit DNA concentration extracted from this method is higher, the quality of DNA is equal and the cost is lower. The optimal DNA extraction method based on phenol in this study is useful tool which may apply for further molecular analysis studies using dried blood spot samples.

Typing of Multiple Single-Nucleotide Polymorphisms by a Microsphere-Based Rolling Circle Amplification Assay

The combination of suspension array with rolling circle amplification can lead to a sensitive and specific assay for single-nucleotide polymorphisms (SNPs) detection, as demonstrated in this study. A circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase on microspheres. The elongation products were labeled with fluorochrome-tagged probes and detected in a flow cytometer, indicating the mutation occurrence. As low as 10 amol of mutated strands was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10 000:1, which could be attributed to the high amplification efficiency of Phi29, the high binding capacity of the microspheres, and the remarkable precision of DNA ligase in distinguishing mismatched bases at the ligation site. A novel design of using two differently labeled detection probes on the same microsphere to target both the wild-type and mutant samples allowed parallel determination of the heterozygosity for two SNPs (K-ras G12C and TP53 R273H) in PCR amplicons prepared from human genomic DNA extracts. This ability lays the groundwork for further enhancing the assay throughput by using multiple fluorophores and microspheres with distinct properties.

A comparison of four methods for extraction of human fecal DNA by using real time PCR

目的 比较国际主要微生态实验室4种常用的核酸抽提方法获得的粪便DNA的质量.方法 采用针对目的细菌(总细菌、类杆菌-普氏杆菌属组、双歧杆菌、肠杆菌科细菌及肠球菌)16s rDNA的特异性引物及实时定量PCR方法,分析QIAamp(R)粪便DNA试剂盒法(QIA法)、FastDNA(R) Kit试剂盒法(Fast法)、酚-氯仿-玻璃珠击打法(酚-氯仿-玻珠法)及QIAamp(R) DNA粪便试剂盒+玻璃珠击打法(QIA+玻珠法)4种方法抽提获得的10份人粪便DNA的质与量.结果通用引物特异性扩增粪便总DNA,发现4/10份由酚-氯仿-玻珠法获得的粪便DNA中存在PCR抑制剂,过柱纯化后PCR抑制现象消失.实时定量PCR检测粪便总细菌量,以QIA法与QIA+玻珠法获得的量最高(分别为12.16±0.18,12.05±0.48),而Fast法与酚-氯仿-玻珠法获得的粪便细菌量(分别为11.26±0.40,11.21±0.16)与QIA法相比显著低下(均P＜0.05).采用QIA法(11.63±0.23,8.68±0.51)及QIA+玻珠法(11.67±0.56,8.77±0.56)抽提获得的粪便类杆菌-普氏杆菌及肠杆菌科细菌的DNA量显著高于Fast法(10.67±0.47,7.39±0.51)及酚-氯仿-玻珠法(10.65±0.33,7.40±0.18)(均P＜0.05).对于双歧杆菌,酚-氯仿-玻珠法获得的量显著低下.但对肠球菌而言,以Fast法获得的量最高.结论 实时定量PCR检测结果表明,QIA法及QIA+玻珠法获得的粪便DNA质与量总体上优于Fast法及酚-氯仿-玻珠法,但根据不同的目的细菌群对象可以选择不同的方法。