Deoxyribonucleic Acid Yield Research Articles

High-efficient separation of deoxyribonucleic acid from pathogenic bacteria by hedgehog-inspired magnetic nanoparticles microextraction

Efficient separation of deoxyribonucleic acid (DNA) through magnetic nanoparticles (MN) is a widely used biotechnology. Hedgehog-inspired MNs (HMN) possess a high-surface-area due to the distinct burr-like structure of hedgehog, but there is no report about the usage of HMN for DNA extraction. Herein, to improve the selection of MN and illustrate the performance of HMN for DNA separation, HMN and silica-coated Fe3O4 nanoparticles (Fe3O4@SiO2) were fabricated and compared for the high-efficient separation of pathogenic bacteria of DNA. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are typical Gram-negative and Gram-positive bacteria and are selected as model pathogenic bacteria. To enhance the extraction efficiency of two kinds of MNs, various parameters, including pretreatment, lysis, binding and elution conditions, have been optimized in detail. In most separation experiments, the DNA yield of HMN was higher than that of Fe3O4@SiO2. Therefore, a HMN-based magnetic solid-phase microextraction (MSPE) and quantitative real-time PCR (qPCR) were integrated and used to detect pathogenic bacteria in real samples. Interestingly, the HMN-based MSPE combined qPCR strategy exhibited high sensitivity with a limit of detection of 2.0 × 101 CFU mL−1 for E. coli and 4.0 × 101 CFU mL−1 for S. aureus in orange juice, and 2.8 × 102 CFU mL−1 for E. coli and 1.1 × 102 CFU mL−1 for S. aureus in milk, respectively. The performance of the proposed strategy was significantly better than that of commercial kit. This work could prove that the novel HMN could be applicable for the efficient separation of DNA from complex biological samples.

Integrated Three-Dimensional Microdevice with a Modified Surface for Enhanced DNA Separation from Biological Samples.

Functional interfaces and devices for rapid adsorption and immobilization of nucleic acids (NAs) are significant for relevant bioengineering applications. Herein, a microdevice with poly(acrylic acid) (PAA) photosensitive resin was integrated by three-dimensional (3D) printing, named DPAA for short. Precise microscale structures and abundant surface carboxyl functional groups were fabricated for fast and high-throughput deoxyribonucleic acid (DNA) separation. Surface modification was then done using polydopamine (PDA) and poly(ethylene glycol) (PEG) to obtain modified poly(acrylic acid) (PAA)-based devices DPDA-PAA and DPEG-PAA rich in amino and hydroxyl groups, respectively. The fabricated device DPAA possessed superior printing accuracy (40-50 μm). Functionalization of amino and hydroxyl was successful, and the modified devices DPDA-PAA and DPEG-PAA maintained a high thermal stability like DPAA. Surface potential analysis and molecular dynamics simulation indicated that the affinity for DNA was in the order of DPDA-PAA > DPEG-PAA > DPAA. Further DNA separation experiments confirmed the high throughput and high selectivity of DNA separation performance, consistent with the predicted affinity results. DPDA-PAA showed relatively the highest DNA extraction yield, while DPEG-PAA was the worst. An acidic binding system is more favorable for DNA separation and recovery. DPDA-PAA showed significantly better DNA extraction performance than DPAA in a weakly acidic environment (pH 5.0-7.0), and the average DNA yield of the first elution was 2.16 times that of DPAA. This work validates the possibility of modification on integrated 3D microdevices to improve their DNA separation efficiency effectively. It also provides a new direction for the rational design and functionalization of bioengineering separators based on nonmagnetic methods. It may pave a new path for the highly efficient polymerase chain reaction diagnosis.

The Integrity of Deoxyribonucleic Acid (DNA) Extracted from Whole Teeth Samples Burnt with Different Accelerants Using Two Extraction Protocols for Forensic Sex Determination

Sex determination is usually the first step in forensic identification of victim(s) in disasters, jungle justice and arsonscenarios for many medical and legal reasons. The use of DNA in forensic analysis offers a good method in sexdetermination and the quality of extracted DNA is very important for downstream PCR. The aim of this study is toinvestigate the viability of DNA obtained from burnt teeth for forensic sex-determination. Two DNA extractionmethods consisting of silica based commercial kit and phenol-chloroform organic method, followed by polymerasechain amplification of amelogenin gene for sex determination were employed. Based on the DNA yield and opticaldensity values, the kit extraction method performed better than the phenol-chloroform method with 100% and85.71% success respectively. A Mann-Whitney U test of 260/280 absorbance showed no statistically significantdifference in the median absorbance for aviation fuel (median = 1.34) and gasoline (median =1.32) burnt samples.Amplification of AMEL genes using the commercial kit and phenol-chloroform method were 52.38% and 22.22%positive respectively compared to pre-extraction sex determination. This study shows that the use of the silica basedcommercial kit technique yielded higher DNA quality and quantity from whole teeth burnt with gasoline andaviation fuel as accelerants for downstream PCR amplification of AMEL genes compared to organic phenolchloroform method.

Selection of a suitable viral DNA extraction method for Sheeppox virus in cell culture

A suitable method for the extraction of nucleic acids should be efficient, sensitive, rapid and simple. Moreover, ideally, good method should yield pure nucleic acid-free from any contaminant inhibitors. Several methods have been reported for viral deoxyribonucleic acid (DNA) isolation but limited information is available on quick and simple isolation of Sheeppox virus (SPPV) genomic DNA in cell culture. In this study, the healthy Vero cells and primary lamb testis cells were infected with SPPV strains such as SPPV-Jaipur, SPPV-Ranipet and SPPV-Roumanian Fanar (RF) and harvested when it exhibited clear cytopathic effect (CPE) in culture. Four different DNA extraction methods i.e., (i) Phenol/chloroform/Isoamyl alcohol method, (ii) Cell lysis buffer method, (iii) Proteinase-k method, and (iv) commercial nucleic acid extraction kit was used to extract optimum yield of viral genomic DNA from clarified culture supernatant of harvested SPPV virus. The DNA sample was characterized using the Nanodrop spectrophotometer and agarose gel electrophoresis. Significantly (p<0.05) higher yield of SPPV genomic DNA was obtained in proteinase-k method which was about 3-5 times more than other methods. Among these methods, proteinase-k protocol was found to be comparatively very effective method in terms of yield of viral genomic DNA, and was free from PCR inhibitors.

Evaluation of Maximum DNA Yield from a New Noninvasive Buccal Collection Device Following Various Extraction Protocols.

Background: As the demand in genetic testing increases, various fields look toward collection methods that are noninvasive and efficient in recovering deoxyribonucleic acid (DNA) for testing that will allow for high first-pass success rates. Objective: Two extraction methods (PrepFiler™ Express Forensic Extraction and the Maxwell® RSC Buccal Swab DNA Kits) were optimized to increase DNA yield from a buccal cell collection device (Gentueri's CollectEject™ Swab). Materials and Methods: Buccal swabs were processed under varying incubation parameters using a forensic workflow. The PrepFiler method was adjusted to test longer incubation times and more aggressive agitation. The Maxwell method was adjusted to test incubation temperatures and duration. Results: Quantitative results showed that increased agitation can yield more DNA through the PrepFiler extraction, but longer incubation times did not increase DNA recovery. The results from the Maxwell study showed no significant difference between incubation temperatures or times. Conclusions: The results indicate that various applied genetic fields can utilize a noninvasive, simple collection method using the CollectEject device in conjunction with extraction methods already implemented in laboratories to collect 5000 ng of DNA or greater from a buccal cell collection.

Shelf life of whole blood samples in a biobank and the yield of deoxyribonucleic acid during genetic testing

Aim. To study the effect of the shelf life of frozen whole blood samples in a biobank on the amount of released deoxyribonucleic acid (DNA).Material and methods. The study included whole blood samples placed in tubes with the anticoagulant EDTA (ethylenediaminetetraacetic acid at a concentration of 1,8 mg/ml) from participants in the epidemiological study ESSE-RF-1 and ESSE-RF-2 and cohort studies conducted at the National Medical Research Center for Therapy and Preventive Medicine. The samples were stored in the biobank of the National Medical Research Center for Therapy and Preventive Medicine at temperature from -22О C to -32О C. The shelf life from blood collection to DNA extraction ranged from several weeks to 11 years. DNA was extracted using QIAamp DNA Blood Mini Kit (250) and 96 Blood Kit (Qiagen, Germany). Statistical analysis was performed using the R 3.6.1 software. To analyze the association of blood storage time with the logarithm of DNA concentration, a linear regression was used.Results. The analysis included data on the DNA concentration of 5405 samples. Multivariate regression showed that the blood shelf life was significantly associated with a decrease in concentration by 3,92% (3,16-4,68) for each year of storage (p <0,0001). For 509 samples, the DNA concentration was measured twice, immediately after isolation and after 4,5 years of DNA storage at -32О C. During storage, the concentration of DNA increased by an average of 2% (p=0,046).Conclusion. Long-term storage of whole blood samples at temperature from -22О C to -32О C is associated with a decrease in the DNA yield. Long-term storage of the isolated DNA at a temperature of -32О C is not associated with a decrease in its concentration.

SUITABILITY OF VARIOUS BUFFERS FOR GENOMIC DNA EXTRACTION OF PINEAPPLE (Ananas comosus) VAR. MD2

Methods for DNA extraction are of paramount importance to obtain high yield and high purity nucleic acids for molecular characterisation downstream. However, there is no specific extraction protocol developed for Ananas comosus var. MD2. Here, we compare the efficiency of five selected DNA extraction buffers which are extensively used for plant deoxyribonucleic acid (DNA) extraction with absence usage of phenol. The suitability of the extraction buffers was assessed based on both DNA yield and its quality. In this study, DNA extracts were quantified using ultraviolet (UV) spectrophotometry, spectrophotometric profiles and gel electrophoresis. Since interfering background substances are not visible in gel electrophoresis, examining PCR products of the crude DNA is recommended. To summarise, all the buffers yielded sufficient DNA of an approximate 50-200 μg from 1 g leaf tissue for downstream applications with different quality level. Out of five extraction methods, two give high yield and high-quality genomic DNA using Dellaporta-based method (213.5 μg/g) and Doyle & Doyle-based method (172.5 μg/g). Among these extraction methods, the exclusion of detergents in extraction buffer served as the best extraction buffer for MD2 genomic DNA extraction. Also, from an economic point of view, the extraction buffer is cheaper compared to commercial DNA extraction kits.

Development of a High Quality, Rapid, Efficient and Economical DNA Extraction Protocol from Climate Resilient Pearl Millet Crop Without Liquid Nitrogen

Extraction of good quality genomic deoxyribonucleic acid (DNA) from plants is a major prerequisite for molecular biology experiments. An efficient genomic DNA protocol must be simple, fast and cost effective with high yield and purity. Presence of polyphenols, polysaccharides and secondary metabolites in some plants hamper with DNA extraction making it a very laborious, difficult and time consuming procedure. Here, we portrayed a modified protocol based on the cetyl trimethyl ammonium bromide (CTAB) method to isolate DNA from climate resilient pearl millet leaf tissues having higher amount of polysaccharides. It also excludes the use of expensive chemicals and equipments like proteinase K, liquid nitrogen and tissue lyser. This method includes extraction of DNA using a buffer (pH 8.0) containing 200 mM Tris-HCl, 20 mM ethylenediamine tetracetic acid (EDTA),1.4 M NaCl, 2% CTAB, 2% sodium dodecyl sulphate (SDS) and 1.0 % β-mercaptoethanol followed by purification of DNA with phenol, chloroform, isoamyl alcohol and finally precipitation of DNA by sodium acetate and isopropanol. Good quality genomic DNA with sharp and clear bands was obtained from 48 pearl millet genotypes using this protocol. The yield of DNA varied from 105.2 to 328.3 ng/μl. The purity of DNA sample ranged from 1.74 to 1.95 based on the absorbance at A260/A280 ratio indicating that it’s free from ribonucleic acid (RNA) and protein contamination. PCR analysis using simple sequence repeat (SSR) primers exhibited consistent and reliable amplification products ranging from 150 to 650 bp.This study reveals a fast, simple, efficient, specific, reproducible, reliable and cost effective method for extraction of DNA from small to large number of plant samples amenable to PCR amplification and could be stored for longer duration.

DNA Extraction from Archived Paraffin Embedded Tissues: A Comparative Study Using Three Different Extraction Method-Extraction of DNA from Paraffin Embedded Tissues

Objective: Formalin fixed paraffin embedded (FFPE) tissue is a valuable source of nucleic acids requiredfor various molecular studies. The purpose of the present study was to extract Deoxyribonucleic acid (DNA)from archival retrieved FFPE oral squamous cell carcinoma tissues using three different extraction method(salting out, commercial kit and microwave method) and to compare the yield of DNA extracted by thesemethod.Method and Material: 30 oral squamous cell carcinoma paraffin embedded tissue blocks were categorizedinto three groups based on the duration of storage as Group A(<2 years), B(2-4 years) and C(4-6years). Thetissue sections made from these blocks were subjected to three DNA extraction method and the DNA yieldwas measured using a spectrophotometer. The extracted DNA was checked by successful amplification ofp53 gene using Polymerase Chain Reaction followed by gel electrophoresis. The values of DNA yield andpurity were statistically analysed and the level of significance was set at P?0.05.Results: The DNA yield was above 3?g in all the three extraction method. On multiple comparison, themean DNA yield was statistically significant (P value<0.05) among the three method in Group A. Also, theDNA yield was found to be higher in salting out method though successful amplification was obtained fromthe extracted DNA in other extraction method as well.Conclusions: DNA was successfully extracted from all the tissue samples using all the three method. Saltingout method yielded the maximum amount of DNA compared to the other method and shall be a good costeffective alternative.

Extraction of DNA from plastinated tissues

IntroductionPlastination allows anatomical samples to be preserved in excellent condition for an indefinite period, free of formalin, and in a format that allows biosafe manipulation by students, academics, and researchers. As with other tissue preservation techniques, it is important to establish the level of conservation of deoxyribonucleic acid (DNA) for use in future applications. The object of the present work was to extract and evaluate DNA from plastinated tissues. MethodsWe used samples of liver from Canis lupus familiaris and skeletal muscle from Rattus norvegicus, Sprague-Dawley strain, extracted from specimens plastinated with silicone at room temperature. The tissue samples were deplastinated by incubation in 5% sodium methoxide dissolved in methanol for 24 or 48 h. The samples were divided into two equal parts and DNA was extracted using two different protocols. After extraction, the DNA was quantified by fluorometry and its integrity was assessed by electrophoresis in a 1% agarose gel. Results and discussionA high yield of DNA was obtained from the deplastinated samples and the DNA was intact. Plastinated tissues have proven to be stable and easily managed. They can also be used for examination under light and electron microscopes. The DNA extraction technique used here allowed us to obtain intact DNA from samples plastinated with silicone at room temperature, without previous fixing. This technique may allow tissue specimens to be preserved for retrospective studies of archived samples of normal and pathological anatomy in the fields of basic, clinical, forensic, and epidemiological sciences. ConclusionsThe extracted DNA was intact and suitable for use in subsequent applications. Obtaining whole DNA from plastinated samples using tissue preservation protocols that preserve the tissue for use in subsequent applications, like real-time PCR, opens up many possibilities, with applications in the basic and clinical sciences, epidemiology, and forensic science.

DNA extraction from placental, fetal and neonatal tissue at autopsy: what organ to sample for DNA in the genomic era?

Incorporation of genome and exome sequencing into fetal and neonatal autopsy investigations has been shown to improve diagnostic yield. This requires deoxyribonucleic acid (DNA) to be extracted from either the placenta or autopsy tissue for molecular testing. However, the sources and quality of DNA obtained are highly variable and there are no adequate published data on what tissue is most ideal to sample for DNA extraction in this setting. Here we compare the quality of DNA extracted from sampling the placenta and various solid organs at fetal and neonatal autopsy, thereby determining the optimal tissue from which to source DNA for ancillary testing as part of the modern perinatal autopsy. A total of 898 tissue samples were obtained at autopsy from 176 fetuses (gestational ages 17-40 weeks) and 44 neonates (age range 0-28 days) at our tertiary institution. Fetal tissue was processed using the QIAsymphony DSP DNA Mini kit and placental tissue was extracted using the New iGENatal Kit. DNA concentration was quantified using the Qubit dsDNA BR Assay Kit. DNA integrity, as stratified by gel electrophoresis was classified as high (≥5 kb) or low quality (<5 kb). Genome sequencing was performed on the extracted DNA, together with respective parental DNA from blood samples, and confirmed absence of maternal contamination in all cases. Analyses used logistic mixed models to test for associations between tissue types, intrauterine retention times, delivery to autopsy and death to autopsy intervals with DNA quality. In the fetal cohort, the placenta had the highest proportion of high quality DNA samples (93.1%), and liver had the lowest proportion (35.3%). Among the neonates, all tissue samples with the exception of liver had over 88% high DNA quality with the placenta also yielding the highest quality (100%). There was statistically significant deterioration in DNA quality with prolonged time interval between demise and autopsy (≥5 days). In the 726 fetal samples, the odds of obtaining higher quality DNA from the placenta, thymus, and spleen were 70.4 [95% confidence interval (CI) 29.2-169.6], 3.6 (95% CI 2.0-6.6) and 3.3 (95% CI 1.8-6.1) times, respectively, more likely than samples from the liver (p values <0.001). DNA yield from other fetal solid organs investigated was not significantly superior to that from the liver. This study shows that, when available, refrigerated unfixed placenta is the most suitable source of high quality DNA during perinatal investigations. Of the solid fetal organs sampled at autopsy, lymphocyte-rich, lytic enzymes-poor organs such as thymus and spleen were significantly more likely to yield good quality DNA than the liver.

Heat Inactivation of Coccidioides posadasii and Coccidioides immitis for Use in Lower Biosafety Containment.

The difficulty involved in obtaining sufficient intact genomic deoxyribonucleic acid (DNA) from Coccidioides spp for downstream applications using published protocols prompted the exploration of inactivating mycelia and arthroconidia using heat under biosafety level 3 containment. This was followed by optimizing DNA extraction from mycelia using various methods at lower containment. Various exposure times and temperatures were examined to identify an effective heat inactivation procedure for arthroconidia and mycelia from both C immitis and C posadasii. Heat inactivation of mycelia was followed by DNA extraction using 2 commercially available kits, as well as a phenol:chloroform-based extraction procedure to determine DNA integrity and quantity among extraction methods using both live and heat-inactivated mycelia. Ten-minute and 30-minute exposure times at 80°C were sufficient to inactivate Coccidioides spp arthroconidia and mycelia, respectively. DNA yield between live versus heat-inactivated mycelia was similar for each extraction procedure. However, DNA obtained using phenol:chloroform was of higher quantity and integrity compared with DNA obtained using the commercially available kits, which was highly fragmented. The ability to heat-inactivate Coccidioides cultures for processing at a lower level of containment greatly increased the efficiency of DNA extractions. Therefore, this is an ideal method for obtaining Coccidioides spp DNA and inactivated arthroconidia.

Adsorption of DNA by using polydopamine modified magnetic nanoparticles based on solid-phase extraction

A polydopamine magnetic composite (PDA@Fe3O4) was prepared for the extraction of human genomic DNA and characterized by transmission electron microscopy, X-ray diffraction, FT-IR spectrometer, zeta potential and vibrating sample magnetometry. PDA@Fe3O4 based on magnetic solid phase extraction (MSPE) method have highly efficient capture of genomic deoxyribonucleic acid (DNA)and gene fragments ranging from about 100 bp to 200 bp. Compared with commercial beads (Shenggong, China) and spin column nucleic acid extraction kit (Tiangen, China), the PDA coated magnetic nanoparticles display superior genomic DNA extraction capacity (116 mg/g) and yield (90.2%). The isolation protocol used the solutions (composed of PEG and NaCl) with a specific pH for the binding and release of DNA. The procedure can be attributed to the charge switch of amino and hydroxyl groups on surface of the magnetic particle. The extracted DNA with high quality (A260/A280 = 1.82 ± 0.04) can be directly used as template for polymerase chain reaction (PCR) followed by agarose gel electrophoresis. The results showed the new composite to be an ideal adsorbent for separation of DNA which had the advantage of its low cost, high extraction capacity and yield.

Rapid, simple and potentially universal method for DNA extraction from Opuntia spp. fresh cladode tissues suitable for PCR amplification.

In Opuntia spp., the cladode tissues contain many polysaccharides and secondary metabolites that interfere with obtaining high-quality deoxyribonucleic acid (DNA), using currently available methods. To circumvent this problem, three commercial kits, three modified versions of the conventional cetyltrimethylammonium bromide method (CTAB) method and one combined method were tested in Opuntia ficus-indica, O. robusta, O. dillenii and O. elata species. We obtained a rapid and simple protocol that allows the extraction of DNA from all the tested species with good DNA yield and purity, namely, the combined method. With this method (DNeasy® Plant Mini Kit combined with the CTAB method), DNA yields from 13.2 ± 7.8 to 15.9 ± 11.3µgg-1 of fresh tissue were obtained in the four Opuntia species. The purity, evaluated by the ratio A260/A280 ratio, ranged from 1.67 ± 0.12 to 2.01 ± 0.25, revealing low levels of problematic metabolites. The extracted DNA quality was confirmed by amplifying a set of nuclear microsatellites obtained for the genus. Reliable reproducible bands and electropherogram profiles were obtained. The combined method has potential to be universal for good-quality DNA extraction in cacti, particularly in the Opuntia genus and other difficult-to-extract species.

Influence of storage methods of whole blood samples on DNA integrity in epidemiological studies

ObjectivesTo investigate the influence of storage methods of whole blood samples on Deoxyribonucleic acid (DNA) quality. Method and materialsWhole blood samples were collected from 90 patients reported to the Government Tertiary-care Dental College and Hospital, Tamil Nadu, India. The blood samples were stored at three different temperatures −80°C, −20°C, 4°C which were analyzed for DNA quality immediately within 4h and after a storage period of 60 days. Quantification of DNA in the sample was recorded using a spectrophotometer. ResultsThe average concentration of isolated DNA ranged from 0.5ng/μl to 154.2ng/μl with purity A260/A280 ratio ranging from 0.7 to 2.0. The mean DNA concentration H=97.2 (p=0.001) and purity H=80.1 (p=0.001) obtained from whole blood stored at 4°C were statistically significant. Thus samples stored at 4°C did not affect on DNA yield and quality. ConclusionWe conclude that storage at 4°C temperature for whole blood does not substantially affect the molecular assay measurements. This study enlightens the influence of storage temperatures and duration in genetic epidemiology.

Assessment of the most reliable sites in mandibular bone for the best deoxyribonucleic acid yield for expeditive human identification in forensics.

Background:In recent years, the techniques used to identify human remains post accidents, trauma or in case of criminal investigation have been expanded, improved and rendered more complex by the emergence of technologies based on deoxyribonucleic acid (DNA) analysis. In the head and neck area, tooth has been proven to be the best quantitative source for DNA but in certain cases where the mandible specimen is edentulous or the tooth is extensively destroyed with caries, large dental restorations, mobile, or if they show any perimortem or postmortem fractures, sampling of such tooth specimen is usually avoided. In such situations, bone is considered the next best site for DNA analysis. Mandible being the largest, strongest and dense cortical bone is the most prominent facial bone that can be easily disarticulated. It can be analyzed for the best short tandem repeat (STR) segment qualitative amplification using polymerase chain reaction (PCR) technique for forensic analysis which can be used for gender and age determination.Aim:The aim of this study is to determine the best site for optimum quantitative and qualitative yield of DNA for amplification using specific and standard STR segment by conventional PCR technique.Methodology:Fifteen mandibular samples exposed to different environmental conditions were collected. Bone pieces of 1 cm × 1 cm were cut from each mandible from three sites, i.e., the ramus, angle and body, wherein the genomic DNA was isolated and was subjected to PCR using restricted number of 25 cycles.Results:The STR segment D3S1358 from clone RP11-438F9 used for the study showed very good amplification in restricted number of PCR cycles in the ramus region with number of repeats in every 15th genomic region.Conclusion:This study highlights the use of mandibular bone for the expeditive human identification. As per the study, the ramus of the mandible gave high quantitative and qualitative yield of DNA with thick amplification band of the STR segment as compared to the body and angle of the mandible. Thus ramus of the mandible can be preferred over other sites for molecular forensic investigations.

Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study.

AimTo determine polymerase chain reaction (PCR)-based blood group on tooth pulp obtained from teeth stored for 1 month, 6 months, and 1 year following extraction and to evaluate the stability of deoxyribonucleic acid (DNA) in primary tooth subjected to a temperature of 200°C ± 5°C for 15 minutes.Materials and methodsDental pulp tissue was collected from 40 exfoliated primary teeth stored for various time durations and temperature and preserved at 4°C till DNA extraction was carried out. Deoxyribonucleic acid was extracted using silica membrane-based spin-column procedure of QIAamp DNA minikit from BioRad. Deoxyribonucleic acid was subjected to PCR amplification and monoplex allele-specific PCR primers for ABO genotyping.Statistical analysis usedThe data were analyzed by comparison (based on percentage).ResultsIn our study, overall, 85% samples showed a DNA yield. Cent percent results were obtained for samples studied at the end of 1 month followed by 90 and 80% for samples studied for 6 months and 1 year respectively. Heated samples showed 70% result.ConclusionPolymerase chain reaction was found to be an effective method for blood group determination for teeth stored at various time durations and temperatures. However, as the time interval increased, the number of positive results obtained decreased.How to cite this articlePai RK, Bhat SS, Salman A, Hegde S. Blood Group Determination using DNA extracted from Exfoliated Primary Teeth at Various Time Durations and Temperatures: A PCR Study. Int J Clin Pediatr Dent 2016;9(4):308-312.

PH-responsive deoxyribonucleic acid capture/release by polydopamine functionalized magnetic nanoparticles

Polydopamine functionalized magnetic nanoparticles (PDA@Fe3O4) were prepared and characterized by transmission electron microscopy, scanning electron microscopy, zeta potential and vibrating sample magnetometry. They were found to enable highly efficient capture of genomic deoxyribonucleic acid (DNA). The adsorption capacity of PDA@Fe3O4 for genomic DNA can reach 161mgg−1. The extraction protocol used aqueous solutions for DNA binding to and releasing from the surface of the magnetic particles based on the pH inducing the charge switch of amino and phenolic hydroxyl groups on PDA@Fe3O4. The extracted DNA with high quality (A260/A280=1.80) can be directly used as templates for polymerase chain reaction (PCR) followed by capillary electrophoresis (CE) analysis. None of the toxic chemical reagents and PCR inhibitors was used throughout the whole procedure. PDA@Fe3O4 based magnetic solid phase extraction (MSPE) method was superior to those using commercial kit and traditional phenol–chloroform extraction methods in yield of DNA. The developed PDA@Fe3O4 based MSPE-PCR-CE method was applied for simultaneous and fast detection of Listeria monocytogenes and Escherichia coli O157:H7 in milk.

Quality and Quantity of Extracted Deoxyribonucleic Acid (DNA) from Preserved Soft Tissues of Putrefied Unidentifiable Human Corpse

Context:The appropriate collection and preservation of soft tissues from putrefied unidentifiable human corpse for the purpose of identification using DNA profiling technique is critically important especially in developing countries like India having different levels of health-care set ups with largely varying facilities and varying climatic conditions.Aims:The present study was carried out, mainly focusing on quality and quantity of extracted DNA from the soft tissues of putrefied unidentifiable human corpse stored upto 4 weeks at 4°C and at −80°C for DNA analysis.Materials and Methods:The present study was conducted on 16 different putrefied unidentifiable human corpses after getting approval from institutional ethical committee. Around 2 g of four different tissues (brain, kidney, heart and muscle) were collected and preserved for one month followed by DNA extraction using the organic method, the quality and quantity of high molecular weight-DNA was estimated using the spectrophotometer and gel electrophoresis. Further, the amplification polymerase chain reaction (PCR) was also performed (AmpFLSTR® Indentifiler™ PCR Amplification kit for multiple loci, of Applied Biosystems, Lab India) and was checked using continuous PAGE.Results:The yield of DNA was significantly higher at −80°C for all the four tissues collected and was best for brain followed by heart, kidney and worst for muscles in all cases.Conclusions:It is suggested that the brain tissue preserved at −80°C is the best among soft issues for DNA extraction. Refrigeration or deep freezing facility should be available at all the centers.

Four methods of nondestructive DNA sampling from freshwater pearl mussels Margaritifera margaritifera L. (Bivalvia:Unionoida)

Nondestructive tissue sampling is desirable for genetic or physiological studies of endangered freshwater mussels. We used the freshwater pearl mussel (Margaritifera margaritifera) to evaluate 4 sampling methods (haemolymph extraction, foot scraping, mantle biopsy, and viscera swabbing) with regard to their effectiveness for deoxyribonucleic acid (DNA) analyses and their effects on sampled mussels. One hundred twenty-eight days subsequent to tissue sampling (1 June–7 October), all sampled individuals were alive, and average growth of sampled individuals was not significantly different from unsampled, control individuals, except that the viscera-swabbing group had lower growth. The magnitude of decreased growth in viscera-swabbed individuals was small (∼0.5 mm less than controls), and the biological significance of this result is unclear. DNA yields from haemolymph extraction and foot scraping were significantly lower and more variable than yields from the other methods. Genotyping success was lowest for haemolymph extraction and mantle biopsy, but was high for the other methods. Viscera-swab samples stored in lysis buffer at room temperature prior to DNA extraction had higher DNA yield than samples stored in buffer at 4°C or samples stored dry, but genotyping success was equivalent among storage methods. On the basis of these results, we recommend use of the noninvasive viscera-swabbing method.