2 Metagenome Analysis
41
2.3.3 Isolation and Purification of Genomic DNA
The method applied for the isolation of metagenomic DNA depends largely on
the downstream metagenomic analysis. Metagenomic DNA for library independent
analysis as well as small insert and large insert shotgun libraries can be retrieved
by “liquid phase-based” methods. This means, that the environmental sample is
directly mixed with an appropriate lysis buffer. The maximum fragment size generally obtained is about 150–200 bp. The method is hardly suitable for the construction
of BAC libraries with average insert sizes exceeding 50 kbp (Rondon et al. 2000,
MacNeil et al. 2001). For the generation of BAC libraries, the environmental samples are generally embedded in a matrix, such as agarose, prior to cell lysis. This
prevents excessive shearing of the DNA.
The “liquid phase-based” method is the most straightforward DNA isolation
technique. It is applicable to nearly every environmental sample. One of the commercially available DNA isolation kits can be used for this purpose. However, if
silica-based purification kits are used, this is often at the expense of a smaller average fragment size and a lower yield of DNA. If larger fragment sizes for large
insert library construction are needed or the sample is limited, other protocols are
preferable. One such protocol was published by Zhou et al. (1996). Cells are lysed
in a high salt buffer containing Proteinase K, cetyl trimethyl ammonium bromide
(CTAB), and sodium dodecyl sulfate (SDS). The lysis of gram-positive bacteria is
enhanced by several freeze and thaw cycles. DNA extracted from plankton samples may be used directly for downstream sequencing or library construction. DNA
from other samples, such as sediment samples, may need further purification in
order to be suitable for downstream enzymatic manipulation. This purification can
be accomplished by gel electrophoresis (Rondon et al. 2000, Quaiser et al. 2003),
anion exchange chromatography (Krüger et al. 2003), density gradient centrifugation (MacNeil et al. 2001, Courtois et al. 2003), or by using commercially available
kits.
Isolation of high quality genomic DNA fragments larger than 200 kbp is more
difficult. DNA fragments of more than 100 kbp are easily sheared by pipetting or
sample mixing. Therefore, the sample needs to be embedded in agarose, and dialysed against different buffers supplemented with enzymes and detergents to remove
proteins and lipids from the embedded cells, leaving naked DNA behind (Green
et al. 1997, Sambrook and Russel 2001). After these treatments, plankton samples
can be used directly for BAC library construction (Beja et al. 2000). The use of
this method with other, more contaminated environmental samples, such as sediment samples, resulted in agarose plugs with partly degraded genomes and enzyme
inhibitors, which could not be removed by the lysis procedure. Further purification of the DNA, for example by electrophoresis through conventional agarose gels
or two-phase agarose gels containing polyvinylpyrrolidone (Quaiser et al. 2002),
or dialysis against a high salt and formamide containing buffer (Liles et al. 2008),
was necessary in these cases. However, this procedure often leads to a reduction
in quantity and shearing of the DNA, which may be afterwards inadequate for the
construction of large insert BAC libraries.
41
2.3.3 Isolation and Purification of Genomic DNA
The method applied for the isolation of metagenomic DNA depends largely on
the downstream metagenomic analysis. Metagenomic DNA for library independent
analysis as well as small insert and large insert shotgun libraries can be retrieved
by “liquid phase-based” methods. This means, that the environmental sample is
directly mixed with an appropriate lysis buffer. The maximum fragment size generally obtained is about 150–200 bp. The method is hardly suitable for the construction
of BAC libraries with average insert sizes exceeding 50 kbp (Rondon et al. 2000,
MacNeil et al. 2001). For the generation of BAC libraries, the environmental samples are generally embedded in a matrix, such as agarose, prior to cell lysis. This
prevents excessive shearing of the DNA.
The “liquid phase-based” method is the most straightforward DNA isolation
technique. It is applicable to nearly every environmental sample. One of the commercially available DNA isolation kits can be used for this purpose. However, if
silica-based purification kits are used, this is often at the expense of a smaller average fragment size and a lower yield of DNA. If larger fragment sizes for large
insert library construction are needed or the sample is limited, other protocols are
preferable. One such protocol was published by Zhou et al. (1996). Cells are lysed
in a high salt buffer containing Proteinase K, cetyl trimethyl ammonium bromide
(CTAB), and sodium dodecyl sulfate (SDS). The lysis of gram-positive bacteria is
enhanced by several freeze and thaw cycles. DNA extracted from plankton samples may be used directly for downstream sequencing or library construction. DNA
from other samples, such as sediment samples, may need further purification in
order to be suitable for downstream enzymatic manipulation. This purification can
be accomplished by gel electrophoresis (Rondon et al. 2000, Quaiser et al. 2003),
anion exchange chromatography (Krüger et al. 2003), density gradient centrifugation (MacNeil et al. 2001, Courtois et al. 2003), or by using commercially available
kits.
Isolation of high quality genomic DNA fragments larger than 200 kbp is more
difficult. DNA fragments of more than 100 kbp are easily sheared by pipetting or
sample mixing. Therefore, the sample needs to be embedded in agarose, and dialysed against different buffers supplemented with enzymes and detergents to remove
proteins and lipids from the embedded cells, leaving naked DNA behind (Green
et al. 1997, Sambrook and Russel 2001). After these treatments, plankton samples
can be used directly for BAC library construction (Beja et al. 2000). The use of
this method with other, more contaminated environmental samples, such as sediment samples, resulted in agarose plugs with partly degraded genomes and enzyme
inhibitors, which could not be removed by the lysis procedure. Further purification of the DNA, for example by electrophoresis through conventional agarose gels
or two-phase agarose gels containing polyvinylpyrrolidone (Quaiser et al. 2002),
or dialysis against a high salt and formamide containing buffer (Liles et al. 2008),
was necessary in these cases. However, this procedure often leads to a reduction
in quantity and shearing of the DNA, which may be afterwards inadequate for the
construction of large insert BAC libraries.