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What’s in a genome? J. Shawn Roach
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ne of the most ambitious scientific undertakings ever conceived, the Human Genome Project (HGP), was completed on June 26. The HGP is a grand biological experiment, and it has led to a revolution in the way that molecular biology is conducted. It will also strongly impact the scientific endeavors and careers of many analytical chemists. As with all large-scale, concerted scientific projects, two important side benefits of the HGP are the advancement of present technologies and the development of new ones. Many of these technologies are highly specialized applications of analytical chemistry, so they have immediate uses. The HGP also has long-term career implications. The prospects of an entirely new era of molecular medicine open the doors to many career opportunities in the pharmaceutical and biotechnology areas. In addition, most major agrochemical companies have begun large-scale plant genomics efforts. So, how does an analytical chemist become educated about the HGP and its associated technologies? What are the latest developments? Where are the opportunities for analytical chemists?
HGP background
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Great places to start are the home page of the HGP, which is supported by the Department of Energy (www.er. doe.gov/production/ober/hug_top. html), and the home page of the National Human Genome Research Institute (NHGRI; www.nhgri.nih.
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gov/HGP), which is a division of the National Institutes of Health. In these pages, you can find a timeline, a list of goals, and the project’s status. Also helpful is the Human Genome Project Information site (http://www.ornl.gov/ hgmis), hosted by Oak Ridge National Laboratory. This site includes a “Primer on Molecular Genetics” (www.ornl.gov/ hgmis/publicat/primer/intro.html), which explains the concepts behind the science while cutting through the field’s notorious jargon, and a “Genome Glossary” (http://www.ornl.gov/ hgmis/publicat/glossary.html). Other useful Web sites include the Genomes Online Database (http://wit. integratedgenomics.com/GOLD), which tracks various prokaryotic and eukaryotic genome mapping projects, and the National Center for Biotechnology Information’s (NCBI) Gene
microarrays; and MS applications. To learn about the first three technologies, a useful starting point is a 1999 review published by J. M. Jaklevic and colleagues (1), which explains the state of the art and discusses anticipated developments in each area.
Robotics and laboratory automation
Genetic sequencing involves many, many repetitive tasks. Sequencing an entire genome requires so much repetition that it has spurred the development of robots to automate as much of the mundane work as possible, freeing the researchers to do more interesting, creative tasks and reducing the potential for human errors. Although robotics may not be classified as analytical chemistry per se, it is certainly relevant to many researchers. Most analytical chemists are accustomed to dealMicroarrays usually measure ing with high volumes of samples and with autosamplers of various kinds, gene expression, but they can and there is always a need for new ways to perform de novo sequencing. reduce sample sizes and make specimens more amenable to robotic handling. To Map site (www.ncbi.nlm.nih.gov/ learn about some of the commercial genemap99), which is devoted to the robotics systems, try the Web sites human genome. For that matter, the of Beckman Coulter (www.beckman. NCBI home page (www.ncbi.nlm. com/Beckman/biorsrch/prodinfo/ nih.gov) is a great place to start for automated_solutions/a_systems.asp), general biotechnology information. A Genetix (www.genetix.co.uk), and nonprofit research institute, The Institute for Genomic Research, also known Qiagen (www.qiagen.com/catalog/ chapter_10/chap10.asp). All three as TIGR (www.tigr.org), is another major database site. Of particular inter- companies sell widely used laboratory automation platforms to the biotechest is the microbial genome database. Finally, to keep track of the private sec- nology and pharmaceutical industries. tor’s research efforts, visit Celera GenDNA-sequencing technologies omic’s home page (www.celera.com). The heart of the HGP, or any genomics Use caution when entering any of effort, is the rapid determination of these sites, though, as information the genetic sequence of a given DNA overload is a common side effect! sample. Because current sequencing The analytical technologies previtechnologies rely on a polymerase enously mentioned are broadly categorzyme that incorporates tagged monoized into four areas: high-throughput mers as it rebuilds fragments of the automation for sample preparation and handling; DNA-sequencing meth- original sample, the process can be viewed on a simplistic level as a molecods; microarrays, especially DNA
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ular size separation and tagging technique. The differences among the technologies lie in the means of performing the separation and the tags used to distinguish the fragments. The technique of preference is fast becoming capillary gel electrophoresis (CE) separation, with fluorescent dye terminator chemistry to identify fragments. This technology has proven superior to traditional slab-gel sequencing because it is faster and more amenable to automation and because it provides better sample tracking (1). Side-by-side comparisons of several commercially available systems for automated, high-throughput, CE-based DNA sequencing are available (2, 3), with the most recent being in the February 7 issue of The Scientist (www.the-scientist.com/yr2000/feb/ profile1_000207.html). Among the systems discussed are those produced by Amersham Pharmacia Biotech (www. apbiotech.com), Beckman Coulter (www.beckmancoulter.com), and PE Biosystems (www.pebio.com/ga), which is now called Applied Biosystems.
Microarray technologies Microarrays are a new technology that offers the ability to measure relative levels of gene expression (expression profiling) or to test for the presence of a particular sequence within sample DNA (resequencing) (4). Microarrays also have the capability to perform de novo sequencing, but they are not as widely used for this purpose. The principle behind microarrays is simple but elegant. A known array of either cDNA—which is synthesized from a messenger RNA template and, thus, omits any noncoding sequences—or DNA oligonucleotides is deposited or synthesized in situ on a solid support, which is typically a coated-glass microscope slide or a membrane. Microarrays composed of deposited DNA or oligonucleotides are known as “spotted” arrays. Microarrays composed of DNA that has been chemically synthesized on the surface are known as “in situ” arrays.
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For analysis, labeled DNA is passed across the surface of the microarray and allowed to hybridize to the complementary DNA on the array. After appropriate treatments, including washes to remove nonspecifically bound DNA, the labeled slide is analyzed—typically by fluorescence analysis—and the relative amount of target DNA present is quantitated. The presence or absence of target DNA or relative amounts of DNA present may be used to distinguish between healthy and diseased states or help researchers to understand the function of the genes. Microarrays are also useful for analyzing genetic variations known as single nucleotide polymorphisms (www.snps.com/whatis. htm) or SNPs (pronounced “snips”). Two very useful sites to learn more about microarray technologies are pioneer Pat Brown’s home page (http:// cmgm.stanford.edu/pbrown) at Stanford University and a site maintained by the Louisiana State University (LSU) Health Science Center (www.medschool. lsumc.edu/bioc/WebSites/DNA_ MicroarraySites-top.html). Information for starting a spotted microarray facility can be found on Brown’s Web site. The LSU site is devoted to the various aspects of microarray technology, including raw materials, technology vendors, and protocols. Additionally, the Microarray Core Facility (http://pompous. swmed.edu/exptbio/microarrays/ index.htm) at the University of Texas Southwestern Medical Center has published a protocol manual that is designed to assist researchers in working with spotted arrays and is available for downloading. Finally, several companies—including Affymetrix (www. affymetrix.com), Incyte (www.incyte. com), and Rosetta InPharmatics (www. rii.com)—have been founded around either the sale of microarray technology or the application of microarray technology to perform fundamental biological research, such as gene discovery.
MS applications MS offers considerable promise as a genetic analysis tool because of its high
sensitivity and ability to unambiguously confirm analyte identity. Techniques such as MALDI are also amenable to automation. Indeed, direct on-chip analysis methodologies to analyze microarrays have been published by researchers at Johns Hopkins University Medical School and Sequenom (www. sequenom.com/corpinfo/content2. html), among others (5, 6).
general bioanalytical applications—can be found in the 1998 and 1999 issues of Current Opinion in Biotechnology, respectively (7, 8).
Outlook From the beginning, the HGP has been a very interdisciplinary effort employing all scientific fields. The future of science lies in these interdis-
MS techniques show great promise for resequencing and SNP analysis. Unfortunately, the short read lengths (the number of consecutive bases read at one time) and the complex sample-preparation methods for genetic samples have limited the widespread use of MS for de novo sequencing to date. However, MS techniques—particularly MALDI and electrospray ionization (ESI)—show great promise for resequencing and SNP analysis. The latter application is more suitable than de novo sequencing because it only involves detecting the presence or absence of short, specific sequences. And MS may be in even greater demand now that the rough draft of the genome has been completed, as the burgeoning fields of functional genomics (linking genes to their biological functions) and proteomics (correlating genetic sequences to the proteins that they encode) become the focus of attention. Both of these fields require the analysis of ultra-low levels of peptides and proteins—an area where MALDI time-of-flight MS and ESI MS excel. Moreover, MALDI and ESI permit pattern matching to identify digested proteins. Coupling this pattern analysis with online databases and automated software opens the door to true highthroughput proteomics. Two useful review articles on MS—the first covers the analysis of oligonucleotides and nucleic acids, and the second covers
ciplinary research efforts, whether they are conducted in academia, private industry, or the public sector. Analytical chemists have expertise in problem solving, separation and detection technologies, and laboratory instrumentation that can play key roles in genomic and proteomic research projects. What lies in a genome for analytical chemists? A world of new opportunities.
References (1) (2) (3) (4) (5)
(6) (7) (8)
Jaklevic, J. M.; Garner, H. R.; and Miller, G. A. Annu. Rev. Biomed. Eng. 1999, 1, 649–678. Swanson, D. The Scientist Feb. 7, 2000, 14, 23. Henry, C. Anal. Chem. 1996, 68, 493 A–497 A. Henry, C. Anal. Chem. 1999, 71, 462 A–464 A. Little, D. P.; Cornish, T. J.; O’Donnell, M. J.; Braun, A.; Cotter, R. J.; Köster, H. Anal. Chem. 1999, 69, 4540–4546. Little, D. P.; Braun, A.; O’Donnell, M. J.; Köster, H. Nat. Med. 1997, 3, 1413–1416. Crain, P. F.; McCloskey, J. A. Curr. Op. Biotechnol. 1998, 9, 25–34. Costello, C. E. Curr. Op. Biotechnol. 1999, 10, 22–28.
J. Shawn Roach is a chemist who focuses on analytical instrumentation. He is currently a research fellow at the Center for Biomedical Inventions at the University of Texas Southwestern Medical Center–Dallas, where he works on laboratory automation and instrumentation in support of the Human Genome Project.
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