scholarly journals DNA recovery from microhymenoptera using six non-destructive methodologies with considerations for subsequent preparation of museum slides

Genome ◽  
2017 ◽  
Vol 60 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Adriana J. Guzmán-Larralde ◽  
Alba P. Suaste-Dzul ◽  
Adrien Gallou ◽  
Kenzy I. Peña-Carrillo
Keyword(s):  
Dna Extraction ◽  
Mitochondrial Gene ◽  
Dna Amplification ◽  
Morphological Identification ◽  
Direct Pcr ◽  
Dna Recovery ◽  
Require Time ◽  
Gene Coi ◽  
Permanent Slide ◽  
Non Destructive

Because of the tiny size of microhymenoptera, successful morphological identification typically requires specific mounting protocols that require time, skills, and experience. Molecular taxonomic identification is an alternative, but many DNA extraction protocols call for maceration of the whole specimen, which is not compatible with preserving museum vouchers. Thus, non-destructive DNA isolation methods are attractive alternatives for obtaining DNA without damaging sample individuals. However, their performance needs to be assessed in microhymenopterans. We evaluated six non-destructive methods: (A) DNeasy® Blood & Tissue Kit; (B) DNeasy® Blood & Tissue Kit, modified; (C) Protocol with CaCl2 buffer; (D) Protocol with CaCl2 buffer, modified; (E) HotSHOT; and (F) Direct PCR. The performance of each DNA extraction method was tested across several microhymenopteran species by attempting to amplify the mitochondrial gene COI from insect specimens of varying ages: 1 day, 4 months, 3 years, 12 years, and 23 years. Methods B and D allowed COI amplification in all insects, while methods A, C, and E were successful in DNA amplification from insects up to 12 years old. Method F, the fastest, was useful in insects up to 4 months old. Finally, we adapted permanent slide preparation in Canada balsam for every technique. The results reported allow for combining morphological and molecular methodologies for taxonomic studies.

scholarly journals Molecular tools for phytoplankton monitoring samples

2018 ◽  
Author(s):  
Bárbara Frazão ◽  
Alexandra Silva
Keyword(s):  
Dna Extraction ◽  
Dna Polymerases ◽  
Life Sciences ◽  
Dna Amplification ◽  
Cell Number ◽  
Molecular Tools ◽  
Direct Pcr ◽  
Tissue Samples ◽  
Thermofisher Scientific ◽  
Phytoplankton Monitoring

AbstractHABs can have severe impacts in fisheries or human health by the consumption of contaminated bivalves. Monitoring assessment (quantitative and qualitative identification) of these organisms, is routinely accomplished by microscopic identification and counting of these organisms. Nonetheless, molecular biology techniques are gaining relevance, once these approaches can easily identify phytoplankton organisms at species level and even cell number quantifications. This work tests 12 methods/kits for genomic DNA extraction and seven DNA polymerases to determine which is the best method for routinely use in a common molecular laboratory, for phytoplankton monitoring samples analyses. From our work, Direct PCR master mix for tissue samples, proved to be the most adequate by its velocity of processivity, practicability, reproducibility, sensitiveness and robustness. However, brands such as Omega Biotek, GRISP, Qiagen and MP Biomedicals also showed good results for conventional DNA extraction as well as all the Taq brands tested (GRISP, GE Healthcare Life Sciences, ThermoFisher Scientific and Promega). Lugol’s solution, with our tested kits did not show negative interference in DNA amplification. The same can be said about mechanical digestion, with no significant differences among kits with or without this homogenization step.

scholarly journals Validating DNA Extraction Protocols for Bentonite Clay

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Katja Engel ◽  
Sara Coyotzi ◽  
Melody A. Vachon ◽  
Jennifer R. McKelvie ◽  
Josh D. Neufeld
Keyword(s):  
Microbial Community ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Bentonite Clay ◽  
Dna Recovery ◽  
Blocking Agents ◽  
Clay Matrix ◽  
Microbial Dna

ABSTRACT Bentonite clay is an integral component of the engineered barrier system of deep geological repositories (DGRs) that are planned for the long-term storage of high-level radioactive waste. Although nucleic acid extraction and analysis can provide powerful qualitative and quantitative data reflecting the presence, abundance, and functional potential of microorganisms within DGR materials, extraction of microbial DNA from bentonite clay is challenging due to the low biomass and adsorption of nucleic acids to the charged clay matrix. In this study, we used quantitative PCR, gel fingerprinting, and high-throughput sequencing of 16S rRNA gene amplicons to assess DNA extraction efficiency from natural MX-80 bentonite and the same material “spiked” with Escherichia coli genomic DNA. Extraction protocols were tested without additives and with casein and phosphate as blocking agents. Although we demonstrate improved DNA recovery by blocking agents at relatively high DNA spiking concentrations, at relatively low spiking concentrations, we detected a high proportion of contaminant nucleic acids from blocking agents that masked sample-specific microbial profile data. Because bacterial genomic DNA associated with casein preparations was insufficiently removed by UV treatment, casein is not recommended as an additive for DNA extractions from low-biomass samples. Instead, we recommend a kit-based extraction protocol for bentonite clay without additional blocking agents, as tested here and validated with multiple MX-80 bentonite samples, ensuring relatively high DNA recoveries with minimal contamination. IMPORTANCE Extraction of microbial DNA from MX-80 bentonite is challenging due to low biomass and adsorption of nucleic acid molecules to the charged clay matrix. Blocking agents improve DNA recovery, but their impact on microbial community profiles from low-biomass samples has not been characterized well. In this study, we evaluated the effect of casein and phosphate as blocking agents for quantitative recovery of nucleic acids from MX-80 bentonite. Our data justify a simplified framework for analyzing microbial community DNA associated with swelling MX-80 bentonite samples within the context of a deep geological repository for used nuclear fuel. This study is among the first to demonstrate successful extraction of DNA from Wyoming MX-80 bentonite.

Non‐destructive DNA recovery from handwritten documents using a dry vacuum technique

2021 ◽  
Author(s):  
Patrick McLaughlin ◽  
Christian Hopkins ◽  
Eliot Springer ◽  
Mechthild Prinz
Keyword(s):  
Dna Recovery ◽  
Handwritten Documents ◽  
Non Destructive

scholarly journals Direct PCR from whole blood, without DNA extraction

1990 ◽  
Vol 18 (19) ◽  
pp. 5908-5909 ◽  
Author(s):  
B. Mercier ◽  
C. Gaucher ◽  
O. Feugeas ◽  
C. Mazurier
Keyword(s):  
Dna Extraction ◽  
Whole Blood ◽  
Direct Pcr

scholarly journals Detection of Bacteria in Environmental Samples by Direct PCR Without DNA Extraction

BioTechniques ◽  
2001 ◽  
Vol 31 (3) ◽  
pp. 598-607 ◽  
Author(s):  
Kimberly A. Fode-Vaughan ◽  
Charles F. Wimpee ◽  
Charles C. Remsen ◽  
Mary Lynne Perille Collins
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Direct Pcr

scholarly journals Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales

Hereditas ◽  
2003 ◽  
Vol 138 (3) ◽  
pp. 161-165 ◽  
Author(s):  
ADRIANE P. WASKO ◽  
CESAR MARTINS ◽  
CLAUDIO OLIVEIRA ◽  
FAUSTO FORESTI
Keyword(s):  
Dna Extraction ◽  
Improved Method ◽  
Genetic Sampling ◽  
Fish Fins ◽  
Non Destructive

A duplex ddPCR assay for simultaneously detecting Ips sexdentatus and Ips typographus (Coleoptera: Curculionidae) in bulk trap samples

2019 ◽  
Vol 49 (8) ◽  
pp. 903-914 ◽  
Author(s):  
Frida A. Zink ◽  
Luke R. Tembrock ◽  
Alicia E. Timm ◽  
Todd M. Gilligan
Keyword(s):  
Dna Extraction ◽  
Bark Beetles ◽  
Digital Pcr ◽  
The United States ◽  
Ips Typographus ◽  
Morphological Identification ◽  
Efficient Manner ◽  
Large Numbers ◽  
The Family ◽  
Ips Sexdentatus

Bark beetles in the family Curculionidae present a growing hazard to forests worldwide. Like native bark beetles, introduced exotic species can pose a serious threat to North American forests. Ips typographus (Boerner) and Ips sexdentatus (Linnaeus), both native to Europe, are two such pests that have caused widespread forest loss in their native ranges. International trade has led to increased interceptions of Scolytine beetles at ports of entry to the United States. Most intercepted individuals are not identified to species due to lack of expert identifiers, poor specimen quality, or incomplete taxonomy. These same problems affect identification for domestic surveys. Therefore, development of molecular methods for identification of potentially invasive Ips species is essential. Because of the need to scrutinize large numbers of beetles in an efficient manner, we describe a duplex droplet digital PCR (ddPCR) assay to identify I. typographus and I. sexdentatus simultaneously in bulk trap samples containing 500 Scolytinae specimens using a scalable, two-step DNA extraction. This ddPCR method is highly effective for processing the entire contents of beetle traps and identifying these potentially invasive species in a timely and definitive manner. We also describe a nondestructive DNA extraction technique that preserves specimens for morphological identification.

scholarly journals Effects of DNA Extraction and Purification Methods on Real-Time Quantitative PCR Analysis of Roundup Ready Soybean

2009 ◽  
Vol 92 (4) ◽  
pp. 1136-1144 ◽  
Author(s):  
Tigst Demeke ◽  
Indira Ratnayaka ◽  
Anh Phan
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Roundup Ready ◽  
Dna Recovery ◽  
Real Time Quantitative Pcr ◽  
Extraction And Purification ◽  
Purification Methods ◽  
Accuracy And Repeatability

Abstract The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

From population genetics to phylogeny: Uses and limits of mitochondrial DNA

1990 ◽  
Vol 3 (1) ◽  
pp. 111 ◽  
Author(s):  
RH Crozier
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Gene ◽  
Single Gene ◽  
Dna Amplification ◽  
Species Tree ◽  
Population Subdivision ◽  
Evolutionary Divergence ◽  
Gene Trees ◽  
Species Trees ◽  
Effective Population

Mitochondrial DNA (mtDNA) is clonally and maternally inherited in all animals and in most plants. Mitochondrial gene content is similar although not identical in all eukaryotes. Because of these characteristics, mtDNA has a number of features useful to systematists for all levels of evolutionary divergence. Clonal inheritance leads to unusual confidence in constructing gene trees which are useful in population-level studies, such as in the detection of population subdivision. Maternal inheritance presents the opportunity to distinguish paternal from maternal gene flow. The clonal, or single-gene, nature of mtDNA inheritance leads to consideration of the expected convergence between gene- and species-trees. For closely related populations or species, it is desirable to use several genes to be sure that the correct species-tree is discovered; this means that, although mtDNA will be the most precise guide to the species tree because of its lower effective population size, nuclear genes should also be used in such studies. Although restriction fragment length polymorphisms dominated the field until recently, sequencing following DNA amplification using the polymerase chain reaction is now easier and opens up the use of preserved specimens to molecular systematists. Because mitochondria1 genes evolve at different rates, one of appropriate rate can be selected for almost any phylogenetic problem.

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