TY - JOUR
T1 - Comparison of six methods for the recovery of PCR-compatible microbial DNA from an agricultural biogas plant
AU - Stagnati, Lorenzo
AU - Soffritti, Giovanna
AU - Lanubile, Alessandra
AU - Busconi, Matteo
PY - 2017
Y1 - 2017
N2 - Six different commercial methods were compared to evaluate their efficiency in recovering high quantity/quality
PCR compatible microbial DNA from an agricultural biogas plant. Within the last two decades, biogas plants have been
developed to produce energy from organic wastes and from devoted biomass. The complex biotransformations are performed
by a diverse consortium of microorganisms that is an important reserve of genes and enzymatic activities with a
huge range of applications in various commercial fields. In this respect, the ability to isolate DNA from a complex matrix
is of high importance. Important parameters of the recovered DNA are good yield, purity, and quality. The methods examined
showed considerable differences about quantity and quality of the recovered DNA and, usually, it was observed that a
higher amount was accompanied by more degradation. DNA purity was determined by its PCR amplificability. Only two
methods were able to provide DNA pure enough to be directly amplified. For the rest of the methods, a few intermediate
steps such as dilution and/or the addition of polyvinylpyrrolidone were necessary to remove the inhibitors present and to
amplify the DNA. Real-time PCR analysis evidenced that, as expected, prokaryotic DNA was much more abundant than
eukaryotic DNA, but some methods were more suited to recovering prokaryotic or eukaryotic DNA. The digestion
analysis of ribosomal DNA amplicons confirmed the influence of the methods on the final output, allowing the recovery
of only a fraction of the present species as determined by sequencing a small prokaryotic and eukaryotic ribosomal
library.
AB - Six different commercial methods were compared to evaluate their efficiency in recovering high quantity/quality
PCR compatible microbial DNA from an agricultural biogas plant. Within the last two decades, biogas plants have been
developed to produce energy from organic wastes and from devoted biomass. The complex biotransformations are performed
by a diverse consortium of microorganisms that is an important reserve of genes and enzymatic activities with a
huge range of applications in various commercial fields. In this respect, the ability to isolate DNA from a complex matrix
is of high importance. Important parameters of the recovered DNA are good yield, purity, and quality. The methods examined
showed considerable differences about quantity and quality of the recovered DNA and, usually, it was observed that a
higher amount was accompanied by more degradation. DNA purity was determined by its PCR amplificability. Only two
methods were able to provide DNA pure enough to be directly amplified. For the rest of the methods, a few intermediate
steps such as dilution and/or the addition of polyvinylpyrrolidone were necessary to remove the inhibitors present and to
amplify the DNA. Real-time PCR analysis evidenced that, as expected, prokaryotic DNA was much more abundant than
eukaryotic DNA, but some methods were more suited to recovering prokaryotic or eukaryotic DNA. The digestion
analysis of ribosomal DNA amplicons confirmed the influence of the methods on the final output, allowing the recovery
of only a fraction of the present species as determined by sequencing a small prokaryotic and eukaryotic ribosomal
library.
KW - DNAextractionmethods compariso, PCR-grade DNA, Inhibitors, Prokaryotic/eukaryoticDNAratio, Metagenomics
KW - DNAextractionmethods compariso, PCR-grade DNA, Inhibitors, Prokaryotic/eukaryoticDNAratio, Metagenomics
UR - http://hdl.handle.net/10807/102973
U2 - 10.1007/s00253-017-8152-5
DO - 10.1007/s00253-017-8152-5
M3 - Article
SN - 0175-7598
VL - 101
SP - 3907
EP - 3917
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
ER -