scholarly journals Optimization of subsampling, decontamination, and DNA extraction of difficult peat and silt permafrost samples

2020 ◽  
Author(s):  
Alireza Saidi-Mehrabad ◽  
Patrick Neuberger ◽  
Maria Cavaco ◽  
Duane Froese ◽  
Brian Lanoil
Keyword(s):  
Dna Extraction ◽  
Soil Texture ◽  
Complete Removal ◽  
Extraction Methods ◽  
Chemical Parameters ◽  
The Core ◽  
Free Dna ◽  
Microbial Genomic ◽  
Dna Extraction Methods ◽  
Genomic Material

ABSTRACTThis study aims to act as a methodological guide for contamination monitoring, decontamination, and DNA extraction for peaty and silty permafrost samples with low biomass or difficult to extract DNA. We applied a biological tracer, either only in the field or both in the field and in the lab, via either spraying or painting. Spraying in the field followed by painting in the lab resulted in a uniform layer of the tracer on the core sections. A combination of bleaching, washing, and scraping resulted in complete removal of the tracer leaving sufficient material for DNA extraction, while other widely used decontamination methods did not remove all detectable tracer. In addition, of four widely used commercially available DNA extraction kits, only a modified ZymoBIOMICS™ DNA Microprep kit was able to acquire PCR amplifiable DNA. Permafrost chemical parameters, age, and soil texture did not have an effect on decontamination efficacy; however, the permafrost type did influence DNA extraction. Based on these findings, we developed recommendations for permafrost microbiologists to acquire contaminant-free DNA from permafrost with low biomass.IMPORTANCEPermafrost has the capacity to preserve microbial and non-microbial genomic material for millennia; however, major challenges are associated with permafrost samples, including decontamination of samples and acquiring pure DNA. Contamination of samples during coring and post coring handling and processing could affect downstream analyses and interpretations. Despite the use of multiple different decontamination and DNA extraction methods in studies of permafrost, the efficacy of these methods is not well known. We used a biological tracer to test the efficacy of previously published decontamination methods, as well as a bleach-based method we devised, on two chemically and structurally different permafrost core sections. Our method was the only one that removed all detectable tracer. In addition, we tested multiple DNA extraction kits and modified one that is able to acquire pure, PCR amplifiable DNA from silty, and to some extent from peaty, permafrost samples.

OPTIMIZATION OF DNA EXTRACTION METHODS FROM BIOLOGICAL MATERIALS OF HONEY BEES IN A DIFFERENT METAMOTPHOSIS PHASE

2016 ◽  
Vol 52 ◽  
pp. 171-176
Author(s):  
M. Palkina ◽  
O. Metlitska
Keyword(s):  
Ion Exchange ◽  
Dna Extraction ◽  
Honey Bees ◽  
Biological Material ◽  
Exchange Resin ◽  
Ion Exchange Resin ◽  
Extraction Methods ◽  
Chelex 100 ◽  
Drone Brood ◽  
Dna Extraction Methods

The aim of the research – adaptation, optimization and using of existing DNA extraction methods from bees’ biological material with the reagent «Chelex-100" under complex economic conditions of native laboratories, which will optimize labour costs and improve the economic performance of DNA extraction protocol. Materials and methods. In order to conduct the research the samples of honey bees’ biological material: queen pupae exuviae, larvae of drone brood, some adult bees’ bodies (head and thorax) were selected. Bowl and drone brood were obtained from the experimental bee hives of Institute of Apiculture nd. a. P. I. Prokopovich of NAAS. DNA extraction from biosamples of Apis mellifera ssp. was carried out using «Chelex-100®» ion exchange resin in different concentrations and combinations. Before setting tests for determination of quantitative and quality indexes, dilution of DNA samples of the probed object was conducted in ratio 1:40. The degree of contamination with protein and polysaccharide fractions (OD 260/230), quantitative content of DNA (OD 260/280) in the extracted tests were conducted using spectrophotometer of «Biospec – nano» at the terms of sample volume in 2 µl and length of optical way in 0,7 mm [7]. Verification of DNA samples from biological material of bees, isolated by «Chelex-100®», was conducted after cold keeping during 24 hours at 20°C using PСR with primaries to the fragment of gene of quantitative trait locus (QTL) Sting-2 of next structure [8]:  3' – CTC GAC GAG ACG ACC AAC TTG – 5’; 3' – AAC CAG AGT ATC GCG AGT GTT AC – 5’ Program of amplification: 94 °C – 5 minutes – 1 cycle; 94 °C – 1 minute, 57°C – 1 minute, 72 °C – 2 minutes – 30 cycles; elongation after 72°C during 2 minutes – 1 cycle. The division of obtained amplicons was conducted by gel electrophoresis at a low current – 7 µÀ, in 1,5 % agarose gel (Sigma ®) in TAE buffer [7]. The results. At the time of optimization of DNA isolation methods, according to existing methods of foreign experts, it was found optimal volume of ion exchange resin solution was in the proposed concentration: instead of 60 µl of solution used 120 µl of «Chelex-100®», time of incubation was also amended from 30 minutes to 180 minutes [9]. The use of the author's combination of method «Chelex-100®» with lysis enzymes, proteinase K and detergents (1M dithiothreitol), as time of incubation was also amended, which was reduced to 180 minutes instead of the proposed 12 hours [10]. Changes in quality characteristics of obtained DNA in samples after reduction in incubation time were not found. Conclusions. The most economical method of DNA isolation from bees’ biological material is 20% solution of «Chelex-100» ion exchange resin with the duration of the incubation period of 180 minutes. It should also be noted that the best results can be obtained from exuviae, selected immediately after the queen’s exit from bowl, that reduces the likelihood of DNA molecules destruction under the influence of nucleases activation, but not later than 12 hours from release using the technology of isolated obtain of queens.

scholarly journals Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1989
Author(s):  
Laura Téblick ◽  
Severien Van Keer ◽  
Annemie De Smet ◽  
Pierre Van Damme ◽  
Michelle Laeremans ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Internal Control ◽  
Extraction Methods ◽  
Urine Collection ◽  
Hpv Dna ◽  
Non Invasive ◽  
Herpesvirus 1 ◽  
Dna Extraction Methods ◽  
Detection Of Biomarkers ◽  
High Risk Hpv

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

scholarly journals A Rapid Bacteriophage DNA Extraction Method

2018 ◽  
Vol 1 (3) ◽  
pp. 27 ◽  
Author(s):  
Džiuginta Jakočiūnė ◽  
Arshnee Moodley
Keyword(s):  
Dna Extraction ◽  
Illumina Miseq ◽  
Extraction Methods ◽  
Resistant Bacteria ◽  
Simple Method ◽  
Antibiotic Resistant ◽  
Phage Dna ◽  
Specialized Equipment ◽  
Miseq Platform ◽  
Dna Extraction Methods

Bacteriophages (phages) are intensely investigated as non-antibiotic alternatives to circumvent antibiotic resistance development as well as last resort therapeutic options against antibiotic resistant bacteria. As part of gaining a better understanding of phages and to determine if phages harbor putative virulence factors, whole genome sequencing is used, for which good quality phage DNA is needed. Traditional phage DNA extraction methods are tedious and time consuming, requiring specialized equipment e.g., an ultra-centrifuge. Here, we describe a quick and simple method (under four hours) to extract DNA from double stranded DNA (dsDNA) phages at titers above 1.0 × 1010 plaque-forming units (PFU)/mL. This DNA was suitable for library preparation using the Nextera XT kit and sequencing on the Illumina MiSeq platform.

131-P: Protein contamination in manual DNA extraction methods

2007 ◽  
Vol 68 (1) ◽  
pp. S80
Author(s):  
Victoriano J. Leon ◽  
Alberto J. Leon ◽  
Juan Luis Garcia
Keyword(s):  
Dna Extraction ◽  
Extraction Methods ◽  
P Protein ◽  
Protein Contamination ◽  
Dna Extraction Methods

scholarly journals Optimizing DNA extraction methods for Nanopore sequencing of Neisseria gonorrhoeae direct from urine samples

2019 ◽  
Author(s):  
Teresa L. Street ◽  
Leanne Barker ◽  
Nicholas D. Sanderson ◽  
James Kavanagh ◽  
Sarah Hoosdally ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Reference Genome ◽  
Extraction Methods ◽  
Urine Samples ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Human Dna ◽  
Whole Genomes ◽  
Dna Extraction Methods ◽  
Multiple Antibiotics

AbstractBackgroundEmpirical gonorrhoea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance.MethodsWe investigated if Nanopore sequencing can detect sufficient N. gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae spiked urine samples and samples from gonorrhoea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced whilst minimizing contaminating host DNA.ResultsIn simulated infections the Qiagen UCP Pathogen Mini kit provided the highest ratio N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections, but decreased yields in clinical samples. In ten urine samples from men with symptomatic urethral gonorrhoea, ≥87% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥92% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR Media tubes and from urethral swabs, and in the presence of simulated Chlamydia co-infection.ConclusionUsing Nanopore sequencing of urine samples from men with urethral gonorrhoea sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

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