scholarly journals The Effect of DNA Extraction Methods on Observed Microbial Communities from Fibrous and Liquid Rumen Fractions of Dairy Cows

2018 ◽  
Vol 9 ◽  
Author(s):  
Jueeli D. Vaidya ◽  
Bartholomeus van den Bogert ◽  
Joan E. Edwards ◽  
Jos Boekhorst ◽  
Sanne van Gastelen ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Dna Extraction Methods

scholarly journals Effect of DNA Extraction Methods on the Apparent Structure of Yak Rumen Microbial Communities as Revealed by 16S rDNA Sequencing

2015 ◽  
Vol 64 (1) ◽  
pp. 29-36 ◽  
Author(s):  
YA-BING CHEN ◽  
DAO-LIANG LAN ◽  
CHENG TANG ◽  
XIAO-NONG YANG ◽  
JIAN LI
Keyword(s):  
Microbial Community ◽  
Bacterial Community ◽  
Microbial Communities ◽  
16S Rdna ◽  
Dna Extraction ◽  
Extraction Methods ◽  
16S Rdna Sequencing ◽  
Bead Beating ◽  
Rdna Sequencing ◽  
Dna Extraction Methods

To more efficiently identify the microbial community of the yak rumen, the standardization of DNA extraction is key to ensure fidelity while studying environmental microbial communities. In this study, we systematically compared the efficiency of several extraction methods based on DNA yield, purity, and 16S rDNA sequencing to determine the optimal DNA extraction methods whose DNA products reflect complete bacterial communities. The results indicate that method 6 (hexadecyltrimethylammomium bromide-lysozyme-physical lysis by bead beating) is recommended for the DNA isolation of the rumen microbial community due to its high yield, operational taxonomic unit, bacterial diversity, and excellent cell-breaking capability. The results also indicate that the bead-beating step is necessary to effectively break down the cell walls of all of the microbes, especially Gram-positive bacteria. Another aim of this study was to preliminarily analyze the bacterial community via 16S rDNA sequencing. The microbial community spanned approximately 21 phyla, 35 classes, 75 families, and 112 genera. A comparative analysis showed some variations in the microbial community between yaks and cattle that may be attributed to diet and environmental differences. Interestingly, numerous uncultured or unclassified bacteria were found in yak rumen, suggesting that further research is required to determine the specific functional and ecological roles of these bacteria in yak rumen. In summary, the investigation of the optimal DNA extraction methods and the preliminary evaluation of the bacterial community composition of yak rumen support further identification of the specificity of the rumen microbial community in yak and the discovery of distinct gene resources.

scholarly journals Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities

PLoS ONE ◽  
2013 ◽  
Vol 8 (9) ◽  
pp. e74787 ◽  
Author(s):  
Gemma Henderson ◽  
Faith Cox ◽  
Sandra Kittelmann ◽  
Vahideh Heidarian Miri ◽  
Michael Zethof ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Dna Extraction ◽  
Extraction Methods ◽  
Sampling Techniques ◽  
Sheep Rumen ◽  
Dna Extraction Methods

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.

Sign in / Sign up

Export Citation Format

Share Document