Whole Genome Amplification: Extending Beyond the Limitations of Traditional PCR

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: tfavello@sial.com. The Whole Genome Amplification kit is available from Sigma-Aldrich Corp. at 800-325-3010 or sigma-aldrich.com/wga.

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