scholarly journals DNA Analysis Using Noninvasive Samples: Methods of Sample Collection, DNA Extraction, PCR Amplification and Kinship Analysis

2015 ◽  
Vol 31 (1) ◽  
pp. 3-18 ◽  
Author(s):  
Eiji INOUE
Keyword(s):  
Dna Extraction ◽  
Dna Analysis ◽  
Pcr Amplification ◽  
Sample Collection ◽  
Kinship Analysis ◽  
Noninvasive Samples

scholarly journals Genetic DNA Identification from Bone Remains in Kinship Analysis Using Automate Extraction System

2021 ◽  
Author(s):  
Raluca Dumache ◽  
Talida Cut ◽  
Camelia Muresan ◽  
Veronica Ciocan ◽  
Emanuela Stan ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Dna Analysis ◽  
Pcr Amplification ◽  
Human Identification ◽  
Kinship Analysis ◽  
Disaster Victims ◽  
Buccal Swabs ◽  
Bone Powder ◽  
Pcr Products ◽  
Bone Remains

The first ever human identification through DNA analysis was done in the year 1987. Since then, this test has been used, not only in the ruling of civil and juridical cases, but also for human identification of missing persons and mass disaster victims. In this chapter we will present the usefulness of genetic DNA testing of skeletonized remains for human identification, by using automate DNA extraction from three different human bone types: tooth, femur and petrous pyramid. For each case, we obtained saliva samples on buccal swabs from relatives. After the bones were washed and cleaned, Bead Balls Mill Mix 20 (Tehtnica Domel, Slovenia), was used to obtain the bone powder. The DNA extraction from bone samples was performed on the automate Maxwell RSC 48 Instrument (Promega, USA), using the Maxwell FSC DNA IQ Casework Kit (Promega, USA). Power Quant System (Promega, USA) was used for DNA quantification of the samples. The DNA samples were amplified on a Pro Flex PCR System (Thermo Fischer, USA), using the Global Filer PCR Amplification Kit (Applied Biosystems, USA). PCR products were run on a 3500 Genetic Analyzer (Thermo Fischer, USA). Data analysis was performed by Gene Mapper 1.4. Considering that these cases involved DNA extraction from teeth, bones and old human remains, automate system was felt to be the best option to reduce handling errors and increase the possibilities of obtaining good quality DNA.

Extracting DNA from submerged pine wood

Genome ◽  
2004 ◽  
Vol 47 (5) ◽  
pp. 994-997 ◽  
Author(s):  
M Megan Reynolds ◽  
Claire G Williams
Keyword(s):  
Dna Extraction ◽  
Dna Analysis ◽  
Pcr Amplification ◽  
Pinus Palustris ◽  
Proteinase K ◽  
High Molecular Weight Dna ◽  
Bisphosphate Carboxylase ◽  
Extraction Protocol ◽  
High Sequence Homology ◽  
Reaction Sequencing

A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser®. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.Key words: conifers, wood, polymerase chain reaction, sequencing.

Sample collection and eDNA extraction from Sterivex filter units v1

2021 ◽  
Author(s):  
Oscar E Chiang ◽  
Pedro Inostroza
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Dna Analysis ◽  
Water Systems ◽  
Sample Collection ◽  
Extraction Protocol ◽  
Water Collection ◽  
Sterile Filter

The following workflow covers several steps in the DNA analysis of environmental samples, from the water collection to the analysis back in the lab. The samples can be taken from several water systems (i.e. sea, lakes, rivers, streams) and collected in triplicate (1 L) in Sterivex sterile filter units (Merck, cat. no. SVGP01050). The DNA extraction protocol modifies the Dneasy PowerWater Sterivex kit (Qiagen, cat. no. 14600-50-nf).

scholarly journals EQUAL-qual: A European Program for External Quality Assessment of Genomic DNA Extraction and PCR Amplification

2007 ◽  
Vol 53 (7) ◽  
pp. 1349-1357 ◽  
Author(s):  
Claudio Orlando ◽  
Paolo Verderio ◽  
Ronald Maatman ◽  
Jan Danneberg ◽  
Simon Ramsden ◽  
...  
Keyword(s):  
Quality Assessment ◽  
Dna Extraction ◽  
Dna Analysis ◽  
External Quality Assessment ◽  
Clinical Chemistry ◽  
Pcr Amplification ◽  
Molecular Techniques ◽  
The European Union ◽  
External Quality ◽  
Pcr Efficiency

Abstract Background: Despite the rapid transition into routine clinical practice of molecular techniques based on PCR, external quality assessment (EQA) is still not widely available. The European Union and European Communities Confederation of Clinical Chemistry have supported the EQUAL project as a series of 3 different EQA programs for the assessment of molecular methods independently from analytes. We present the results from the EQUAL-qual program designed to evaluate the analytical aspects of DNA analysis by means of a conventional qualitative PCR experiment. Methods: The EQUAL-qual program provided DNA, blood samples, and primer sets to participant laboratories to assess DNA extraction and PCR amplification. We have developed statistical procedures to identify laboratories performing poorly in DNA extraction (quality and quantity), PCR efficiency, and data interpretation after electrophoresis. Results: An application to participate was obtained from 213 laboratories (from 25 countries), and 175 (82%) of laboratories submitted results for assessment. Questionable results in terms of quality and/or quantity of DNA derived from blood extractions were returned by 27% of laboratories (46 of 166). PCR efficiency showed high variability, with 3% of laboratories (5 of 163) showing a consistently low rate of amplification and 10% (18 of 175) not reporting the expected number of bands of the amplified targets. Conclusions: The results showed considerable variability in all phases of the experiment. The approach confirms the validity of EQA as a method for evaluating analytical aspects of PCR-based tests.

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

2014 ◽  
Vol 6 (01) ◽  
pp. 031-035 ◽  
Author(s):  
Shashank Pooniya ◽  
Sanjeev Lalwani ◽  
Anupuma Raina ◽  
Tabin Millo ◽  
Tirath Das Dogra
Keyword(s):  
Dna Extraction ◽  
Dna Analysis ◽  
Soft Tissues ◽  
Pcr Amplification ◽  
Climatic Conditions ◽  
High Molecular Weight Dna ◽  
Human Corpse ◽  
Multiple Loci ◽  
Polymerase Chain ◽  
The Brain

ABSTRACT 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.

scholarly journals Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

2018 ◽  
Vol 13 (3) ◽  
pp. 115
Author(s):  
Dwiyitno Dwiyitno ◽  
Stefan Hoffman ◽  
Koen Parmentier ◽  
Chris Van Keer
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Blue Mussel ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Seafood Products ◽  
Polymerase Chain ◽  
Commercial Kit

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

scholarly journals The Fellowship of the Ring Test: DNAqua-Net WG2 initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardisation

2021 ◽  
Vol 4 ◽  
Author(s):  
Valentin Vasselon ◽  
Éva Ács ◽  
Salomé Almeida ◽  
Karl Andree ◽  
Laure Apothéloz-Perret-Gentil ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Aquatic Ecosystems ◽  
Quality Index ◽  
Ecological Status ◽  
Pcr Amplification ◽  
Ring Test ◽  
Dna Metabarcoding ◽  
Ecological Status Assessment ◽  
Status Assessment

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces

2008 ◽  
Vol 72 (2) ◽  
pp. 124-132 ◽  
Author(s):  
Jordan M. Nechvatal ◽  
Jeffrey L. Ram ◽  
Marc D. Basson ◽  
Phanramphoei Namprachan ◽  
Stephanie R. Niec ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Pcr Amplification ◽  
Human Feces ◽  
Fecal Collection

Microbial characteristics of a methanogenic phenol-degrading sludge

2005 ◽  
Vol 52 (1-2) ◽  
pp. 73-78 ◽  
Author(s):  
T. Zhang ◽  
S. Z. Ke ◽  
Y. Liu ◽  
H.P. Fang
Keyword(s):  
Dna Analysis ◽  
Pcr Amplification ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Chain Reaction ◽  
Microbial Characteristics ◽  
Polymerase Chain ◽  
Anaerobic Sludge Blanket

Microbial properties of a methanogenic granular phenol-degrading sludge were characterized using the 16S rRNA/DNA-based techniques, including polymerase chain reaction (PCR) amplification, cloning, DNA sequencing, and fluorescence in situ hybridization (FISH). The sludge was sampled from an upflow anaerobic sludge blanket reactor, which removed 98% of phenol (up to 1260 mg/l) in wastewater at 26°C with 12 hours of hydraulic retention. Based on DNA analysis, the Eubacteria in the sludge was composed of 13 operational taxonomy units (OTUs). Two OTUs, one resembling Clostridium and the other remotely resembling Desulfotomaculum, were likely responsible for the conversion of phenol to benzoate, which was further degraded by five Syntrophus-resembling OTUs to acetate and H2/CO2; methanogens lastly converted acetate and H2/CO2 into methane. The role of six remaining OTUs remains unclear. Overall, the sludge was composed of 26±6% Eubacteria and 74±9% methanogens, of which 54±6% were acetotrophic Methanosaetaceae, 14±3% and 3±2% were hydrogenotrophic Methanomicrobiales and Methanobacteriaceae, respectively.

scholarly journals Variability of D2/D3 segment sequences of several populations and pathotypes of potato cyst nematodes (Globodera rostochiensis, Globodera pallida)

2010 ◽  
Vol 46 (No. 4) ◽  
pp. 171-180 ◽  
Author(s):  
O. Douda ◽  
M. Zouhar ◽  
E. Nováková ◽  
J. Mazáková ◽  
P. Ryšánek
Keyword(s):  
Dna Analysis ◽  
Pcr Amplification ◽  
Globodera Rostochiensis ◽  
Globodera Pallida ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes ◽  
Routine Diagnostics ◽  
Pair Comparisons ◽  
Single Fragment ◽  
Direct Management

Potato cyst nematodes (Globodera rostochiensis, Globodera pallida) remain a key pest in the main potato growing regions of the Czech Republic. Due to difficult direct management and presence of diverse pathotypes attacking different potato cultivars the rapid and reliable diagnostics is of crucial importance. Currently, efforts are aimed at a description of different pathotypes based on DNA analysis. The main objective of this study was to evaluate the homogeneity of sequences of D2/D3 segments of the 28S rDNA gene obtained from 3 populations of G. rostochiensis and 5 populations of G. pallida and estimate their value for diagnostic purposes. PCR amplification yielded a single fragment of the length of 700 bp approximately in all populations. The alignment score of the vast majority of all pair comparisons of G. rostochiensis and G. pallida populations varied from 98 to 99. In total 14 point deletions and 3 substitutions were observed. The variability of D2/D3 segments of potato cyst nematodes is rather low and this DNA region can be used for diagnostics on a species level because more differences were found after comparing with G. tabacum and G. millefolii sequences obtained from Gene Bank; however the applicability of D2/D3 sequences to routine diagnostics of potato cyst nematodes could be complicated by its similarity to corresponding sequences of the nematode G. artemisiae.

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