To extract bacterial DNA for the creation of clone libraries (metagenome projects; Rondon et al. 2000; Lorenz and Schleper 2002), it is desirable to achieve high molecular weight and unbiased DNA (Lorenz and Schleper 2002; Berry et al. 2003). The necessity of large DNA inserts is particularly important when using vectors which can carry large (100 kb) inserts (BAC, Cosmid) which are used to construct libraries suitable for sequence homology-based screening. Thus, Ginolhac et al. (2004) use a DNA extraction protocol which ensures a minimum of physical DNA shearing; cells are first extracted from the soil (on Nycodenz), then mounted in an agarose gel plug prior to lysis (by lysozyme and achromopeptidase) and extraction (100 mM EDTA, 1% lauryl sarcosyl, proteinase K).
However, even for libraries used for functional screening (using plasmids or lambda vectors with strong promoters), the size of the insert will affect the chances of expression (Gabor et al. 2004). Lee et al. (2004) used the > 25 kb size fraction of soil DNA (extracted by the freeze/thaw enzymatic method; Zhou et al. 1996) to construct a metagenome library (in Escherichia coli with acosmidvector), which wasscreenedfor lipolyticactivity. Gaboret al. (2003) used somewhat smaller inserts (4-6 kb, prepared by mechanical shearing of soil DNA) inserted with the pZero-2 vector (Invitrogen) to create a gene bank (E. coli) screened for novel amidases.
Based on the foregoing discussion of cell lysis, it appears practically impossible to achieve an unbiased DNA extract from entire communities without shearing the DNA. The conflict between efficient lysis and minimum shearing was demonstrated by Kauffmann et al. (2004) who compared two extraction methods, the method of Zhou et al. (1996; freeze thaw, proteinase K and SDS) and the bead beating procedure of Moré et al. (1994; bead beating, SDS), for extraction of DNA from a Gram-negative (Pseudomonas LU2023) and two Gram-positive species (Arthrobacter LU9144, Rhodococcus LU9002). Pseudomonas DNA was efficiently extracted with both methods but the bead beating resulted in substantially more shearing of its DNA. In contrast, bead beating was the only method able to lyse and extract DNA from the Gram-positive cells. By using the two methods in sequence (bead beating extraction of cell remaining after Zhou extraction), they achieved two "biased" DNA extracts, one dominated by high molecular weight DNA from Pseudomonas (after Zhou extraction), and one dominated by DNA from the Gram-positive cells (bead beating extraction of the cells remaining after the Zhou extraction).
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