2 Metagenome Analysis
49
or glycerol (Epicentre Biotechnologies 2007). Also, the recombinant cells may be
stored in glycerol at –80 ◦ C immediately after transformation.
The method used for long-term storage of plated libraries depends on many factors: the uniqueness of the sample, the library size, and the method chosen for the
subsequent screening. The storage of several copies of a library is generally recommended. The libraries can be stored at –80 ◦ C in pools of recombinant clones that
have been washed off their agar plates using culture medium containing cryoprotectant (e.g. 5–7% glycerol). In cases where approaches such as insert-end sequencing
are planned, picking and arraying of individual clones is generally conducted.
2.3.5.5 Screening of Metagenomic Libraries
Metagenomic libraries can either be screened by sequence-based or function-based
approaches.
Sequence-based approaches include (i) PCR-screening, (ii) hybridisation, and
(iii) insert-end sequencing. PCR-screening is the fastest method to screen a library,
especially, when DNA pools or pools of recombinant clones have been prepared.
Examples for complex pooling schemes have already been published (Kim et al.
1996, Asakawa et al. 1997). Several strategies have been developed to prevent
amplification of the host’s chromosomal DNA by cross-reacting primers. In one
study, the chromosomal DNA of the host was selectively hydrolysed with an ATPdependent DNase prior to PCR-screening (Beja et al. 2000, Liles et al. 2003).
Host specific, terminally modified oligonucleotides have also been included in the
PCR reaction (Goodman and Liles 2001, Liles et al. 2003). Another study added
restriction fragment length polymorphism (RFLP) analysis to the PCR protocol in
order to identify positive clones (Liles et al. 2003). Screening by hybridisation has
been performed using colony blots (Asakawa et al. 1997, Osoegawa et al. 2000),
high density DNA arrays (Rondon et al. 1999), and microarrays (Park et al. 2008).
Finally, insert-end sequences can be determined to find certain genes (Kube et al.
2005) or overlapping clones for genome walking (Meyerdierks et al. 2005), and also
to characterise and compare microbial assemblages (DeLong et al. 2006).
Function-based screening is based on heterologous expression of the cloned
genes by the host cell. This approach is predominantly applied to identify enzymes
for biotechnological purposes. If the transcription and translation machinery of the
host and donor strain are incompatible, shuttle vectors may be used to transfer the
library into a different host strain (Handelsman et al. 2002, Riesenfeld et al. 2004).
2.3.6 Library Independent Metagenome Analysis
The dideoxynucleotide-based DNA sequencing method was for decades the standard method for DNA sequencing. Improvements to this method, which was first
described by Sanger et al. (1977), have included the use of fluorescent- instead of
radioactive-labelled dideoxynucleotides as chain terminators (Prober et al. 1987).
The use of this method in (meta)genome sequencing requires the construction of
49
or glycerol (Epicentre Biotechnologies 2007). Also, the recombinant cells may be
stored in glycerol at –80 ◦ C immediately after transformation.
The method used for long-term storage of plated libraries depends on many factors: the uniqueness of the sample, the library size, and the method chosen for the
subsequent screening. The storage of several copies of a library is generally recommended. The libraries can be stored at –80 ◦ C in pools of recombinant clones that
have been washed off their agar plates using culture medium containing cryoprotectant (e.g. 5–7% glycerol). In cases where approaches such as insert-end sequencing
are planned, picking and arraying of individual clones is generally conducted.
2.3.5.5 Screening of Metagenomic Libraries
Metagenomic libraries can either be screened by sequence-based or function-based
approaches.
Sequence-based approaches include (i) PCR-screening, (ii) hybridisation, and
(iii) insert-end sequencing. PCR-screening is the fastest method to screen a library,
especially, when DNA pools or pools of recombinant clones have been prepared.
Examples for complex pooling schemes have already been published (Kim et al.
1996, Asakawa et al. 1997). Several strategies have been developed to prevent
amplification of the host’s chromosomal DNA by cross-reacting primers. In one
study, the chromosomal DNA of the host was selectively hydrolysed with an ATPdependent DNase prior to PCR-screening (Beja et al. 2000, Liles et al. 2003).
Host specific, terminally modified oligonucleotides have also been included in the
PCR reaction (Goodman and Liles 2001, Liles et al. 2003). Another study added
restriction fragment length polymorphism (RFLP) analysis to the PCR protocol in
order to identify positive clones (Liles et al. 2003). Screening by hybridisation has
been performed using colony blots (Asakawa et al. 1997, Osoegawa et al. 2000),
high density DNA arrays (Rondon et al. 1999), and microarrays (Park et al. 2008).
Finally, insert-end sequences can be determined to find certain genes (Kube et al.
2005) or overlapping clones for genome walking (Meyerdierks et al. 2005), and also
to characterise and compare microbial assemblages (DeLong et al. 2006).
Function-based screening is based on heterologous expression of the cloned
genes by the host cell. This approach is predominantly applied to identify enzymes
for biotechnological purposes. If the transcription and translation machinery of the
host and donor strain are incompatible, shuttle vectors may be used to transfer the
library into a different host strain (Handelsman et al. 2002, Riesenfeld et al. 2004).
2.3.6 Library Independent Metagenome Analysis
The dideoxynucleotide-based DNA sequencing method was for decades the standard method for DNA sequencing. Improvements to this method, which was first
described by Sanger et al. (1977), have included the use of fluorescent- instead of
radioactive-labelled dideoxynucleotides as chain terminators (Prober et al. 1987).
The use of this method in (meta)genome sequencing requires the construction of