scholarly journals Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 146
Author(s):  
Catarina Xavier ◽  
Mayra Eduardoff ◽  
Barbara Bertoglio ◽  
Christina Amory ◽  
Cordula Berger ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Ancient Dna ◽  
Extraction Method ◽  
Fragment Size ◽  
Molecular Genetic ◽  
Extraction Methods ◽  
Degraded Dna ◽  
Size Analysis ◽  
Dna Fragments ◽  
Dna Amount

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

scholarly journals Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

2017 ◽  
Author(s):  
Maria A. Nieves-Colón ◽  
Andrew T. Ozga ◽  
William J. Pestle ◽  
Andrea Cucina ◽  
Vera Tiesler ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Ancient Dna ◽  
Extraction Methods ◽  
Optimal Choice ◽  
Skeletal Remains ◽  
Tissue Type ◽  
Dna Fragments ◽  
Illumina Hiseq ◽  
Tropical Environments ◽  
Significant Difference

ABSTRACTObjectivesThe tropics harbor a large part of the world’s biodiversity and have a long history of human habitation. However, paleogenomics research in these climates has been constrained so far by poor ancient DNA yields. Here we compare the performance of two DNA extraction methods on ancient samples of teeth and petrous portions excavated from tropical and semitropical sites in Tanzania, Mexico, and Puerto Rico (N=12).Materials and MethodsAll samples were extracted twice, built into double-stranded sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an extended digestion and decalcification step.ResultsNo significant difference was found in overall ancient DNA yields or post-mortem damage patterns recovered from samples extracted with either method, irrespective of tissue type. However, Method H samples had higher endogenous content and more mapped reads after quality-filtering, but also higher clonality. In contrast, samples extracted with Method D had shorter average DNA fragments.DiscussionBoth methods successfully recovered endogenous ancient DNA. But, since surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our results suggest that Method D is the optimal choice for working with samples from warm and humid environments. Additional optimization of extraction conditions and further testing of Method H with different types of samples may allow for improvement of this protocol in the future.

scholarly journals Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

2020 ◽  
Vol 5 (2) ◽  
pp. 95 ◽  
Author(s):  
Rajashree Chowdhury ◽  
Prakash Ghosh ◽  
Md. Anik Ashfaq Khan ◽  
Faria Hossain ◽  
Khaledul Faisal ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Methods ◽  
Recombinase Polymerase Amplification ◽  
National Guidelines ◽  
Diagnostic Efficacy ◽  
Rapid Extraction ◽  
Kala Azar ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

scholarly journals DNA extraction technique for different rice varieties grown in Sri Lanka

2015 ◽  
Vol 3 (3) ◽  
pp. 398-401
Author(s):  
Ranganathan Kapilan
Keyword(s):  
Sri Lanka ◽  
Dna Extraction ◽  
Extraction Method ◽  
Extraction Methods ◽  
Rice Varieties ◽  
Modified Method ◽  
Dna Extraction Methods ◽  
Very High ◽  
Different Parts

Extraction of DNA is very important nowadays in bio-molecular researches. Extracted DNA should be purified and the quality of DNA should also be very high. The objective of the study was to develop a simple efficient method to isolate DNA from the rice varieties in an open laboratory environment, and to eliminate the usage of expensive chemicals and tools. The DNA extraction methods developed by the DNeasy plant kit method supplied by QIAGEN, Cheng et al., Doyle et al. and Michiels et al. were applied to five different rice varieties grown in different parts of Sri Lanka. Based on the quantity and quality of the extracted DNA tested by measuring the absorbance of DNA at 260 nm using Nanodrop® ND-1000 spectrophotometer and measuring the ratio of A260 / A280 and gel running on agarose, the efficiency of the extraction method chosen varied among rice varieties. Among the methods used, the methods introduced by DNeasy plant kit method supplied by QIAGEN and Cheng et al, yielded good and amplifiable quality DNA with satisfactory concentration for all the rice varieties tested. Therefore the modified method of Cheng et al, 1987 could be used to extract DNA from rice varieties instead of the commercially available expensive and hazardous DNeasy plant kit method supplied by QIAGEN.Int J Appl Sci Biotechnol, Vol 3(3): 398-401

scholarly journals Toward standards in clinical microbiome studies: comparison of three DNA extraction methods and two bioinformatic pipelines

2019 ◽  
Author(s):  
Q.R. Ducarmon ◽  
B.V.H. Hornung ◽  
A.R. Geelen ◽  
E.J. Kuijper ◽  
R.D. Zwittink
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Bacterial Biomass ◽  
Extraction Methods ◽  
Rrna Gene ◽  
Method Choice ◽  
Advantages And Disadvantages ◽  
Dna Extraction Method ◽  
Dna Extraction Methods ◽  
Negative Controls

ABSTRACTWhen studying the microbiome using next generation sequencing, DNA extraction method, sequencing procedures and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing, and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with maximum three-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for estimating richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs and variation induced by DNA extraction methods was lower than inter-subject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we demonstrate the importance of including negative controls when analyzing low bacterial biomass samples.IMPORTANCEMethod choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls, and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiome studies. Our methods were not only applied to commonly studied samples for microbiota analysis, e.g. feces, but also for more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g. urine and tumor biopsies) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiome studies, especially when low bacterial biomass is expected.

scholarly journals Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of Vitis vinifera L.

OENO One ◽  
2013 ◽  
Vol 47 (4) ◽  
pp. 227 ◽  
Author(s):  
Gemma Marsal ◽  
Núria Boronat ◽  
Joan Miquel Canals ◽  
Fernando Zamora ◽  
Francesca Fort
Keyword(s):  
Vitis Vinifera ◽  
Dna Extraction ◽  
Woody Plants ◽  
Extraction Method ◽  
Low Cost ◽  
Handling Time ◽  
Extraction Methods ◽  
Original Method ◽  
Dna Extraction Method ◽  
Functional Dna

<p style="text-align: justify;"><strong>Aim</strong>: To compare different methods for extracting DNA from non-recalcitrant and recalcitrant tissues of <em>Vitis vinifera</em> woody plants and propose a modification of a previously published method to reduce the time and cost of extraction.</p><p style="text-align: justify;"><strong>Methods and results</strong>: DNA was extracted from young and mature leaves as well as from stems and seeds using some of the most common methods of DNA isolation and two commercial kits. Another commercial kit, which does not require DNA extraction prior to PCR, was also used. Only two methods provided adequate results in all tissues. Other methods were only applicable to some tissues and some did not yield any functional DNA in any tissue. A modification of the method reported by Marsal <em>et al</em>. (2011) is proposed to reduce handling time and cost.</p><p style="text-align: justify;"><strong>Conclusion</strong>: All of the methods studied here use a surfactant to improve the extractions. For DNA extraction from recalcitrant tissues to be optimal, it is best to use a combination of dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). The changes made to the protocol reported by Marsal <em>et al</em>. (2011) enable functional DNA to be obtained from leaves in only 90 minutes and at very low cost (17 €/8 samples). However, this method cannot adequately isolate DNA from recalcitrant tissues (stems and seeds) and so, for this type of sample, we would recommend using the original method.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: Nowadays, handling time and cost are key factors in selecting the most suitable DNA extraction method. This study compares not only the effectiveness of the various methods but also the handling time and cost. It also proposes a modification of the fastest and most economic DNA extraction method for leaves so that handling time and processing cost will be reduced even further.</p>

scholarly journals Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Anthony Ablordey ◽  
Evans Ahotor ◽  
Charles A. Narh ◽  
Sandra A. King ◽  
Isra Cruz ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Point Of Care ◽  
Buruli Ulcer ◽  
Extraction Methods ◽  
Isothermal Amplification ◽  
Mycobacterium Ulcerans ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Extraction Method ◽  
Dna Extraction Methods

Abstract Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

scholarly journals A comparison of different DNA extraction methods and molecular techniques for the detection and identification of foodborne pathogens

2021 ◽  
Vol 7 (3) ◽  
pp. 304-319
Author(s):  
Spyridon Andreas Papatheodorou ◽  
◽  
Panagiotis Halvatsiotis ◽  
Dimitra Houhoula ◽  
Keyword(s):  
Dna Extraction ◽  
Foodborne Pathogens ◽  
Extraction Method ◽  
Pathogenic Bacteria ◽  
Low Cost ◽  
Isoamyl Alcohol ◽  
Extraction Methods ◽  
Molecular Techniques ◽  
Bacterial Dna ◽  
Dna Extraction Method

<abstract> <p>Foodborne infections continue to plague Europe. Food safety monitoring is in crisis as the existing techniques for detecting pathogens do not keep up with the global rising of food production and consumption. Thus, the development of innovative techniques for detecting and identifying pathogenic bacteria has become critical. The aim of the present study was firstly to develop an innovative simple and low cost method of extracting bacterial DNA from contaminated food and water samples with <italic>Salmonella enteric</italic> subsp. <italic>enteric</italic> serovar Typhimurium and <italic>Listeria monocytogenes</italic> and its comparison with two commercial DNA extraction kits (Qiagen, Macherey-Nagel). Finally, pathogens' detection using two molecular techniques (PCR-electrophoresis, LAMP), in order to evaluate the best combination of DNA extraction and identification based on their sensitivity, cost, rapidity and simplicity. Considering the above criteria, among them, best was proved an in-house bacterial DNA extraction method, based on the chloroform-isoamyl alcohol protocol, with certain modifications. This technique showed statistically similar results in terms of sensitivity, compared to the commercial kits, while at the same time maintained high rapidity and much lower cost. Lastly, between the molecular techniques, LAMP was found more promising considering its simplicity, high rapidity and sensitivity. Conclusively, the in-house DNA extraction method along with the LAMP technique, was proven to be the best among the presented combinations.</p> </abstract>

scholarly journals Optimized Batrachochytrium dendrobatidis DNA extraction of swab samples results in imperfect detection particularly when infection intensities are low

2020 ◽  
Vol 139 ◽  
pp. 233-243
Author(s):  
LA Brannelly ◽  
DP Wetzel ◽  
M West ◽  
CL Richards-Zawacki
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Batrachochytrium Dendrobatidis ◽  
Cost Benefit Analysis ◽  
Cost Benefit ◽  
Extraction Methods ◽  
Imperfect Detection ◽  
Spin Column ◽  
Column Extraction ◽  
Detection And Quantification

Accurate detection of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) is critical for wildlife disease research; however, false negatives in detection do occur. Here we compared different DNA extraction methods to determine the threshold for Bd detection and identify an optimal extraction method to improve detection and quantification of the pathogen. We extracted both lab-created cell suspension standards using PrepMan Ultra, Chelex resin, and 3 spin column DNA extraction kits (Qiagen DNeasy Blood and Tissue, Zymo Quick DNA miniprep, and IBI gMAX mini kit), and further compared extraction methods using field-collected samples. We found that when extracting Bd DNA from cells in lab-created culture, the spin column extraction methods and PrepMan Ultra were equivalent, while the resin method detected higher Bd DNA quantities, especially at higher loads. However, when swabs from live animals were analyzed, low Bd quantities were more than twice as likely to be detected using a spin column extraction than with the PrepMan Ultra extraction method. All tested spin column extraction methods performed similarly across both field and lab samples. Samples containing low Bd quantities yielded inconsistent detection and quantification of Bd DNA copies regardless of extraction method. To manage imperfect detection of Bd, we suggest that presence/absence analyses are more informative than attempting to quantify Bd DNA when quantities are low. Overall, we recommend that a cost-benefit analysis of target species susceptibility and epidemiology be taken into consideration when designing an experiment to determine the most appropriate DNA extraction method to be used, because sometimes detecting low Bd quantities is imperative to the study, whereas in other situations, detecting low DNA quantities is less important.

scholarly journals Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Q. R. Ducarmon ◽  
B. V. H. Hornung ◽  
A. R. Geelen ◽  
E. J. Kuijper ◽  
R. D. Zwittink
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Bacterial Biomass ◽  
Extraction Methods ◽  
Rrna Gene ◽  
Method Choice ◽  
Advantages And Disadvantages ◽  
Dna Extraction Method ◽  
Dna Extraction Methods ◽  
Negative Controls

ABSTRACT When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples. IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.

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