Isolation Of Deoxyribonucleic Acid Research Articles

Isolation of genomic DNA from groundnut plant by modified, rapid and efficient protocol

DNA isolation is prerequisite to study the crop at molecular level. However, DNA (Deoxyribonucleic Acid) isolation is difficult in groundnut due to presence of large amount of polyphenols and polysaccharides. The age and growth stage of the plant also affect the DNA purity during isolation. In this study, newly expanded leaves and stems of five days old seedlings and matured leaves of 20 days old seedlings were used for optimization of extraction protocol. High quality DNA was obtained from all plant materials by this modified CTAB (Cetyltrimethylammonium bromide) protocol without separate purification. Optimized protocol was used to isolate DNA from newly expanded leaves of 5 days old seedlings of 100 groundnut genotypes. DNA obtained ranged from 6.3-22.1 µg/ g of sample when quantified by Nano Drop spectrophotometer and was able to amplify with Simple sequence repeats (SSR) primers during Polymerase Chain Reaction (PCR).

ANALYSIS OF APOLIPOPROTEIN-B (APO-B) GENE IN ATHEROSCLEROSIS MICE GIVEN CURCUMINOID EXTRACT OF ZANTHORRIZA IN ORAL

The aim of this study was to investigate the apolipoprotein-B (apo-B) gene in atherosclerosis mice which were orally given curcuminoid extract of Curcuma xanthorriza. A total number of 30 white mice were split into 6 groups, the first group considered as control (without any treatment), second group as atherogenic feed control, the third group as extract control, while the fourth, fifth and sixth groups as atherogenic feed and curcuminoid Curcuma xanthorriza extract group treated with 5 mg/mouse, 10 mg/mouse and 15 mg/ mouse, respectively for three months. The blood samples were taken from all six groups for the deoxyribonucleic acid (DNA) analysis using total DNA isolation, DNA amplification with polymerase chain reaction (PCR), and DNA sequencing. The data analysis showed that 374 bp nucleotide sequence gen of apo-B from Rattus norvegicus in groups B, C, D, E, and F did not cause any changes in genes. The analysis showed the sequence of apo-B Rattus norvegicus gene in the treatment group was apparently identical with that of Rattus norvegicus group A as the control group without treatment. As conclusion, the administration of curcuminoid zanthorrizza to atherosclerosis mice did not change the gene structure of apo-B 100.

Comparison of Methods for Genomic Deoxyribonucleic Acid (DNA) Extraction Suitable for Whole-Genome Genotyping in Traditional Varieties of Rice

The utilization and conservation traditional rice genotypes have attracted global attention. Optimization of DNA isolation protocol for genetic characterization of plants is a necessary and primary step. Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research, with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated DNA is prerequisite for successful and reliable large-scale genotyping analysis. Therefore, standardization of DNA isolation is a basic requirement. Here we employed three methods of DNA isolation namely, Dellaporta, Hi-purA and modified CTAB techniques for isolation of genomic DNA from 25 indigenous rice genotypes. From the results, it was found that genomic DNA isolated by modified CTAB method to be the most appropriate for extracting high quality and maximum quantity DNA suitable for genotyping. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements and gel electrophoresis.

Deoxyribonucleic Acid Extraction and Quantification from Human Saliva Deposited on Foods with Bitemarks

The aim of this study is to evaluate the potential of deoxyribonucleic acid (DNA) recovery from bite marks in foods, in different collection types, from DNA quantification. The sample consisted of 80 swabs, obtained from 20 cheese pieces, bitten by the same person, using the double-swab technique in the center and the periphery of the bite. A statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS) statistical software version 20.0, with values of p < 0.05 being considered statistically significant. The DNA was recovered in all cheese pieces, regardless of the collection types and the bite region. However, the comparative analysis of DNA recovery potential in the four swabs allowed us to infer that the collections in the central region of the bite (DC and WC) were the ones that presented better precision, in addition to extracting a higher DNA concentration, the dry swab being in the center of the bite which presented better results. The results proved the effectiveness of the double-swab technique for collecting genetic materials in bite marks; however, in the food used, a single collection at the center of the bite would be enough, optimizing the resources and time needed for the analysis. Due to the difficulties of physically comparing a site of a skin lesion and the dental arches of the suspect, the evidence of DNA in saliva has been used to indicate the perpetrator of the bite. In addition, the collection, preservation, and isolation of saliva DNA can be done at low cost and provide flexibility for clinical and laboratory workflow.

Optimalization of deoxyribonucleic acid extraction using various types of magnetic particles

Isolation of deoxyribonucleic acid is an important step in the molecular diagnostics of microorganisms. A high quality of isolated deoxyribonucleic acid is necessary for deoxyribonucleic acid amplification by the polymerase chain reaction. The conventional deoxyribonucleic acid isolation using phenol–chloroform extraction and deoxyribonucleic acid precipitation in ethanol is time-consuming and requires the use of toxic phenol. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solid-phase deoxyribonucleic acid extraction. The amounts of deoxyribonucleic acid in separation mixtures were measured using ultraviolet spectrophotometry. The first experimental conditions were tested on chicken erythrocytes deoxyribonucleic acid. Phosphate buffer (pH 7, 7.6 and 8) was used for the adsorption of deoxyribonucleic acid on magnetic particles. Tested values of pH have no effects on deoxyribonucleic acid adsorption. It was shown that approximately almost one half of deoxyribonucleic acid was adsorbed to the particles. A number of different elution conditions (temperature, time and pH value of elution buffer) were investigated to determine their effect on elution efficiency. It was shown that only a small fraction of the bound DNA was released at 22 °C, while the release was more effective as the temperature was increased also the amount of elution deoxyribonucleic acid was more effective when time was increased. The higher amounts of deoxyribonucleic acid were eluted with TE buffer pH 9.0. Second, bacterial deoxyribonucleic acid was tested. This deoxyribonucleic acid eluted from the particles was in polymerase chain reaction ready quality.

Evaluation of collagenase-3 matrix metalloproteinase-13 gene-associated polymorphisms 11A/12A and -77A/G and its associated alleles with and without periodontitis.

Context:Connective tissue devastation in periodontitis and other chronic inflammatory diseases is a major concern. There are several inflammatory mediators associated with this process among which matrix metalloproteinases (MMPs) play a predominant role. Collagen degradation is primarily mediated by the collagenases. MMP-13 is familiar as collagenase-3, which has the aptitude to humiliate fibrillar collagen.Aims:This study aims to evaluate MMP-13 promoter polymorphism, 11A/12A, and −77A/G and associated alleles in patients with and without chronic periodontitis (CP).Settings and Design:This was an observational case–control study.Materials and Methods:Of the total 100 patients, 50 with CP (test group) and 50 without CP (Control group), blood was collected for deoxyribonucleic acid isolation. The 11A/12A and −77A/G polymorphisms of the MMP-13 gene were picked out by polymerase chain reaction (PCR)- single-strand conformation polymorphism analysis method and PCR-restriction fragment length polymorphism by BseNI restriction enzyme, respectively.Statistical Analysis Used:Association between MMP-13 genotype (GTs) (11A/12A, 11A/11A, 12A/12A) and (AA, AG, GG) was assessed by Chi-square and Student's t-test for intergroup comparison.Results:11A/12A GT was seen in 24% and 20%, 11A/11A 64% and 72%, 12A/12A 12% and 8% in test and control groups, respectively. However, the association was not statistically significant. −77A/G polymorphism associated GT s AA was 56% and 62%, AG 24% and 28%, GG 20% and 10% in test and controls, respectively. An association of GG was statistically significant.Conclusion:The present study results indicated that MMP-13 −77A/G gene polymorphisms, GG GT may be predicted intensive ability for CP. On the other hand, there was no significant association between MMP-13 11A/12A gene polymorphisms with CP.

Nucleotide incorporated magnetic microparticles for isolation of DNA

For the comprehensive understanding of many diseases, especially genetic disease, the highly purified deoxyribonucleic acid (DNA) is needed to perform in-detail studies. There are various methods to obtain highly purified DNA, but low in number. In this study, Fe&Ni-mc-poly (2-hydroxyethyl methacrylate-adenine methacrylate) [poly(HEMA-AdeM)] microparticles were synthesized to get high purification ratio because of natural and thus selective interaction between adenine on the microparticles and thymine on the DNA molecule and also high amount of DNA purified. For the characterization of microparticles scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), elemental analysis, swelling ratio and zeta size analysis methods were performed. All experiments were performed batch-wise for the determination of optimum conditions such as pH, initial DNA concentration, temperature and interaction time. At the end of the all study, the desorption yield was obtained as 97.7% and after 5 adsorption-desorption cycle, the adsorption capacity was decreased only 3%.

Gender Determination by Isolation of Cell-free Fetal DNA from the Maternal Circulation

ABSTRACT Objectives Early identification of fetal gender is important in management of X-linked and other metabolic disorders. Since ultrasound may not predict gender accurately during the first trimester, noninvasive fetal gender determination using deoxyribonucleic acid (DNA) amplification has been proposed. The aim of this study is to evaluate the feasibility of noninvasive prenatal gender determination by examining cell-free fetal DNA (cffDNA) from maternal plasma. Materials and methods Blood samples were collected from 49 pregnant women of gestational ages ranging from 12 to 41 weeks. Deoxyribonucleic acid was extracted from maternal plasma using a QIAamp DNA Blood Mini Kit. Real-time quantitative polymerase chain reaction (PCR) was performed to amplify the male specific DNA marker sex-determining region Y (SRY). Results From a total of 49 subjects, fetal gender was correctly determined in 13 out of 14 male fetuses and 32 out of 35 female fetuses, giving an overall accuracy of 92%. The sensitivity and specificity of the test to detect male fetuses was 93 and 91% respectively. There were three false-positive cases and one false-negative case. Conclusion Identification of fetal gender from maternal plasma using real-time PCR technique is feasible in a developing country, like Pakistan, and appears to be a promising tool for noninvasive prenatal diagnosis. How to cite this article Mohammed N, Nuruddin R, Hoodbhoy Z. Gender Determination by Isolation of Cell-free Fetal DNA from the Maternal Circulation. J South Asian Feder Obst Gynae 2016;8(1):29-32.

The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability.

Background:Nowadays, definitive diagnosis of numerous diseases is based on the genetic and molecular findings. Therefore, preparation of fundamental materials for these evaluations is necessary. Deoxyribonucleic acid (DNA) is the first material for the molecular pathology and genetic analysis, and better results need more pure DNA. Furthermore, higher concentration of achieved DNA causes better results and higher amplifying ability for subsequent steps. We aim to evaluate five DNA extraction methods to compare DNA intimacy including purity, concentration, and amplifying ability with each other.Materials and Methods:The lymphoid tissue DNA was extracted from formalin-fixed, paraffin embedded (FFPE) tissue through five different methods including phenol-chloroform as the reference method, DNA isolation kit (QIAamp DNA FFPE Tissue Kit, Qiagen, Germany), proteinase K and xylol extraction and heat alkaline plus mineral oil extraction as authorship innovative method. Finally, polymerase chain reaction (PCR) and real-time PCR method were assessed to compare each following method consider to DNA purity and its concentration.Results:Among five different applied methods, the highest mean of DNA purity was related to heat alkaline method. Moreover, the highest mean of DNA concentration was related to heat alkaline plus mineral oil. Furthermore, the best result in quantitative PCR was in proteinase K method that had the lowest cycle threshold averages among the other extraction methods.Conclusion:We concluded that our innovative method for DNA extraction (heat alkaline plus mineral oil) achieved high DNA purity and concentration.

Association of luteinizing hormone chorionic gonadotropin receptor gene polymorphism (rs2293275) with polycystic ovarian syndrome.

Polycystic ovaries and irregular menstruation/anovulation are important diagnostic criteria along with hyperandrogenism as per the Androgen Excess Society-2006 criteria for polycystic ovarian syndrome (PCOS). In the etiopathogenesis of PCOS, one of the candidate genes causing ovarian failure is the luteinizing hormone (LH) chorionic gonadotropin hormone receptor (LHCGR). Our aim was to study the association of LHCGR polymorphism (rs2293275) with PCOS in our study population. Genetic case-control study from multiple gynecological centers from Hyderabad, a cosmopolitan city in South India. The study involved 204 women with PCOS and 204 healthy, sex-, and age-matched controls. Anthropometric and biochemical profiles were taken in a well-designed pro forma. Isolation of deoxyribonucleic acid (DNA) and genotype analysis were done for the entire study population using the polymerase chain reaction-restriction fragment length polymorphism method followed by 12% polyacrylamide gel electrophoresis. In this study, we have demonstrated an association between LHCGR (rs2293275) polymorphism and PCOS. The frequency of the G allele was 0.60 in PCOS and 0.49 in controls (odds ratio [OR] 1.531, confidence interval [CI] 1.16-2.01, and p-value=0.0026), which indicates that the G allele is associated with PCOS in our population. The GG genotype conferred a significant risk of developing PCOS (OR 3.36, CI 1.96-5.75, and p-value<0.0001). We found a significant association of the GG allele with body-mass index, waist to hip ratio, insulin resistance, LH, and LH/follicle-stimulating hormone (FSH) ratio in PCOS when compared with controls. The AA allele showed high basal FSH levels. This study suggests that LHCGR (rs2293275) polymorphism is associated with PCOS and could be used as a relevant molecular marker to identify women with the risk of developing PCOS in our population and may provide an understanding about the etiology of PCOS.

DNA Barcode of Thief Ant Complex (Hymenoptera: Formicidae)

The thief ant, Solenopsis molesta (Say), a common nuisance species found throughout the United States is genetically related to red imported fire ants, S. invicta Buren. Therefore, its identification at the molecular level is very important. The deoxyribonucleic acid (DNA) barcoding, a recent technique was used to identify thief ant complex at species and subspecies levels using a short DNA sequence from the cytochrome oxidase subunit 1 (COI) mitochondrial region. The DNA from thief ants collected from 9 states was extracted using Qiagen's Gentra PUREGENE® DNA Isolation Kit. The polymerase chain reactions (PCR) were run on the extracted DNA to amplify partial sequence of COI using primers Lep-F1 (forward) and Lep-R1 (reverse). The resulting DNA products were concentrated, purified and sequenced. The 600 bp sequences of the COI generated were submitted to GenBank that issued accessions numbers from HM179641 to HM179653. The sequences associated with these accession numbers were used as DNA barcodes for distinguishing species and subspecies. Based on this molecular analysis, thief ants collected from New York, Indiana and 1 location in Nebraska were separated in 1 group as S. molesta validiuscula (Emery) and another with ants from Louisiana identified as S. carolinensis (Forel). The third group was comprised of ants from South Dakota, Washington, New Jersey, Tennessee, Kansas and 2 other locations in Nebraska was identified as S. molesta molesta (Say).

A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples

Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H′=2.30 and 1.27) and kit QS (H′=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed.

Influence of various environmental conditions on DNA isolation from dental pulp for sex determination using polymerase chain reaction

Objective: To determine the reliablility of sex determination by polymerase chain reaction Materials and Methods: Extracted teeth were subjected to the following treatment: Varying temperature (100, 200, 300, and 400°C), immersing in sea water (20-36 days), and burying at 30-cm depth. After subjecting to specific environmental stress condition, DNA (Deoxy Ribonucleic acid) was extracted from the tooth pulp. Primers Y-chromosome specifi c alphoid centromeric repeat sequences DYZ3, X-chromosome specifi c alphoid centromeric repeat sequence DXZ1 were used for sex determination by PCR. Results: Out of 15 samples subjected to various environmental conditions, DNA was isolated in 13 samples and sex determination was carried out. There was no DNA yield when teeth were subjected to high temperatures (300, 400°C). The DNA analysis after the respective PCR showed accurate sex determination of the 13 samples. Conclusion: our study showed that teeth are reliable source for sex determination even when subjected to different environmental conditions except during extreme high temperatures. Hence, sex determination by PCR analysis is the most reliable method in markedly decayed or preadolescent bodies.

Rapid isolation and detection of aquaculture pathogens in an integrated microfluidic system using loop-mediated isothermal amplification

Aquaculture is an economically important activity for maritime countries. However, the spread of infectious diseases among aquaculture species have a serious impact on this industry. Therefore, the early detection of pathogens is crucial for protecting aquaculture species with a premium commercial value. This study reports an integrated microfluidic system for the rapid detection of aquaculture pathogens. In this new device, the DNA (deoxyribonucleic acid) of the target pathogen was first isolated by magnetic beads coated with specific nucleotide probes after cell lysis. Then the extracted DNA fragments were amplified by the loop-mediated isothermal amplification (LAMP) process and the amplified products were detected optically. This device contained normally closed microvalves, micropumps, LAMP reaction chambers and wash units such that the entire process for isolation of pathogen DNA, nucleic acid amplification and optical detection was performed automatically in the developed microfluidic system. Experimental data showed that the developed system can automatically complete the entire process within 65min with a detection limit of 20 copies. Another advantage of this device is that the chip is comprised of four identical modules. Therefore, this device could carry out detection of four different pathogens simultaneously. In this study, four pathogens common to cultured ornamental fishes, namely Streptococcus agalactiae, koi herpes virus, Iridovirus and Aeromonas hydrophila were analyzed by this integrated device. This device successfully completed four simultaneous detections from infected fishes in a short period of time. Compared to previous studies, this microfluidic system could complete all processes involved in pathogen detection including sample pre-treatment, nucleic acid amplification and optical detection without any manual operation. As a whole, the developed microfluidic system is promising for rapid isolation and detection of aquaculture pathogens.

Advances in DNA-Based Techniques for the Detection of Seafood Species Substitution on the Commercial Market

Increased worldwide trade and processing of seafood has increased the potential for species substitution on the commercial market. To detect and prevent species substitution, several deoxyribonucleic acid (DNA)-based methods have been developed that can be used to identify species in a variety of food types. For large-scale applications, such as regulatory screening, these methods must be rapid, cost-effective, reliable, and have high potential for automation. This review highlights recent technological advances in DNA-based identification methods, with a focus on seafood species identification in automated, high-throughput settings. Advances in DNA isolation methods include silica-based columns for use in high-throughput operations and magnetic bead particles for increased and targeted recovery of DNA. The three most widely used methods for seafood species identification (polymerase chain reaction [PCR] sequencing, PCR-restriction fragment length polymorphism, and species-specific PCR) will be discussed, with a focus on the incorporation of technologies such as rapid PCR cycling, microfluidic chips, and real-time PCR. Emerging methods, including DNA microarrays and next-generation sequencing will also be explored for their potential to identify seafood species on a large scale. Overall, many of the technological advances discussed here offer complementary properties that will enable species identification in a variety of settings and with a range of products.

Virtual models for interactive e-learning in Medical Biochemistry

The aim is to produce a collection of own and free virtual models and useful links to Web-sites. Models found illustrate the structure, function and purification of deoxyribonucleic acids (DNA). Selected expensive, complex and dangerous experiments were modeled. Short videos and animations were created as illustrations of real processes. These virtual models cover the techniques for isolation of DNA from cells, the polymerase chain reaction and the steps for direct DNA sequencing. The virtual models and links are useful for self-work, traditional lecture-based and problem-based learning. They are appropriate for regular and distance e-learning in Medical Biochemistry.

Optimalization of DNA isolation from oral epithelial mucous cell with smear method

Deoxyribonucleic acid (DNA) is a genetic material which is found in all living organisms. On the human cell or eukaryotes cell, the DNA is found in the nucleus cell and the mitochondria. The DNA arrangement on each cell in human body is the same, that is why, for the analysis meaning, DNA can be isolated from any cell in the body. The source of DNA to be analyzed usually coming from the blood sample by an injection method, such a way resulting in pain and bringing about constraint. Therefore, a study was carried out to look for an alternative of DNA isolation. The aim of this experimental study was to get an optimal DNA isolation method by using oral mucous smear method with a purpose to get a quick and easy DNA isolation. The investigation materials were in the form of samples which were taken from the oral epithelial mucous cells out of three different subjects. The epithelial cells were obtained by the oral mucous smear method which in a variation of two, four and six times of smear applications, respectively. The DNA was then isolated using buffer extraction method. The concentrations of DNA were measured by using ultraviolet spectrophotometer at 260 nm wavelength. The results of DNA isolation were analyzed by Polymerase Chain Reaction (PCR) technique. The optimal DNA isolation could be analyzed by PCR technique. The experimental results show that from three different subjects of study, DNA can be isolated optimally by oral mucous smear method with six times of smear applications.

Mycosphaerella dearnessii and Mycosphaerella pini

<i>Mycosphaerella dearnessii</i> and <i>Mycosphaerella pini</i>

Microfluidic systems for extracting nucleic acids for DNA and RNA analysis

This paper is to review the differences in the developments of microfluidic chips for extracting genomic deoxyribonucleic acid (DNA) and viral ribonucleic acid (RNA) from blood by the Biosensor Focus Interest Group (BFIG) in Singapore. DNA was extracted in a multi-step process by isolating and lysing white blood cells (WBC), typically ∼10 μm in diameter. Viral RNA was extracted directly from the submicron viruses in the blood. In terms of basic microfluidic components required, both DNA and RNA extractions used similar mixers for mixing reagents, filters for capturing or separating the blood cells, and a binder for capturing and purifying the DNA/RNA molecules. The designs of the filters were adapted to either capture WBC for DNA isolation or capture all virus particles for RNA isolation. The designs of these two kinds of filters had to be different. Besides the differences in the sizes of WBC and viruses, the concentration of the virus particles is usually much lower than WBC. Thus, a much higher volume of blood for filtering would be required for extracting viral RNA, especially for the intention to detect the viruses at early onset of infection. With proper modifications of the protocols, it has been demonstrated that both genomics DNA and viral RNA could be extracted successfully in these microfluidic chips. The quality of the extracted samples was verified by polymerase chain reaction (PCR) and gel-electrophoresis after the extractions.

Microchip-Based Macroporous Silica Sol−Gel Monolith for Efficient Isolation of DNA from Clinical Samples

Effective microchip extraction of deoxyribonucleic acid (DNA) from crude biological matrixes has been demonstrated using silica beads or hybrid phases composed of beads and sol-gel. However, the use of monolithic sol-gels alone for extraction of human genomic DNA has been more difficult to define. Here we describe, for the first time, the successful use of monolithic tetramethyl orthosilicate-based sol-gels for effective micro-solid-phase extraction (muSPE) of DNA in a glass microchip format. A functional monolithic silica phase with micrometer-scale pores in the silica matrix resulted from addition of poly(ethylene glycol), a poragen, to the precursor mixture. This allowed a monolithic sol-gel bed to be established in a microchip channel that provided large surface area for DNA extraction with little flow-induced back pressure. DNA extraction efficiencies for simple systems (lambda-phage DNA) were approximately 85%, while efficiencies for the reproducible extraction of human genomic DNA from complex biological matrixes (human blood) were approximately 70%. Blockage of the sol-gel pores by components in the lysed blood was observed in repeat extraction on a single device as a decrease in the extraction efficiency. The developed muSPE protocol was further evaluated to show applicability to clinical samples and bacterial cultures, through extraction of PCR-amplifiable DNA.