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Methods Protoc., Volume 1, Issue 3 (September 2018) – 12 articles

Cover Story (view full-size image): Generation of locus-specific genomic editing using CRISPR/Cas9 in cell lines and primary cells is becoming routine lab practice. However, identification of specifically induced mutations and sequence changes can require laborious screening of hundreds of clones. This protocol permits high-throughput genotyping of edited cells using next generation sequencing. In addition to allowing for the screening of hundreds of clones simultaneously, this approach provides the edited sequence of each allele and can be applied to multiple loci in a single sequencing run. View this paper.
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6 pages, 708 KiB  
Protocol
Two-Step Concentration of Complex Water Samples for the Detection of Viruses
by Kata Farkas, James E. McDonald, Shelagh K. Malham and Davey L. Jones
Methods Protoc. 2018, 1(3), 35; https://doi.org/10.3390/mps1030035 - 10 Sep 2018
Cited by 20 | Viewed by 6138
Abstract
The accurate detection and quantification of pathogenic viruses in water is essential to understand and reduce the risk of human infection. This paper describes a two-step method suitable for concentrating viruses in water and wastewater samples. The method involves a tangential flow ultrafiltration [...] Read more.
The accurate detection and quantification of pathogenic viruses in water is essential to understand and reduce the risk of human infection. This paper describes a two-step method suitable for concentrating viruses in water and wastewater samples. The method involves a tangential flow ultrafiltration step that reduces the sample volume of 1–10 L to approximately 50 mL, followed by secondary precipitation using polyethylene glycol 6000, which reduces the volume to 1–4 mL. For method validation, water samples were spiked with different concentrations of enteric viruses, and viral recovery in the concentrates exceeded 10% in all experiments. The method is suitable for water samples with high and low salinity and turbidity, allowing an accurate comparison of viral titers in a diverse range of water types. Furthermore, the method has the potential to concentrate other pathogens, e.g., bacteria or protozoa. Hence, the use of this method can improve the holistic assessment of risks associated with wastewater-contaminated environments. Full article
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10 pages, 4126 KiB  
Benchmark
Histological Studies of Mycorrhized Roots and Mycorrhizal-Like-Structures in Pine Roots
by Carla Ragonezi and Maria Amely Zavattieri
Methods Protoc. 2018, 1(3), 34; https://doi.org/10.3390/mps1030034 - 5 Sep 2018
Cited by 1 | Viewed by 5259
Abstract
Several studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses [...] Read more.
Several studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses of plants because of a weak root system that is incapable of water and nutrient absorption. During a series of mycorrhization experiments, cryostat and ultrafine cuts were used to study the morpho-histological transformation of the symbiotic roots. To obtain cryostat cuts from pine roots a method frequently used for animal tissue was adopted. Molecular methods allowed fungi identification in all the mycorrhization phases and in the acclimation of derived plants. Mycorrhizal-like-structures derived from in vitro culture and axenic liquid cultures of roots were microscopically analyzed and compare with mycorrhizal roots. Full article
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11 pages, 2324 KiB  
Technical Note
Improved Detection and Fragmentation of Disulphide-Linked Peptides
by Evelyne Maes, Stefan Clerens, Jolon M. Dyer and Santanu Deb-Choudhury
Methods Protoc. 2018, 1(3), 33; https://doi.org/10.3390/mps1030033 - 3 Sep 2018
Cited by 1 | Viewed by 3436
Abstract
Characterisation of peptides containing intact disulphide bonds (DSBs) via mass spectrometry is challenging. Our study demonstrates that the addition of aniline to alpha-cyano-4-hydroxycinnamic acid improves detection and fragmentation of complex DSB peptides by matrix-assisted laser desorption/ionization, tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS). This [...] Read more.
Characterisation of peptides containing intact disulphide bonds (DSBs) via mass spectrometry is challenging. Our study demonstrates that the addition of aniline to alpha-cyano-4-hydroxycinnamic acid improves detection and fragmentation of complex DSB peptides by matrix-assisted laser desorption/ionization, tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS). This improved assignment will be a significant new tool when a simple screening to confirm the DSB existence is required. Full article
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7 pages, 824 KiB  
Technical Note
“Mirror” Method to Estimate Mutagenic Activity of DNA Lesions
by Leonid V. Gening, Oleg V. Shevchenko, Konstantin Y. Kazachenko and Vyacheslav Z. Tarantul
Methods Protoc. 2018, 1(3), 32; https://doi.org/10.3390/mps1030032 - 27 Aug 2018
Cited by 1 | Viewed by 3141
Abstract
We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in [...] Read more.
We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered. Full article
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14 pages, 1243 KiB  
Benchmark
Validation of Total Mercury in Marine Sediment and Biological Samples, Using Cold Vapour Atomic Absorption Spectrometry
by Ahmed Abou Elezz, Hassan Mustafa Hassan, Hamood Abdulla Alsaadi, Ahmed Easa, Saeed Al-Meer, Khaled Elsaid, Zafar Khan Ghouri and Ahmed Abdala
Methods Protoc. 2018, 1(3), 31; https://doi.org/10.3390/mps1030031 - 23 Aug 2018
Cited by 13 | Viewed by 5360
Abstract
A method for the measurement of total mercury (T-Hg) in environmental samples using cold vapour atomic absorption spectrometry (CV AAS) has been validated yielding a dynamic range (0.04–10.00 μg/kg) and high certified reference material (CRM) recovery (>90%). The validation was carried out according [...] Read more.
A method for the measurement of total mercury (T-Hg) in environmental samples using cold vapour atomic absorption spectrometry (CV AAS) has been validated yielding a dynamic range (0.04–10.00 μg/kg) and high certified reference material (CRM) recovery (>90%). The validation was carried out according to International Union of Pure and Applied Chemistry (IUPAC) validation and Eurachem Guides. A freeze-dried and homogenised sample was weighed and then digested using Suprapur acids (HNO3, H2SO4, and HF) with potassium dichromate solution in a hot block digestion system. A calibration curve was constructed (R2 > 0.999). Two CRMs (Marine Sediment Reference Material (PACS-3) and Trace Elements in Muscle Tissue (Trace Elements and Methylmercury in Mussel Tissue (NIST2976)) were utilised for quality assurance and control. The limit of quantification (LOQ) calculated as 0.04 µg/kg, and uncertainty (U) calculated as 2%. The obtained results showed the suitability of this method for direct mercury measurement in environmental samples. Additionally, the proficiency of this method was recognised by accreditation under the standard of International Organization for Standardization (ISO/IEC 17025:2017) for competence of testing and calibration laboratories. Full article
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12 pages, 1776 KiB  
Technical Note
Molecular Analysis of PKU-Associated PAH Mutations: A Fast and Simple Genotyping Test
by Manuela Tolve, Cristiana Artiola, Amelia Pasquali, Teresa Giovanniello, Sirio D’Amici, Antonio Angeloni, Antonio Pizzuti, Claudia Carducci, Vincenzo Leuzzi and Carla Carducci
Methods Protoc. 2018, 1(3), 30; https://doi.org/10.3390/mps1030030 - 16 Aug 2018
Cited by 5 | Viewed by 5073
Abstract
Neonatal screening for phenylketonuria (PKU, OMIM: 261600) was introduced at the end of the 1960s. We developed a rapid and simple molecular test for the most frequent phenylalanine hydroxylase (PAH, Gene ID: 5053) mutations. Using this method to detect the 18 [...] Read more.
Neonatal screening for phenylketonuria (PKU, OMIM: 261600) was introduced at the end of the 1960s. We developed a rapid and simple molecular test for the most frequent phenylalanine hydroxylase (PAH, Gene ID: 5053) mutations. Using this method to detect the 18 most frequent mutations, it is possible to achieve a 75% detection rate in Italian population. The variants selected also reach a high detection rate in other populations, for example, 70% in southern Germany, 68% in western Germany, 76% in Denmark, 68% in Sweden, 63% in Poland, and 60% in Bulgaria. We successfully applied this confirmation test in neonatal screening for hyperphenylalaninemias using dried blood spots and obtained the genotype in approximately 48 h. The method was found to be suitable as second tier test in neonatal screening for hyperphenylalaninemias in neonates with a positive screening test. This test can also be useful for carrier screening because it can bypass the entire coding sequence and intron–exon boundaries sequencing, thereby overcoming the questions that this approach implies, such as new variant interpretations. Full article
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14 pages, 1709 KiB  
Protocol
High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates
by Lea Nussbaum, Jelena M. Telenius, Stephanie Hill, Priscila P. Hirschfeld, Maria C. Suciu, The WIGWAM Consortium, Damien J. Downes and Jim R. Hughes
Methods Protoc. 2018, 1(3), 29; https://doi.org/10.3390/mps1030029 - 1 Aug 2018
Cited by 4 | Viewed by 8354
Abstract
The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array [...] Read more.
The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis. Full article
(This article belongs to the Collection Current Advances and Methodologies in Gene Editing)
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17 pages, 2807 KiB  
Protocol
Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors
by Gemma Moir-Meyer, Pak Leng Cheong, Aude-Anais Olijnik, Jill Brown, Samantha Knight, Andrew King, Ryo Kurita, Yukio Nakamura, Richard J. Gibbons, Douglas R. Higgs, Veronica J. Buckle and Christian Babbs
Methods Protoc. 2018, 1(3), 28; https://doi.org/10.3390/mps1030028 - 30 Jul 2018
Cited by 19 | Viewed by 9792
Abstract
The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells [...] Read more.
The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4–7 fold higher than starting cell numbers within 9–12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations. Full article
(This article belongs to the Collection Current Advances and Methodologies in Gene Editing)
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5 pages, 563 KiB  
Protocol
A Rapid Bacteriophage DNA Extraction Method
by Džiuginta Jakočiūnė and Arshnee Moodley
Methods Protoc. 2018, 1(3), 27; https://doi.org/10.3390/mps1030027 - 27 Jul 2018
Cited by 65 | Viewed by 11611
Abstract
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 [...] Read more.
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. Full article
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16 pages, 1881 KiB  
Article
Mass Spectrometric Analysis of Bisphenol A Desorption from Titania Nanoparticles: Ammonium Acetate, Fluoride, Formate, and Hydroxide as Chemical Desorption Agents
by Seyed Mohammad Majedi and Edward P. C. Lai
Methods Protoc. 2018, 1(3), 26; https://doi.org/10.3390/mps1030026 - 19 Jul 2018
Cited by 8 | Viewed by 5437
Abstract
Bisphenol A (BPA) is a widely used chemical in several consumer products and a well-studied environmental toxicant, and therefore, its accurate measurement is highly demanded. However, the co-presence of nanoparticles as an emerging class of contaminants could result in inaccurate determination of BPA [...] Read more.
Bisphenol A (BPA) is a widely used chemical in several consumer products and a well-studied environmental toxicant, and therefore, its accurate measurement is highly demanded. However, the co-presence of nanoparticles as an emerging class of contaminants could result in inaccurate determination of BPA due to binding of BPA onto nanoparticle surface. In this study, mass spectrometry (MS) was used to investigate desorption of BPA bound on the surface of titania (TiO2) nanoparticles in water. Ammonium acetate, fluoride, formate, and hydroxide were evaluated as chemical agents for their desorption capabilities. The percentages of recovery, adsorption, and desorption were determined by this new method without requiring any prior separation of nanoparticles from BPA. MS analysis demonstrated the desorption of BPA by 10–20 mM of ammonium hydroxide for a mixture of 5 µg/mL BPA and 10 µg/mL TiO2 nanoparticles, with a desorption efficiency of 72 ± 1%. Due to adsorption of BPA onto the nanoparticle surface that was inefficient for electrospray ionization, the resulting abundance of target ions could be reduced in the detection of BPA by mass spectrometry. As such, these findings collectively promise an accurate determination of the total BPA concentration in water whether it exists in the free or bound form. Efficient desorption of contaminants from the surface of nanoparticles would improve the accuracy of the contaminant analysis by mass spectrometry. Full article
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12 pages, 3710 KiB  
Article
An Impact Mapping Method to Generate Robust Qualitative Evaluation of Community-Based Research Programs for Youth and Adults
by Melissa D. Olfert, Rebecca L. Hagedorn, Jade A. White, Barbara A. Baker, Sarah E. Colby, Lisa Franzen-Castle, Kendra K. Kattelmann and Adrienne A. White
Methods Protoc. 2018, 1(3), 25; https://doi.org/10.3390/mps1030025 - 17 Jul 2018
Cited by 11 | Viewed by 5788
Abstract
Ripple Effect Mapping (REM) is an evaluation approach that has traditionally been used in community settings to visually map the impact of programming and community interventions. This manuscript utilizes the Community Capitals Framework (CCF) to inform REM and to better highlight the changes [...] Read more.
Ripple Effect Mapping (REM) is an evaluation approach that has traditionally been used in community settings to visually map the impact of programming and community interventions. This manuscript utilizes the Community Capitals Framework (CCF) to inform REM and to better highlight the changes and impact between various levels of a community, following a childhood obesity prevention intervention. The addition of in-depth qualitative analyses makes this approach particularly useful for the evaluation of interventions with a research–community partnership focus. The objective of this study was to describe a CCF-informed REM approach with detailed protocol, training, and application to the community-based, childhood obesity prevention intervention, iCook 4-H, which targeted youth and adult pairs. This protocol includes the steps required to prepare for REM sessions of, ideally, six youth and adult pairs, one facilitator, and one or two evaluators/note takers. REM sessions typically begin with an icebreaker and appreciative inquiry activities that inform the REM mapping process that follows. In-depth qualitative analysis of the notes and map images captured during REM sessions ensure the rigor required for research-related interventions. Researchers, community members, and participants can use CCF-informed REM collectively as a robust evaluation tool to demonstrate, through visual mapping, the positive effects of community-partnered research programs. Full article
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13 pages, 2467 KiB  
Benchmark
A Novel Oligonucleotide Pair for Genotyping Members of the Pseudomonas Genus by Single-Round PCR Amplification of the gyrB Gene
by Betina Cecilia Agaras and Claudio Valverde
Methods Protoc. 2018, 1(3), 24; https://doi.org/10.3390/mps1030024 - 2 Jul 2018
Cited by 11 | Viewed by 4750
Abstract
Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that [...] Read more.
Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species. Full article
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