Metagenomic Libraries for Functional Screening

Improved cultivation and metagenomics as new tools for bioprospecting in cold environments

“…The sequence-based approach includes screening for genes by hybridization with labeled DNA probes or by PCR, both of which are based on sequences of known genes (Aakvik et al 2011 ; Simon and Daniel 2011 ; Lee and Lee 2013 ). Since the price for direct high-throughput sequencing of metagenomic DNA has reached a level affordable for many laboratories (de Pascale et al 2012 ; Hunter et al 2012 ), this is now the standard technology for gene discovery (Ekkers et al 2012 ).…”

“…For both enrichment cultures and pure isolates, isolation of mRNA is more feasible than for complex environmental samples. Also, if eukaryotic genes are of interest for a functional bioprospecting approach using a prokaryotic host for expression, cDNA is necessary to circumvent problems introduced by introns in gDNA (Aakvik et al 2011 ). Direct extraction of DNA from environmental samples involves the in situ lysis of cells within the sample matrix and DNA extraction, whereas indirect sampling involves the isolation of cells from the sample matrix before ex situ lysis.…”

“…Direct extraction can result in co-extraction of inhibitory substances such as humic acids from soil or the matrix may itself adsorb the DNA. Unwanted DNA such as eukaryotic or extracellular DNA can also constitute a significant part of the extracted DNA (Kakirde et al 2010a ; Aakvik et al 2011 ; Williamson et al 2011 ; Akondi and Lakshmi 2013 ; Lee and Lee 2013 ). These problems can be circumvented by indirect extraction.…”

“…Bias is also introduced by the choice of lysis and DNA extraction method and it is likely that an accurate representation of the community will never be achieved. In general, it is advisable to apply multiple extraction methods, and when working with environments with low biomass, as is the case for many extreme environments, DNA yields are often low (Aakvik et al 2011 ; Vester et al 2014a , b ). To obtain sufficient amounts of DNA for further analyses, MDA with the φ29 DNA polymerase can be applied (Spits et al 2006 ).…”

“…To obtain sufficient amounts of DNA for further analyses, MDA with the φ29 DNA polymerase can be applied (Spits et al 2006 ). MDA is, however, heavily biased and will not generate quantitative results (Yilmaz et al 2010 ; Vester et al 2014b ) and there is a risk of introducing point mutations (Aakvik et al 2011 ).…”