scholarly journals A pilot study of DNA yield from bloodstains on various surfaces using Phenol chloroform isoamyl alcohol (PCIA) and Chelex DNA extraction methods

2020 ◽  
Vol 13 (2) ◽  
pp. 124-127
Author(s):  
Ishwar Prasad Dubey ◽  
R. K. Kumawat ◽  
I. P. Tripathi ◽  
Pankaj Shrivastava
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Isoamyl Alcohol ◽  
Extraction Methods ◽  
Forensic Dna ◽  
Dna Yield ◽  
Dna Extraction Methods ◽  
Short Tandem

Presently, Short Tandem Repeats (STRs) based forensic DNA typing technology is being globally used in solving a diverse range of forensic cases such as paternity, identification of unknown dead bodies/skeletal remains, or suspect in a case of rape or mass rape. The technology has invaded its tentacles in almost all areas of criminal investigation in the last few decades. The present forensic DNA technology is based on capillary electrophoresis and utilizes short tandem repeats(STRs).On one hand, the technology is extensively used in the investigation of crime in highly sensitive cases, but on the another hand, obtaining DNA profile from forensic samples are highly challenging many times. Advent of PCR has been a boon for handling the challenging samples in forensic DNA analysis. The quality DNA profiles from challenging samples rely on the yield and quality of DNA, which is mainly dependent upon the method used for DNA extraction. Any specific method can never be thought of to be useful for all variety of samples. Still, Phenol Chloroform Isoamyl Alcohol (PCIA) organic extraction method has been proven to be useful for a wide variety of samples from the simplest saliva/blood to complex teeth and bone samples. In the present study, we compared the yield of DNA from blood stains recovered from various surfaces using the PCIA extraction method and Chelex DNA extraction methods and their compatibility with present-day STR based capillary electrophoresis typing. The mean value of DNA yield was found 50.5 ng/ µl and 32.25 ng/ µl by PCIA and Chelex DNA extraction methods, respectively. Overall, the highest yield was observed from all the tested samples from the PCIA method.

Conditional Genotypic Probabilities for Microsatellite Loci

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1973-1980
Author(s):  
Jinko Graham ◽  
James Curran ◽  
B S Weir
Keyword(s):  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Dirichlet Distribution ◽  
Forensic Dna ◽  
Match Probability ◽  
Microsatellite Genotype ◽  
Allelic State ◽  
Forensic Assessments ◽  
Dna Profiles ◽  
Short Tandem

Abstract Modern forensic DNA profiles are constructed using microsatellites, short tandem repeats of 2–5 bases. In the absence of genetic data on a crime-specific subpopulation, one tool for evaluating profile evidence is the match probability. The match probability is the conditional probability that a random person would have the profile of interest given that the suspect has it and that these people are different members of the same subpopulation. One issue in evaluating the match probability is population differentiation, which can induce coancestry among subpopulation members. Forensic assessments that ignore coancestry typically overstate the strength of evidence against the suspect. Theory has been developed to account for coancestry; assumptions include a steady-state population and a mutation model in which the allelic state after a mutation event is independent of the prior state. Under these assumptions, the joint allelic probabilities within a subpopulation may be approximated by the moments of a Dirichlet distribution. We investigate the adequacy of this approximation for profiled loci that mutate according to a generalized stepwise model. Simulations suggest that the Dirichlet theory can still overstate the evidence against a suspect with a common microsatellite genotype. However, Dirichlet-based estimators were less biased than the product-rule estimator, which ignores coancestry.

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 A modified SDS-based DNA extraction method from raw soybean

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Yimiao Xia ◽  
Fusheng Chen ◽  
Yan Du ◽  
Chen Liu ◽  
Guanhao Bu ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Pcr Amplification ◽  
Isoamyl Alcohol ◽  
Monitoring Methods ◽  
Detection Techniques ◽  
Dna Yield ◽  
Seed Flour ◽  
Dna Extraction Method ◽  
Lysis Buffer

Abstract Soybean is the most important genetically modified (GM) oilseed worldwide. Regulations relating to the approval of biotech soybean varieties and product labeling demand accurate and reliable detection techniques to screen for GM soya. High-quality extracted DNA is essential for DNA-based monitoring methods. Thus, four widely used protocols (SDS, CTAB, DP305, and DNeasy Plant Mini Kit) were compared in the present study to explore the most efficient DNA extraction method for raw soya matrix. The SDS-based method showed the highest applicability. Then crucial factors influencing DNA yield and purity, such as SDS lysis buffer component concentrations and organic compounds used to isolate DNA, were further investigated to improve the DNA obtained from raw soybean seeds, which accounts for the innovation of this work. As a result, lysis buffer (2% SDS (w/v), 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0) and organic reagents including chloroform/isoamyl alcohol (24:1, v/v) (C: I), isopropanol, and ethanol corresponding to the extraction and first and second precipitation procedures, respectively, were used in the optimized SDS method. The optimized method was verified by extracting approximately 2020–2444 ng DNA/mg soybean with A260/280 ratios of 1.862–1.954 from five biotech and non-biotech soybean varieties. Only 0.5 mg of soya was required to obtain enough DNA for PCR amplification using the optimized SDS-based method. These results indicate that the screening protocol in the present study achieves the highest suitability and efficiency for DNA isolation from raw soya seed flour.

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 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 Forensic Genetics and Genotyping

2019 ◽  
Vol 20 (2) ◽  
pp. 75-86
Author(s):  
Katarina Vitoševic ◽  
Danijela Todorovic ◽  
Zivana Slovic ◽  
Radica Zivkovic-Zaric ◽  
Milos Todorovic
Keyword(s):  
Genetic Markers ◽  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Forensic Genetics ◽  
Personal Identification ◽  
Hair Color ◽  
Forensic Dna ◽  
Kinship Analysis ◽  
Dna Profile ◽  
Short Tandem

Abstract Forensic genetics represents a combination of molecular and population genetics. Personal identification and kinship analysis (e.g. paternity testing) are the two main subjects of forensic DNA analysis. Biological specimens from which DNA is isolated are blood, semen, saliva, tissues, bones, teeth, hairs. Genotyping has become a basis in the characterization of forensic biological evidence. It is performed using a variety of genetic markers, which are divided into two large groups: bi-allelic (single-nucleotide polymorphisms, SNP) and multi-allelic polymorphisms (variable number of tandem repeats, VNTR and short tandem repeats, STR). This review describes the purpose of genetic markers in forensic investigation and their limitations. The STR loci are currently the most informative genetic markers for identity testing, but in cases without a suspect SNP can predict offender’s ancestry and phenotype traits such as skin, eyes and hair color. Nowadays, many countries worldwide have established forensic DNA databases based on autosomal short tandem repeats and other markers. In order for DNA profile database to be useful at a national or international level, it is essential to standardize genetic markers used in laboratories.

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 The Plateau Method for Forensic DNA SNP Mixture Deconvolution

2017 ◽  
Author(s):  
Darrell O. Ricke ◽  
Joe Isaacson ◽  
James Watkins ◽  
Philip Fremont-Smith ◽  
Tara Boettcher ◽  
...  
Keyword(s):  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
High Throughput Sequencing ◽  
Nucleotide Polymorphisms ◽  
Forensic Dna ◽  
Dna Mixtures ◽  
Single Nucleotide ◽  
Mixture Deconvolution ◽  
Dna Mixture ◽  
Short Tandem

AbstractIdentification of individuals in complex DNA mixtures remains a challenge for forensic analysts. Recent advances in high throughput sequencing (HTS) are enabling analysis of DNA mixtures with expanded panels of Short Tandem Repeats (STRs) and/or Single Nucleotide Polymorphisms (SNPs). We present the plateau method for direct SNP DNA mixture deconvolution into sub-profiles based on differences in contributors’ DNA concentrations in the mixtures in the absence of matching reference profiles. The Plateau method can detect profiles of individuals whose contribution is as low as 1/200 in a DNA mixture (patent pending)1.

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