A rapid method for the isolation of genomic DNA from citrated whole blood.
Ontology highlight
ABSTRACT:
SUBMITTER: Parzer S
PROVIDER: S-EPMC1149904 | biostudies-other | 1991 Jan
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC4134021 | biostudies-literature
Project description:High quality and sufficient quantity of genomic DNA (gDNA) are the primary requisites of several molecular biologic applications, including clinical studies related to genetics, genomics, gene polymorphism, and DNA fingerprinting. Whole blood is the primary source of gDNA in most of the clinical investigations. Currently, commercial kits are primarily used to achieve these goals. However, the use of kits is limited by the cost and involvement of several centrifugal steps. Other methods reported are either laborious or do not produce high quality or quantity of gDNA or both. Here, we present the data on the development of a centrifugation-free, cost-effective, and user-friendly method for the isolation of human gDNA from the buffy coat of human blood that involves limited numbers of steps with about 15 min of hands-on time per sample.
| S-EPMC5865945 | biostudies-literature
Project description:The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist.
| S-EPMC4486329 | biostudies-literature
Project description:Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates.Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng--3 microg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase cocktail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human beta-globin locus YAC.An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.
| S-EPMC406510 | biostudies-literature
Project description:In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.
| S-EPMC5648668 | biostudies-literature
Project description:BackgroundGenotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis.All samples were obtained from premature newborn infants between 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 μl) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort.ResultsBoth whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3 years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples.ConclusionsgDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies.
| S-EPMC3817355 | biostudies-literature
Project description:Isolation and amplification of nucleic acid (DNA) is considered a vital and potent instrument in molecular biological research. However, its functioning outside of a laboratory setting is difficult because of complex procedures that demand expert personnel and expensive equipment in addition to the fulfillment of several additional requirements. DNA isolation from minute insects is sometimes difficult, making diagnostic and genotyping procedures problematic. Thus, the current work offers a high-throughput, cost-effective, straightforward, and faster approach for isolating DNA from the aphid Myzus persicae. Intriguingly, two-step DNA extraction process yielded a high yield of extremely pure genomic DNA and required only 10 s to complete. PCR investigation aiming at amplifying the non-synonymous R81T region on the loop D site of the nAChR gene of M. persicae was subsequently utilized to successfully validate the recovered DNA. Moreover, the proposed method was compared in terms of yield and purity with conventionally used DNA isolation methods including, phenol:chloroform, salt out, and commercially available kits. In conclusion, this newly developed method would enable researchers to quickly process many biological samples used to analyze genetic diversity, mutant screening, and large spectrum diagnosis both in laboratory and field conditions.
| S-EPMC10497781 | biostudies-literature
Project description:Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.
| S-EPMC4670203 | biostudies-literature
Project description:Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories.
| S-EPMC4831698 | biostudies-literature
Project description:BACKGROUND:Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS:We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 ?L unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS:PCR and DNA sequencing results obtained by DEP from 25 ?L CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. CONCLUSIONS:Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.
| S-EPMC4114224 | biostudies-literature