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The Impact of Multiple Displacement Amplification on Microbial Ecology

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The Impact of Multiple Displacement Amplification on Microbial Ecology The ISME Journal (2008) 2, 233–241 & 2008 International Society for Microbial Ecology All rights reserved 1751-7362/08 $30.00 www.nature.com/ismej MINI REVIEW Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology Erik K Binga1, Roger S Lasken2 and Josh D Neufeld1 1Department of Biology, University of Waterloo, Waterloo, Ontario, Canada and 2J. Craig Venter Institute, La Jolla, CA, USA Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a ‘power tool’ in the molecular toolbox of microbial ecologists. The ISME Journal (2008) 2, 233–241; doi:10.1038/ismej.2008.10; published online 7 February 2008 Subject Category: microbial population and community ecology Keywords: metagenomics; microbial ecology; multiple displacement amplification; phi29 DNA polymerase; whole genome amplification; single-cell microbiology Introduction the ‘rare biosphere’ (Sogin et al., 2006) contain enzyme-encoding genes that hold great promise for Environmental microbial diversity is poorly under- medicine, biotechnology and industry. Fortunately stood and the majority of microbes are inaccessible for microbial ecologists in the early 21st century, by laboratory cultivation. As a result, microbial small yields of DNA from low-biomass communities ecology has sought to develop molecular methods to or individual uncultivated cells are readily retrieved characterize whole communities. The extraction of by the advent of WGA via the multiple displacement community nucleic acids is a typical initial step, amplification (MDA) reaction. Here we highlight the and although cell biomass is often sufficiently high history of MDA and discuss its mechanism, cap- to enable analysis by PCR, gene hybridization or abilities and limitations. The applications of MDA metagenomics, this is not always the case. Because in microbial ecology are reviewed for accessing low- microbial ecologists study challenging environ- abundance DNA from cells or environmental sam- ments, such as insect guts (Broderick et al., 2004), ples. Finally, we discuss the future of MDA and its ice cores (Christner et al., 2001), permafrost (Steven potential integration into additional facets of micro- et al., 2006), deep subsurface sediments (Teske, bial ecology research. 2005) and air (Brodie et al., 2007), high-sensitivity PCR protocols have been required for the analysis of single genes. A whole genome amplification (WGA) A brief history of MDA step provides access to community DNA from these low-biomass environments. In addition, the vast Initial WGA reactions utilized PCR-based techni- number of uncultivated organisms associated with ques such as degenerate oligonucleotide primed PCR (Telenius et al., 1992) and primer extension PCR (Zhang et al., 1992). However, these were Correspondence: JD Neufeld, Department of Biology, University limited by nonspecific artifacts of amplification of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1. (Cheung and Nelson, 1996), strong bias (Paunio E-mail: [email protected] et al., 1996) and short amplification products Published online 7 February 2008 (Telenius et al., 1992; Zhang et al., 1992; Paunio Multiple displacement amplification and microbial ecology EK Binga et al 234 et al., 1996). MDA was the first WGA method based mely high processivity, adding an average of 70 000 on an isothermal reaction (Dean et al., 2001). nucleotides each time it binds the primer template Double-stranded DNA template is initially dena- (Blanco et al., 1989). Use of single-stranded M13 tured before incubating at 30 1C for 2–16 h, depend- DNA as template gave a rolling-circle mode of ing on the amount of starting template and the replication, in which the polymerase repeatedly commercial MDA kit being used (Table 1). MDA copied around the circular template via its strand utilizes target DNA template, buffer, dNTPs, random displacement activity, yielding a product of con- phosphorothioate-modified hexamers and DNA catenated M13 repeats. The DNA polymerase’s polymerase, which is commonly derived from the associated 30–50 exonuclease proofreading activity Bacillus subtilis bacteriophage phi29. The 30 phos- results in a low intrinsic error rate of 10À6–10À7 phorothioate modifications are required for amplifi- (Watabe et al., 1984; Blanco and Salas, 1985) and the cation, as they render the hexamers resistant to the accumulation of mutations in MDA products at a 30–50 exonuclease proofreading activity of the poly- merase (Dean et al., 2001). The phi29 DNA poly- merase extends the random primers and its strong 3’ 5’ ‘strand displacement activity’ allows it to displace existing primer-originated extensions downstream (Figure 1). Continued priming and strand displace- ment generates a branched structure and gives exponential DNA amplification. The reaction yields double-stranded linear DNA, single-stranded forms and some remaining branched intermediate struc- 3’ 5’ tures (Figure 1). Complete denaturation of MDA products and subsequent resolution on denaturing alkaline agarose gels revealed a 12 kb average length of the resulting single-stranded amplified DNA (Dean et al., 2002). The reaction is terminated by heating to 65 1C for 10 min, denaturing the polymerase, and yields up to 40 mg of DNA per 50-ml reaction. 3’ 5’ The phi29 DNA polymerase was first isolated by Figure 1 Sequential diagrammatic representation of branched Blanco and Salas (1984) from Escherichia coli cells structures formed during multiple displacement amplification expressing the P2 gene, which encodes the sole (MDA). The arrowheads represent the location of the phi29 phi29-associated DNA polymerase. It has an extre- polymerase synthesizing DNA in a 50 to 30 direction. Table 1 Several commercially available kits for multiple displacement amplification of DNA Provider Kit Intended template for amplification Incubation Volume (ml) Time (h) Yield (mg) GE Healthcare TempliPhi 100/500 Small circular DNA (for example, 10 4–6 1–1.5 (Baie d’Urfe´, plasmids) Quebec, Canada) GE Healthcare TempliPhi Large Large circular DNA (for example, ND 18 4.5–5 Construct BACs, cosmids and fosmids) GE Healthcare TempliPhi HT Small circular DNA (for example, 20 18 3.5–4 plasmids) GE Healthcare TempliPhi Circular templates containing 10 18 1 Sequence Resolver ‘difficult’ sequence (for example, repeats, inverted sequences and compressions) GE Healthcare GenomiPhi V2 Linear DNA 20 1.5–2 4–7 GE Healthcare GenomiPhi HY Linear DNA (high yield) 50 4 40–50 Qiagen REPLI-g Mini Linear and circular DNA 50 10–16 7–10 (Mississauga, Ontario, Canada) Qiagen REPLI-g Ultrafast Linear and circular DNA 20 1–1.5 7–10 Mini Qiagen REPLI-g Midi Linear and circular DNA 50 8–16 40 Epicentre RepliPHI Customizable amplification ND ND ND (Madison, of linear DNA Wisconsin, USA) Abbreviation: ND: not defined in the manufacturer’s protocol. The ISME Journal Multiple displacement amplification and microbial ecology EK Binga et al 235 rate of only 10À5–10À6 (Esteban et al., 1993; Nelson 3- and 6-fold, respectively (Wu et al., 2006). Other et al., 2002), nearly 1000-fold less than for PCR studies also showed increased bias with decreased using the Taq DNA polymerase (Dunning et al., template copy number (Detter et al., 2002; Bergen 1988; Saiki et al., 1988). et al., 2005; Holbrook et al., 2005; Kalyuzhnaya The first use of MDA for amplifying whole et al., 2006; Neufeld et al., 2008). genomes targeted human DNA (Dean et al., 2002) Chimera formation and amplification bias are and as few as 90 copies of the genome (0.3 ng DNA) concerns for genomic sequencing of DNA generated yielded more than 30 mg of product. For eight loci from MDA. Zhang et al. (2006) discovered that assessed, amplification bias (over- and underrepre- cloned MDA products included up to 50% chimeric sentation of sequences) was several orders of inserts (3 kb average length). They postulated (in- magnitude less than for the PCR-based WGA (Dean correctly) that chimeras were generated by the et al., 2002). Since then, MDA has been applied to cloning step, with E. coli rearranging the cloned many more eukaryotic studies, including the geno- and branched DNA. Remarkably, chimeras were mic sequencing and genotyping of humans and reduced by 80% (to a final rate of 6% of inserts primates (Lovmar et al., 2003; Barker et al., 2004; screened) through three enzymatic treatments of (a) Paez et al., 2004; Dickson et al., 2005; Jiang et al., phi29 DNA polymerase and dNTPs in the absence of 2005; Lu et al., 2005; Ro¨nn et al., 2006), insects primers to ‘debranch’ the DNA, (b) S1 nuclease to (Gorrochotegui-Escalante and Black, 2003), fungi digest remaining single-stranded DNA present in the (Foster and Monahan, 2005; Gadkar and Rillig, 2005; mixture and (c) flow shearing and size selection on Ferna´ndez-Ortun˜ o et al., 2007) and additional agarose gels followed by nick translation with E. coli applications in forensics and medicine (Hosono DNA polymerase I. et al., 2003; Lasken and Egholm, 2003; Barber and The predominant mechanism for chimera forma- Foran, 2006; Spits et al., 2006; Ballantyne et al., tion has now been elucidated and occurs during the 2007).
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