Advancing Whole Genome Amplification

Courtesy of Molecular Staging UNRAVELING MDA: Molecular Staging's MDA technology relies on a highly processive, strand-displacing DNA polymerase. As high-throughput genomics and its postgenomics successor, proteomics, are the technologies of choice for rapid screening of new pharmaceuticals, a company that establishes itself as a technology leader in both fields could be ahead of the pack: New Haven, Conn.-based Molecular Staging (MSI). In November 2002, MSI announced in rapid succession that 1) it received a Small Business Innovation Research (SBIR) grant from the US Department of Energy's Genomes to Life program to develop MSI's multiple displacement amplification (MDA) technology for DNA amplification and identification of microbes; 2) its REPLI-g™ Whole Genome Amplification kits are now available; and 3) it received an SBIR award from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, to develop a chip that detects osteoarthritis protein biomarkers. REPLI-g is a line of kits that make whole genome amplification (WGA) easier and faster. MSI's chief operating officer Stephen Kingsmore explains that the kits come in two configurations. "One allows you to take a tiny amount of DNA and make a lot of it," he says. The other extracts genomic DNA from a sample, such as buccal swabs, buffy coats, or cultured cells, before amplifying it. The kits rely on MDA technology developed by Yale University pathology professor Paul Lizardi1 and licensed to MSI. Most WGA methods rely on PCR and have drawbacks like inconsistent amplification (uneven sequence amplification) and short fragments (generally less than 1 kb). MDA, however, yields a more consistent genomic amplification, with fragments greater than 10 kb on average. Unlike PCR, MDA is isothermal, requiring no heating or cooling steps. A DNA sample, which can include whole genomic DNA, is incubated at 30°C with random hexamer primers and *29 DNA polymerase.2 This polymerase, says Kingsmore, is the secret to the method's success. First, it is highly processive, sometimes extending up to 70 kb from the primer. "Once [the polymerase] sits down on the DNA molecule and begins to amplify it, [the polymerase] does not fall off," he notes. But in addition, the polymerase can displace downstream product strands, enabling multiple concurrent and overlapping rounds of replication. Kingsmore sees the target market for the WGA as those working with DNA-constrained samples: pharmaceutical companies that want to score 200,000 single nucleotide polymorphisms (SNPs), forensics labs trying to genotype five SNPs, or researchers collecting DNA from a buccal swab that only contains a few cells. The procedure is fast--six hours from start to finish. But for those who don't wish to use the kits themselves, Molecular Staging offers a service that will amplify samples submitted in 96-well plates. The Genomes to Life SBIR will support use of MDA to sequence entire microbial genomes of unculturable microbes. Kingsmore notes that the vast majority of environmental microbes are unculturable in the lab. Researchers need to sequence these organisms, but logically, a DNA sequence cannot be obtained if the organism cannot be grown. MDA allows amplification of microbial DNA from an environmental sample. This, says Kingsmore, has another benefit: Scientists can also use this approach to run diagnostic tests on human samples that contain only a few cells, such as amniotic fluid, urine, or sputum. The SBIR award for development of a protein chip that will identify osteoarthritis markers showcases the company's proteomics efforts. "The goal," says Kingsmore, "is simple serum tests that would allow arthritis of different types to be distinguished." The company hopes to collect serum samples from normal individuals and patients with osteoarthritis or rheumatoid arthritis and then quantify hundreds of proteins using protein chips. --Myrna E. Watanabe References 1. P.M. Lizardi, "Multiple displacement amplification," US Pat. 6,124,120, Issued 2000. 2. F.B. Dean et al., "Comprehensive human genome amplification using multiple displacement amplification," Proc Natl Acad Sci, 99:5261-6, 2002.

Advancing Whole Genome Amplification

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