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May 11, 2007 Research Highlight Medicine / Disease

A new tool for tracking the tiniest changes

A robust new method for genomic analysis could extend the reach of personalized medicine within the developing world

Image of SMAP 2 diagnostic assays Figure 1: SMAP 2 diagnostic assays can be performed directly on blood samples, simplifying sample collection and preparation.

Even the smallest genetic variations, known as single nucleotide polymorphisms (SNPs), can have important implications for an individual’s predisposition to certain diseases or response to specific medicines. As such, analyzing the SNPs of a given patient can reveal important health information and make it possible to develop personalized therapeutic strategies.

Most techniques for SNP screening rely on a method called the polymerase chain reaction (PCR), which exploits a DNA-replicating enzyme to rapidly amplify genomic sequences for analysis. The amplified region is determined by a pair of short, single-stranded DNA molecules called ‘primers’, which are designed to match target sequences in the genome. In principle, an identical primer-target match is required for sequences to be amplified, enabling the use of specific primers to identify particular SNP variants.

PCR is a powerful tool for DNA amplification, but also suffers from a number of liabilities that can limit its clinical power. Assays may generate false-positive results due to contamination or the amplification of inappropriate targets, and therefore careful sample preparation is essential—especially for clinical applications. Each PCR reaction is a multi-step process, where samples need to be ‘cycled’ through a series of incubations at different temperatures, and identifying the proper reaction conditions for amplifying a given target sequence can be challenging. Because of these requirements, PCR requires the use of specialized and fairly expensive equipment, which makes it a less practical solution for clinical facilities with limited resources.

Enter SMAP 2—a new tool for point-of-care diagnostics

Image of SMAP 2-based kit Figure 2: Hayashizaki’s group and collaborators in Singapore have developed a SMAP 2-based kit for screening EGFR gene mutations.

“Existing technologies are generally complex in design and consequently more expensive,” explains Yoshihide Hayashizaki of the RIKEN Genomic Sciences Center in Yokohama. “They take longer, and require more preparation.” In a recent article from Nature Methods, Hayashizaki and his colleagues describe an alternative DNA amplification method, smart amplification process version 2 (SMAP 2), which they suggest could provide a strong alternative to PCR for point-of-care diagnostics1.

SMAP 2 uses five primers instead of two; one of these is selected to serve as the ‘discrimination primer’, which is designed to reveal the presence or absence of a target mutation. For each SMAP 2 assay, different versions of the discrimination primer are created, where each version can only bind to a specific SNP variant. By analyzing the amplification that takes place with different discrimination primers, it becomes possible to characterize a patient’s genomic variations.

Advantages of SMAP 2

SMAP 2 is extremely precise, and much of this precision is gained from a unique feature of the assay—the addition of MutS, a purified bacterial protein that eliminates ‘background signal’ resulting from the amplification of inappropriate sequences. As a test of the discriminatory power of SMAP 2, Hayashizaki’s group demonstrated the assay’s ability to accurately detect minor variations in one member of the cytochrome P450 gene family, a group of closely related genes with very similar sequences.

SMAP 2 offers a number of other advantages over PCR as a clinical tool. Unlike the multi-step process of PCR-based detection, SMAP 2 samples need only be incubated at a single temperature for 15 to 30 minutes. Additionally, no DNA purification is required to prepare samples for SMAP 2—assays can be performed directly on raw blood or tissue samples (Fig. 1), making this an ideal tool for rapid patient screening. Most importantly, SMAP 2 integrates the amplification and detection process, so that no signal is generated if amplification has not taken place, and sequence analysis therefore becomes simple and accurate.

The rules governing effective primer design are not entirely clear, and the initial development of a SMAP 2 assay can be somewhat complicated compared to PCR, but Hayashizaki and colleagues have developed a computer program that considerably simplifies this process. “It may take a week to a few months to create an optimized assay for routine diagnostic use,” he says. “But with more experience and the creation of intelligent software algorithms, we expect to simplify this process enormously in the very near future.”

Adapting SMAP 2 for clinical use

Since the initial publication of this work, the development and commercialization of effective diagnostic tools has become a top priority. Hayashizaki’s team has already begun adapting SMAP 2 for clinical use, and recently applied the technology toward the detection and analysis of mutations in the epidermal growth factor receptor (EGFR) gene, a useful diagnostic indicator for lung cancer.

These results will be published later in the year, and Hayashizaki’s group recently announced the launch of a collaboration with Singaporean researchers and doctors at the National University of Singapore and the National University Hospital, with the aim of developing a simple and rapid EGFR screen (Fig. 2). Accurately identifying such mutations can help doctors to apply an appropriate therapeutic strategy, but current diagnostic procedures require about three weeks—and surgery. SMAP 2-based diagnosis could reduce this time to as little as five hours, and would require only a tiny biopsy sample, making screening faster and more comfortable for patients.

Hayashizaki makes it clear that SMAP 2 is not intended as a replacement for PCR as a research tool, but initial findings clearly suggest that this technique could be a powerful method for extending the reach of genetic analysis as a clinical tool. “One of the greatest potentials for using knowledge of genetic information is in the area of personalized medicine,” he says, “and SMAP has the added potential to be employed at point-of-care since the technology is very simple and very robust.”

References

  • 1. Mitani, Y., Lezhava, A., Kawai, Y., Kikuchi, T., Oguchi-Katayama, A., Kogo, Y., Itoh, M., Miyagi, T., Takakura, H., Hoshi, K. et al. Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch suppression technology. Nature Methods 4, 257–262 (2007). doi: 10.1038/nmeth1007

About the Researcher

Yoshihide Hayashizaki

Image of Yoshihide Hayashizaki

Yoshihide Hayashizaki was born in Osaka, Japan, 1957. He received his MD and PhD from Osaka University Medical School in 1982 and 1986, respectively. From 1988 to 1992, he worked as a research scientist at the National Cardiovascular Center Research Institute (Department of Bioscience), in Osaka, and developed a new technology known as the Restriction Landmark Genome Scanning (RLGS) System. In 1992, he joined RIKEN, and was appointed Project Director for the RIKEN Genome Project in 1995. Since then he has been taking part in, and aiming for, the establishment of a Mouse Genome Encyclopedia. His present position is project director of the Genome Exploration Research Group, Genomic Sciences Center, RIKEN. He organized the FANTOM (Functional Annotation of Mouse cDNA) consortium to annotate all of the RIKEN mouse clones. During this work, he has been able to discover a large amount of non-protein coding RNAs, so-called RNA continent. He also developed the DNABookTM, which enables the efficient distribution of a large amount of clones. Currently he works toward an illumination of the gene transcriptional network. In 2001 he was assigned as a foreign adjunctive professor of the Karolinska Institute (Sweden) and an honorary professor of the University of Queensland (Australia).

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