The history of molecular biology is marked by
technological advances that have opened up
the investigation of both fundamental and
applied biological problems. Consequently,
specialized disciplines of molecular biology
emerged along with the need for more powerful
and dynamic analysis tools. A commonality
among these diverse research areas has
been the limitations on the availability of
DNA. In response to the challenge of obtaining
adequate quantities of specific DNA,
researchers began examining various methods
for producing copies of DNA. Polymerase chain reaction (PCR) is one such method that
enabled researchers to exponentially amplify
specific segments of a DNA template and,
ultimately, revolutionized many research areas
ranging from viral identification to transcriptional
regulation.
As PCR became a fundamental research
tool, the apparent limitations of sensitivity,
specificity, and amplification of a limited
target-specific fragment presented certain
challenges. Incremental improvements of PCR technology such as hot-start polymerases
and more advanced instrumentation
have eased some of the constraints.
However, DNA (as a limiting resource) has
impacted the extent of research and understanding
in many areas of molecular analysis,
including pharmacogenomics, target
discovery/validation, and population studies.
Therefore, a technology that allows
unbiased replication of the entire genome
from limited source material is necessary to
support continuous investigative endeavors
and has become a critical focus.
Discussion
Whole genome amplification (WGA) methods
began with the introduction of primer extension
preamplification (PEP), degenerate
oligonucleotide-primed (DOP) PCR, and
tagged PCR (T-PCR) in the early 1990s. Each
method utilized a different strategy in order to
achieve amplification of low starting amounts of
genomic DNA with the ultimate goal of generating
a complete and unbiased representation of
the entire genome.
Although all methods proved promising in their
respective approach, each has had specific drawbacks
that affected its usefulness with many
downstream genetic analyses. These early genome
amplification methods all gave varying degrees of
bias when compared to the representation present
in the starting material. Additionally, integration
of these methods into various analyses highlighted
an inability to produce long products from
very low (nanogram or picogram) quantities of
genomic DNA. Efforts to improve existing technologies
and develop innovative techniques
became necessary.
The delineation of newly developed applications
for WGA is founded on new means to reduce
amplification bias. These methods are easily differentiated
by their respective method of amplification.
The crucial step required by all genome
amplification technologies is the successful
denaturation of the target DNA and competition
of primer annealing over annealing of
the opposite strand. Thermodynamics are
unfavorable for the competition of intermolecular
priming over intramolecular strand
“rebound” for long strands of DNA; thus
priming events occur too distant to allow
effective amplification.
Figure 1 - Graphical representation of the GenomePlex whole genome amplification
kit. Genomic DNA is fragmented, primed, and amplified to generate the
OmniPlex library. This library can be amplified for immediate use or stored for
future studies.
Of the various WGA methods available,
GenomePlex™ WGA (Rubicon Genomics,
Inc., Ann Arbor, MI) best addresses the issues
of amplification biases and primer binding.
The method is based on random fragmentation
of the genome into a series of overlapping,
short templates. The resulting, shorter DNA
strands can be efficiently primed and amplified
to generate a library of DNA fragments with
defined 3′ termini—the OmniPlex® library
(Rubicon Genomics). This library is replicated
using a linear, isothermal amplification
in the initial stages, followed by a limited
round of geometric (PCR) amplifications
(Figure 1). Upon completion of PCR, amplified
DNA may be purified by standard purification
methods and used immediately for
genetic or genomic analysis or it may be
archived for future investigation.
Figure 2 - DNA samples isolated from buccal swabs amplified using
GenomePlex WGA and genotyped using a conventional TaqMan™ assay (Applied
Biosystems, Foster City, CA). The intensities and clustering of the data were comparable
to those from unamplified DNA. (Data provided by Rubicon Genomics.)
GenomePlex WGA technology is compatible
with single-reaction PCR tubes and the high-throughput
format of 96-well PCR plates
(Figure 2). Other existing technologies rely
on a highly processive mesophillic polymerase
to amplify the long regions between the
sparse priming events. The method of amplification
is crucial in determining the time
required to generate microgram quantities of
genomic DNA from subnanogram starting
concentrations. Isothermal strand-displacement
methods carry out amplification at a
static temperature that requires a 6-hr to
overnight incubation. Amplification by traditional
temperature cycling, as demonstrated
with GenomePlex WGA technology, is completed
in approx. 3 hr.
Figure 3 - Use of GenomePlex WGA in cancer cell DNA analysis. Cancer cells were diluted and
the DNA amplified using whole genome amplification. The amplified DNA was screened with a cancer-specific
marker. After screening, the cancer cell underwent DNA analysis and the positive cancerous
cells could be identified.
Figure 4 - Flow chart demonstrating the versatility of the GenomePlex whole genome amplification
system. Various sources of DNA can be amplified to fuel multiple downstream applications. These
applications have utility across a broad range of scientific disciplines.
Many research projects have been significantly
impacted due to the limited availability
of sufficient quantities of genomic DNA.
With continuous efforts focusing on the development
of more sensitive analytical research tools,
this limitation has presented a continuous challenge
for many disciplines of molecular research,
including tumor pathology and patient genotyping.
Access to a constant source of genomic material
is critical for proper identification of potential
genetic markers. GenomePlex WGA technology
bypasses this obstacle by ensuring equal representation
of the whole genome with little or no bias
(Figures 3 and 4). Unlike traditional clinical trial investigations that may require facilities to offer virtually unlimited storage capacity
for samples, WGA provides a platform that minimizes collection and storage by
generating concentrated high-fidelity yields of genomic DNA from human blood
or buccal swabs, thereby enabling researchers to easily store archived material or
distribute it among their collaborators.
Summary
Since the early 1990s, the evolution of whole genome amplification technologies has resulted in
significant improvements that have ultimately eased the constraint of limited genomic DNA.
The technology of GenomePlex WGA allows multiple disciplines of molecular research to
extend beyond the limitations of traditional PCR. In less than 3 hr, nanogram amounts of
genomic DNA from numerous sources such as cultures (blood or even tissue) are amplified into
microgram yields. This DNA can be archived for future use or analyzed by a variety of genetic
tools. The high yield and unlimited potential of the GenomePlex WGA technology make it the
obvious choice for genetic research.
The authors are with Sigma-Aldrich Corp., 3050 Spruce St., St. Louis, MO 63103, U.S.A.; tel.: 314-286-7879; fax: 314-286-7817; e-mail: [email protected]. The Whole Genome Amplification kit is available
from Sigma-Aldrich Corp. at 800-325-3010 or sigma-aldrich.com/wga.