scholarly journals Comparison and Validation of Ichthyoplankton DNA Extraction Methods

2021 ◽  
Vol 4 (4) ◽  
pp. 87
Author(s):  
Diouri Lamia ◽  
Uwiringiyeyezu Théophile ◽  
Abdelouahab Hinde ◽  
Malki Mohamed ◽  
Baibai Tarik ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Dna Extraction ◽  
Classical Method ◽  
Pcr Amplification ◽  
Temporal Distribution ◽  
Extraction Methods ◽  
Direct Pcr ◽  
Fisheries Resources ◽  
Morphological Criteria

Ichthyoplankton is the cluster of planktonic organisms that consists of fish eggs and larvae. These planktonic stages belong to the temporary zooplankton, representing future exploitable stocks. The study of the early ontogenesis of fish plays a key role in the understanding and evaluation of these populations through the study of their abundance and their spatio-temporal distribution. To better understand and protect these fisheries resources, it is essential to identify the different stages of fish embryonic development. This identification is usually performed using the classical method, based on morphological criteria under a binocular magnifying glass; however, this methodology is not always sufficient and is time consuming and, therefore, it is necessary to rely increasingly on molecular tools. The major problem with these tools is the yield and quality of the nucleic acids extracted from ichthyoplankton, especially in the case of eggs, which are small. Several methods have been used for DNA extraction from ichthyoplankton, either automated or manual, but very often from larvae or adults. In the present work, five fish egg DNA extraction protocols were compared based on their DNA yield and extraction quality, verified by agarose gel electrophoresis and quantitative PCR amplification. The results showed that extraction by our heat-protocol for direct PCR (Hp-dPCR) presents the simplest and cheapest protocol of all the kits used in this study, providing a sufficient quantity and quality of nucleic acids to be used for PCR amplification, and being within the reach of third world laboratories that often do not have sufficiently large budgets to obtain automated kits.

scholarly journals Improved direct lysis PCR amplification method of microalgal culture for sequencing and species identification

2021 ◽  
Vol 948 (1) ◽  
pp. 012013
Author(s):  
F Fitriyah ◽  
Y Faramitha ◽  
D A Sari ◽  
I Kresnawaty ◽  
T Panji ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Species Identification ◽  
Dna Barcode ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Rbcl Gene ◽  
Direct Pcr ◽  
Nannochloropsis Gaditana ◽  
Amplification Method ◽  
Microalgal Culture

Abstract Molecular approach plays important role in species identification for microalgae which involves sequencing of specific DNA barcode present in the genome. This approach involved preparation of template DNA for polymerase chain reaction (PCR) which is time consuming and requires large amounts of algal cells. Microalgal direct PCR have been used frequently for species identification, which simplified the DNA isolation procedure. However, the recent attempts to amplify the rbcL gene of microalga using the previously reported protocol led to poor repeatability. In this study, Nannochloropsis gaditana NIES-2587 was cultured in f/2 liquid medium. The culture growth was estimated on optical density value and the lysis process was improved using gradual temperature procedure during the PCR process. The same culture was extracted using manual DNA extraction method for comparison. The DNA obtained from both methods were amplified using RbclN primer pair to amplify 1486 bp partial sequence of Nannochloropsis rbcL gene, followed by the sequencing of the PCR product. Molecular identification based on the sequence result and BLAST analysis indicated that direct PCR and manual DNA extraction methods successfully produced high sequences result and confirmed the identity of microalgae species into N. gaditana strain CCMP527 with a genetic similarity of >99%.

scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

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

Simple ‘universal’ DNA extraction procedure compatible with direct PCR amplification

1996 ◽  
Vol 52 (4) ◽  
pp. 295-295 ◽  
Author(s):  
D. Goldenberger ◽  
I. Perschil ◽  
M. Ritzler ◽  
M. Altwegg
Keyword(s):  
Dna Extraction ◽  
Extraction Procedure ◽  
Pcr Amplification ◽  
Direct Pcr

scholarly journals ALKALINE LYSIS AS AN EFFICIENT AND ECONOMICAL ALTERNATIVE FOR DNA EXTRACTION FROM HORSEHAIR HAIR SAMPLES: COMPARISON BETWEEN DIFFERENT METHODS

2021 ◽  
Vol 9 (02) ◽  
pp. 836-841
Author(s):  
Myriam Janeth Ortega Torres ◽  
◽  
Jessica Almeida Braga ◽  
Camilo Torres ◽  
Ahmed Sami Shaker ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
Extraction Methods ◽  
Forensic Population ◽  
Alkaline Lysis ◽  
Hair Samples ◽  
Genomic Studies ◽  
Dna Extraction Methods ◽  
Short Time

Studies related to DNA extraction are becoming more ambitious in the sense that large studies are intended to be carried out with minimum DNA sources. The DNA extracted must be of quality for genetic, forensic, population and genomic studies, these samples must be easy to obtain and product of efficient manipulation.Samples obtained from horsehair are an important technical challenge since they constitute the preferred non-invasive sample for genetic studies in horses, which has been shown to obtain reliable results in a short time. In this sense, working into effective techniques to optimizeDNA extraction of scarse samples is a pertinent task. In this study, different DNA extraction methods were evaluated from mane samples obtained from a population of wild horses from the Region of Arauca in eastern Colombia.Three DNA extraction methods were evaluated (phenol chloroform, alkaline lysis and twocommercial DNA extraction kit), DNA concentration, purity and qualityweredeterminate and PCR amplification product were obtain using primers for a hypervariable region of DNA mitochondrial. DNA preparation from hair roots using alkaline lysis was the most economical and efficient method with which it was possible to obtain high quality and quantity DNA.

scholarly journals Quantitative and Qualitative Improvements in DNA Extraction Procedures Using a Bronopol™ Tablet in Alpine Goat Milk.

2018 ◽  
Author(s):  
Ashley Newton ◽  
Amber L. Martin ◽  
Yonathan Tilahun ◽  
Steve Zeng
Keyword(s):  
Dna Extraction ◽  
Somatic Cells ◽  
Goat Milk ◽  
Extraction Methods ◽  
Preservation Solution ◽  
Capra Aegagrus ◽  
The Difference ◽  
Dna Extraction Methods

Abstract Preservation of milk is important as it relates to Capra aegagrus hircus (Alpine goat) milk DNA extraction. We examined the difference in concentration and quality of DNA resulting from the use of a preservation tablet (BronopolTM) versus a preservation solution by Norgen Biotek. When examining the concentration and quality of DNA in goat milk for studies using somatic cells from goat milk, it is ideal to use a substance that has a long-term preservation potential. The concentrations and quality of DNA obtained from goat milk was compared. Two separate trial samples of Alpine goat milk were obtained. The preservation tablet commonly known as B-14 or Bronopol™ was dissolved into one sample of milk. Another sample of goat milk without a tablet used a preservation solution from a Norgen Biotek. All DNA extraction methods followed the Norgen Biotek Corp. manufacturer's protocol. DNA quantity and quality was analyzed using a Thermo Scientific NanodropLite spectrophotometer. The study showed that the traditional Bronopol™ was a better method of preserving and maintaining the integrity of DNA in the somatic cells that are present in Alpine goat milk. This is based on the results obtained following determination of quantity and A260/A280 readings for quality assessment. Thus, the use of Bronopol™ is the preferred method of preserving goat milk for DNA extraction.

scholarly journals Sticky problems: extraction of nucleic acids from molluscs

2021 ◽  
Vol 376 (1825) ◽  
Author(s):  
Coen M. Adema
Keyword(s):  
Nucleic Acids ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Biological Properties ◽  
Alternative Methods ◽  
28S Rrna ◽  
Future Directions ◽  
Dna And Rna ◽  
Standard Quality ◽  
Dna Extraction Methods

Traditional molecular methods and omics-techniques across molluscan taxonomy increasingly inform biology of Mollusca. Recovery of DNA and RNA for such studies is challenged by common biological properties of the highly diverse molluscs. Molluscan biomineralization, adhesive structures and mucus involve polyphenolic proteins and mucopolysaccharides that hinder DNA extraction or copurify to inhibit enzyme-catalysed molecular procedures. DNA extraction methods that employ the detergent hexadecyltrimethylammoniumbromide (CTAB) to remove these contaminants importantly facilitate molecular-level study of molluscs. Molluscan pigments may stain DNA samples and interfere with spectrophotometry, necessitating gel electrophoresis or fluorometry for accurate quantification. RNA can reliably be extracted but the ‘hidden break’ in 28S rRNA of molluscs (like most protostomes) causes 18S and 28S rRNA fragments to co-migrate electrophoretically. This challenges the standard quality control based on the ratio of 18S and 28S rRNA, developed for deuterostome animals. High-AT content in molluscan rRNA prevents the effective purification of polyadenylated mRNA. Awareness of these matters aids the continuous expansion of molecular malacology, enabling work also with museum specimens and next-generation sequencing, with the latter imposing unprecedented demands on DNA quality. Alternative methods to extract nucleic acids from molluscs are available from literature and, importantly, from communications with others who study the molecular biology of molluscs. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Alternative Methods of extracting and Amplifying Dna from lichens

2000 ◽  
Vol 32 (2) ◽  
pp. 189-196 ◽  
Author(s):  
Maria P. Martín ◽  
Katarina Winka
Keyword(s):  
Dna Extraction ◽  
Pcr Amplification ◽  
Alternative Methods ◽  
High Quality

AbstractWe have investigated whether DNA extraction protocols designed specifically for fungi and/or lichens perform better on lichens than do corresponding protocols designed for plants and insects. Two different PCR-amplification protocols were used to evaluate the quality of the DNA extracted with each method. The DNA extractions with highest quality were obtained with the protocols designed for insects and plants, and the most successful amplifications were obtained with Ready-To-Go PCR Beads. This indicates that fungal or lichen specific protocols might not be necessary for successful extraction of high quality DNA from lichens.

scholarly journals Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils

2019 ◽  
Author(s):  
Gareth Trubl ◽  
Simon Roux ◽  
Natalie Solonenko ◽  
Yueh-Fen Li ◽  
Benjamin Bolduc ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Cesium Chloride ◽  
Extraction Methods ◽  
Viral Lysis ◽  
Bead Beating ◽  
Dna Extraction Methods ◽  
Dsdna Viruses ◽  
Ssdna Viruses ◽  
The Impact

Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ~1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1–2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ~4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates ‘living’ protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.

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