Get the Basics

Photo: Courtesy of Michelle Lizott Waniewski DROP AND GIVE ME 20! Attendees at the 2002 NEB workshops labor through basic training. If you've ever been confused by the jargon of molecular biology, you are not alone. With biotechnology in the news day by day, it is easy to forget that for many of us--even for some readers of The Scientist--the tools, techniques, and concepts that undergird much of this research are foreign. To help keep up, some scientists are queuing up to learn the basics in a variety of immersive "boot camps." New England Biolabs (NEB) of Beverly, Mass., sponsors one such event, the NEB Molecular Biology and PCR Summer Workshop (www.math.smith.edu/~sawlab/neb.html). At these two-week courses, held at the Clark Science Center, Smith College, Northampton, Mass., "We teach all of the basics, and then some," says course instructor Steven Williams, Gates professor of biological sciences at Smith College and professor of molecular and cellular biology at the University of Massachusetts, Amherst. This intense course covers two semesters' worth of information in two weeks, with lectures and labs that typically begin at 8:30 in the morning and continue until 10:00 at night. Experiments include genomic cloning and DNA sequencing, genome analysis, gene expression analysis, complementary DNA (cDNA) cloning, and PCR library analysis. "We have always felt that hands-on experimentation rather than just lectures was a key to having our students really learn the technology we are teaching," explains coinstructor Barton Slatko, senior scientist at NEB. "The NEB boot camp at Smith was one of the best things I've done in my adult life," asserts Rob Phillips, professor of applied physics at California Institute of Technology. "It was a fantastic venue for scientists to very quickly learn new techniques and concepts." Participants performed 60 to 70 experiments that provided hands-on experience to complement the lectures. This experience is needed because "traditional barriers between disciplines are dissolving," he says, citing the directed collaborations of National Institutes of Health and National Science Foundation grants. NEB's boot camp was developed in the early 1980s after Slatko and Williams coauthored a paper about DNA sequencing.1 "We got calls asking 'How do you do it' and we thought we should do a course on the topic," Slatko explains. "Don Comb, NEB president, agreed to let us offer the courses." The boot camp has grown significantly since then. "The course is financially self-sufficient, but it's not a moneymaker," Slatko says. Nor was it designed to bring customers to NEB. In fact, many companies throughout the industry donate reagents--including enzymes, NEB's lifeblood. "Without the donations, the cost would be prohibitive for many students," Slatko explains. NEB's workshop cost $3,500 (US) per person this past summer, when the company hosted three sessions in June and July. Each session was open to 48 participants, and according to the event's Web site, the represented research fields included "various disciplines of medicine, biochemistry, ecology, immunology, microbiology, pharmacology, plant biology, genetics, physiology, and others. They came from large universities, small colleges, medical schools, hospitals, industry, and private foundations. 75% came from the USA and 25% from overseas." NOT JUST FOR SCIENTISTS ANYMORE Not surprisingly, scientists are not the only ones confounded by all this 'newfangled' molecular biology. Reporters also struggle with the material, and like the scientists they report about, they can attend boot camps, too. Knight Science Journalism Fellowships at the Massachusetts Institute of Technology (MIT) (http://mit.edu/knight-science/) offers a boot camp that is not as intense or as hands-on as the scientists' training; it is more like an introductory course in genomics for nonscience majors. The first two days of this six-day course are an introduction to how cells and genes work. "It's all lectures," says Boyce Rensberger, program director. The rest of the week covers applications: gene therapy, stem cells, cell signaling, the Human Genome Project, proteomics, legal issues, ethics, and so on. Eventually, participants go into an MIT molecular biology lab to carry out a four-hour hands-on experiment in DNA fingerprinting to solve a hypothetical crime. This year's "Genes & Cells" boot camp ("Taking the Genetic Revolution by Storm") will be held Dec. 1-8 and is open to 12 journalists. Aaron Zitner, science reporter on the national staff of the Los Angeles Times, says his boot camp experience nearly two years ago was "truly fantastic." "It gave me a lot more understanding of the science of genes and stem cells," he adds. MOLECULAR CLONING 101 It can be quite useful for molecular biology neophytes to consider the cell as a well-run office, its supply closet filled with enzymes. Every office has several pairs of scissors. For molecular biologists, those cellular scissors are called restriction enzymes. Given the critical role these enzymes play in everyday molecular biology, they have a surprisingly obscure name. Simple though they are, bacteria have evolved a way to resist infection by external sources of genetic material. Long before scientists understood the mechanism, they recognized that if a virus infected one strain of bacteria, it could then infect other organisms of that same strain but not of different strains. In other words, the viruses were restricted to a particular strain. At the heart of this observation is a set of two enzyme classes: methylases, which modify bacterial DNA, marking them as "self"; and restriction enzymes, which digest foreign DNA. Restriction enzymes are sequence-specific; that is, they cut DNA at specific locations, generally palindromic sequences such as GGATCC (the reverse complement of this sequence is also GGATCC). While some restriction enzymes produce blunt ends, others make staggered cuts to yield "sticky" ends. About 250 of these enzymes are commercially available, though more than 3,500 individual enzymes have been found, according to REBASE, the restriction enzyme database (http://rebase.neb.com/rebase/) maintained at NEB. Cloning (in the molecular biology sense of the word) would not be possible without restriction enzymes. Their sticky ends act like molecular Velcro, holding together two pieces of DNA that have complementary ends. To actually suture those fragments into a single piece of DNA requires DNA ligase, the glue in our molecular storage closet. DNA ligase permanently joins the assembled DNA sequences with covalent bonds, producing a recombinant DNA molecule. FOR ALL YOUR DUPLICATION NEEDS The cellular office contains a copy center, too. In this case, the equipment is a collection of enzymes that synthesize nucleic acids. These enzymes are template-driven; in other words, they make a copy of either DNA or RNA from a preexisting sequence. They do not synthesize de novo, random sequences. DNA polymerases duplicate the chromosomes before each cell division so that both daughter cells will have a complete set of genetic instructions. To carry out those instructions, the cell uses an RNA polymerase to transcribe the genes into RNA. If the DNA polymerase is a genetic photocopier, an RNA polymerase is like a genetic fax machine: it produces a copy of the gene for transmission from the cell's nuclear headquarters to its cytoplasmic manufacturing facility. Both DNA and RNA polymerases require a DNA template. But some viruses, like HIV, have an RNA genome. To duplicate that genome and incorporate it into the cell's genetic material, these viruses encode an enzyme, reverse transcriptase, which is capable of making a DNA copy of an RNA template. Extending the analogy of a cellular copy center, reverse transcriptase activity is akin to making a digital copy of a paper document using a scanner; the enzyme produces a storable genetic copy of the RNA text. Molecular biologists have harnessed the power of each of these enzymes in their research. DNA polymerases are used to sequence DNA and to fill in or pare back the sticky ends produced by restriction digestion reactions, among other applications. Researchers use a special type of thermostable DNA polymerase to carry out the polymerase chain reaction (PCR), which is a technique used to mass-produce a particular segment of genetic material. RNA polymerases are used to create RNA transcripts from cloned genes in vitro. Reverse transcriptases are commonly used to create cDNA libraries, which are batteries of DNA molecules, each one representing a single expressed gene. The cDNA libraries can be analyzed to determine which genes are active under different cellular conditions or in different tissues. A typical experiment uses many of these basic molecular biology tools. Consider the act of transferring a particular gene from one plasmid to another, and then expressing the protein the gene encodes in a test tube. To transfer the DNA fragment, the source and destination plasmids are digested with one or two restriction enzymes, and then the desired fragments are joined into a single molecule using DNA ligase. To express this gene in vitro, researchers would incubate this new plasmid construct with an RNA polymerase to make RNA. They would use the RNA transcript to "program" an in vitro translation system, which would then synthesize the encoded protein. Of course, other enzymatic activities critical to daily research also exist, and scientists are discovering new ones regularly. So as biotechnology continues this breakneck evolution, boot camp organizers can assure themselves of one thing: They will have a steady stream of applicants in the years to come. Gail Dutton (gaildutton@gi-wireless.net) is a freelance writer in Mayne Island, British Columbia. Jeffrey M. Perkel can be contacted at jperkel@the-scientist.com. 1. S. Williams et al., "Sequencing in the fast lane: A rapid protocol for a [35S]-dATP dideoxy DNA sequencing," BioTechniques, 4:138-47, 1986.

Get the Basics

Interested in reading more?

Become a Member of