Something from (almost) nothing:
the impact of multiple displacement
amplification on microbial ecology
Erik K Binga1, Roger S Lasken2and Josh D Neufeld1
1Department of Biology, University of Waterloo, Waterloo, Ontario, Canada and2J. 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
Environmental microbial diversity is poorly under-
stood and the majority of microbes are inaccessible
by laboratory cultivation. As a result, microbial
ecology has sought to develop molecular methods to
characterize whole communities. The extraction of
community nucleic acids is a typical initial step,
and although cell biomass is often sufficiently high
to enable analysis by PCR, gene hybridization or
metagenomics, this is not always the case. Because
microbial ecologists study challenging environ-
ments, such as insect guts (Broderick et al., 2004),
ice cores (Christner et al., 2001), permafrost (Steven
et al., 2006), deep subsurface sediments (Teske,
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)
step provides access to community DNA from these
low-biomass environments. In addition, the vast
number of uncultivated organisms associated with
the ‘rare biosphere’ (Sogin et al., 2006) contain
enzyme-encoding genes that hold great promise for
medicine, biotechnology and industry. Fortunately
for microbial ecologists in the early 21st century,
small yields of DNA from low-biomass communities
or individual uncultivated cells are readily retrieved
by the advent of WGAvia the multiple displacement
amplification (MDA) reaction. Here we highlight the
history of MDA and discuss its mechanism, cap-
abilities and limitations. The applications of MDA
in microbial ecology are reviewed for accessing low-
abundance DNA from cells or environmental sam-
ples. Finally, we discuss the future of MDA and its
potential integration into additional facets of micro-
bial ecology research.
A brief history of MDA
Initial WGA reactions utilized PCR-based techni-
ques such as degenerate oligonucleotide primed
PCR (Telenius et al., 1992) and primer extension
PCR (Zhang et al., 1992). However, these were
limited by nonspecific artifacts of amplification
(Cheung and Nelson, 1996), strong bias (Paunio
et al., 1996) and short amplification products
(Telenius et al., 1992; Zhang et al., 1992; Paunio
Published online 7 February 2008
Correspondence: JD Neufeld, Department of Biology, University
of Waterloo, 200 University Avenue West, Waterloo, Ontario,
Canada N2L 3G1.
The ISME Journal (2008) 2, 233–241
& 2008 International Society for Microbial Ecology All rights reserved 1751-7362/08 $30.00
et al., 1996). MDA was the first WGA method based
on an isothermal reaction (Dean et al., 2001).
Double-stranded DNA template is initially dena-
tured before incubating at 301C for 2–16h, depend-
ing on the amount of starting template and the
commercial MDA kit being used (Table 1). MDA
utilizes target DNA template, buffer, dNTPs, random
polymerase, which is commonly derived from the
Bacillus subtilis bacteriophage phi29. The 30phos-
phorothioate modifications are required for amplifi-
cation, as they render the hexamers resistant to the
30–50exonuclease proofreading activity of the poly-
merase (Dean et al., 2001). The phi29 DNA poly-
merase extends the random primers and its strong
‘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-
tures (Figure 1). Complete denaturation of MDA
products and subsequent resolution on denaturing
alkaline agarose gels revealed a 12kb average
length of the resulting single-stranded amplified
DNA (Dean et al., 2002). The reaction is terminated
by heatingto 651C
the polymerase, and yields up to 40mg of DNA per
The phi29 DNA polymerase was first isolated by
Blanco and Salas (1984) from Escherichia coli cells
expressing the P2 gene, which encodes the sole
phi29-associated DNA polymerase. It has an extre-
mely high processivity, adding an average of 70000
nucleotides each time it binds the primer template
(Blanco et al., 1989). Use of single-stranded M13
DNA as template gave a rolling-circle mode of
replication, in which the polymerase repeatedly
copied around the circular template via its strand
displacement activity, yielding a product of con-
catenated M13 repeats. The DNA polymerase’s
associated 30–50exonuclease proofreading activity
results in a low intrinsic error rate of 10?6–10?7
(Watabe et al., 1984; Blanco and Salas, 1985) and the
accumulation of mutations in MDA products at a
Table 1 Several commercially available kits for multiple displacement amplification of DNA
ProviderKit Intended template for amplificationIncubation
Volume (ml)Time (h)Yield (mg)
(Baie d’Urfe ´,
TempliPhi 100/500Small circular DNA (for example,
Large circular DNA (for example,
BACs, cosmids and fosmids)
Small circular DNA (for example,
Circular templates containing
‘difficult’ sequence (for example,
repeats, inverted sequences and
Linear DNA (high yield)
Linear and circular DNA
Linear and circular DNA201–1.57–10
Linear and circular DNA
of linear DNA
Abbreviation: ND: not defined in the manufacturer’s protocol.
structures formed during multiple displacement amplification
(MDA). The arrowheads represent the location of the phi29
polymerase synthesizing DNA in a 50to 30direction.
Sequential diagrammatic representation of branched
Multiple displacement amplification and microbial ecology
EK Binga et al
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