scholarly journals Biased nicking of mitochondrial DNA during extraction can be avoided by choice of isolation method

2021 ◽  
Author(s):  
Bruno Marçal Repolês ◽  
Choco Michael Gorospe ◽  
Phong Tran ◽  
Anna Karin Nilsson ◽  
Paulina H. Wanrooij
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Dna ◽  
Dna Isolation ◽  
Isolation Procedure ◽  
Isolation Method ◽  
Strand Breaks ◽  
Careful Choice ◽  
Isolation Methods ◽  
Solid Tissues ◽  
Long Range Pcr

AbstractThe integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR. We show that a commonly-used DNA isolation procedure preferentially introduces strand breaks into the mtDNA extracted from the skeletal muscle of aged mice, while mtDNA from adult animals is less affected. We present a comparison of mtDNA isolation methods and identify one that avoids this biased loss of muscle mtDNA integrity. Our results highlight the importance of a careful choice of mtDNA isolation method and serve as a resource to researchers planning analysis of mtDNA isolated from solid tissues.

scholarly journals DEVELOPMENT OF DNA BIOSENSOR BASED ON SILVER NANOPARTICLES UV-Vis ABSORPTION SPECTRA FOR Escherichia coli DETECTION

2015 ◽  
Vol 2 (1) ◽  
pp. 382 ◽  
Author(s):  
Ruth Chrisnasari ◽  
Antonius Loren Wijaya ◽  
Maria Goretti Marianti Purwanto
Keyword(s):  
Escherichia Coli ◽  
Silver Nanoparticles ◽  
Absorption Spectra ◽  
Magnetic Beads ◽  
Dna Isolation ◽  
Dna Biosensor ◽  
Isolation Method ◽  
Isolation Methods ◽  
Spectra Properties ◽  
Magnetic Field Condition

<p>In this research we reported the synthesis of oligonucleotide-silver nanoparticle (OSN) conjugates and demonstrated their use along with magnetic beads as biosensor for Escherichia coli detection under magnetic field condition. Oligonucleotide DNA probes were conjugated on silver nanoparticles using alkanethiols linker. Two kinds of alkanethiols linker, 11-mercaptoundodecanoic acid (11-MUDA) and 16-mercaptophexadecanoic acid (16-MHDA) were compared to get the best probe conjugation yield and OSN UV-Vis absorption spectra properties. Three different methods of Escherichia coli DNA isolation i.e. Chen and Kuo (1993), Phenol Chloroform Isoamylalcohol (PCI) extraction and boiling lysis were also compared to explore the performance of the biosensor towards the DNA target purity. Detection process through hybridization between the DNA probe and the target was carried out at 55oC for 1 hour incubation time. The results showed that 16-MHDA gave higher conjugation yield and higher OSN UV-Vis absorption spectra than 11-MUDA. The biosensor was able to detect the presence of the DNA target which was isolated from the three isolation methods. The best detection signal was achieved by Chen and Kuo isolation method in which it could detect the presence of the DNA target up to 1.3 ng/µL.</p><p><br /><strong>Keywords</strong>: DNA biosensor, Silver Nanoparticles, Escherichia coli</p>

scholarly journals Performance evaluation of a new custom, multi-component DNA isolation method optimized for use in shotgun metagenomic sequencing-based aerosol microbiome research

2019 ◽  
Author(s):  
Kari Oline Bøifot ◽  
Jostein Gohli ◽  
Line Victoria Moen ◽  
Marius Dybwad
Keyword(s):  
Microbial Community ◽  
Real World ◽  
Dna Isolation ◽  
New Method ◽  
Metagenomic Sequencing ◽  
Isolation Method ◽  
Air Samples ◽  
Shotgun Metagenomic Sequencing ◽  
Isolation Methods ◽  
Microbiome Research

ABSTRACTBackgroundAerosol microbiome research advances our understanding of bioaerosols, including how airborne microorganisms affect our health and surrounding environment. Traditional microbiological/molecular methods are commonly used to study bioaerosols, but do not allow for generic, unbiased microbiome profiling. Recent studies have adopted shotgun metagenomic sequencing (SMS) to address this issue. However, SMS requires relatively large DNA inputs, which are challenging when studying low biomass air environments, and puts high requirements on air sampling, sample processing and DNA isolation protocols. Previous SMS studies have consequently adopted various mitigation strategies, including long-duration sampling, sample pooling, and whole genome amplification, each associated with some inherent drawbacks/limitations.ResultsHere, we demonstrate a new custom, multi-component DNA isolation method optimized for SMS-based aerosol microbiome research. The method achieves improved DNA yields from filter-collected air samples by isolating DNA from the entire filter extract, and ensures unbiased microbiome representation by combining chemical, enzymatic and mechanical lysis. Benchmarking against two state-of-the-art DNA isolation methods was performed with a mock microbial community and real-world subway air samples. All methods demonstrated similar performance regarding DNA yield and community representation with the mock community. However, with subway air samples, the new method obtained drastically improved DNA yields, while SMS revealed that the new method reported higher diversity and gave better taxonomic coverage. The new method involves intermediate filter extract separation into a pellet and supernatant fraction. Using subway air samples, we demonstrate that supernatant inclusion results in improved DNA yields. Furthermore, SMS of pellet and supernatant fractions revealed overall similar taxonomic composition but also identified differences that could bias the microbiome profile, emphasizing the importance of processing the entire filter extract.ConclusionsBy demonstrating and benchmarking a new DNA isolation method optimized for SMS-based aerosol microbiome research with both a mock microbial community and real-world air samples, this study contributes to improved selection, harmonization, and standardization of DNA isolation methods. Our findings highlight the importance of ensuring end-to-end sample integrity and using methods with well-defined performance characteristics. Taken together, the demonstrated performance characteristics suggest the new method could be used to improve the quality of SMS-based aerosol microbiome research in low biomass air environments.

Rapid Mitochondrial DNA Isolation Method for Direct Sequencing

2015 ◽  
pp. 89-95 ◽  
Author(s):  
Wilber Quispe-Tintaya ◽  
Ryan R. White ◽  
Vasily N. Popov ◽  
Jan Vijg ◽  
Alexander Y. Maslov
Keyword(s):  
Mitochondrial Dna ◽  
Dna Isolation ◽  
Direct Sequencing ◽  
Isolation Method

scholarly journals Genetic of Salak Pondoh, Gading Varieties and Its Hybrids Based on RAPD Markers

2021 ◽  
Vol 1 (1) ◽  
pp. 5
Author(s):  
Nandariyah Nandariyah ◽  
Parjanto Parjanto ◽  
Pinaka Pinasti Ratu
Keyword(s):  
Molecular Marker ◽  
Rapd Markers ◽  
Dna Isolation ◽  
Dna Amplification ◽  
Isolation Method ◽  
Isolation Methods

<p>A molecular marker of parent and offspring is used to find fast and accurate markers influenced by DNA isolation and amplification. This research aims to find the most suitable DNA isolation and DNA  amplification methods. This study used four DNA isolation methods; namely IM01, IM02, IM03, and IM04. DNA amplification used ten protocols (AP01, AP02, AP03, AP04, AP05, AP06, AP07, AP08, AP09, and AP010). The results of the research showed that the most suitable DNA isolation method for salak was  IM0, and the most suitable DNA amplification for salak was AP04 that produces the highest value of DNA bands.</p><p><strong> </strong></p><p>Keywords: DNA isolation; DNA amplification; hybrids.</p>

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