DNA Libraries: Offering Researchers A Genome At A Glance

LIBRARY SHOPPING To obtain a DNA library is like getting your hands on a bagel or a pizza: It's easier to buy one than it is to assemble the ingredients to make your own. "Libraries are difficult to make: Most scientists only make a library once or twice in their careers," and perfecting library-making detracts from other activities, says Lani San Mateo, technical service representative at Clontech Laboratories Inc. in Palo Alto, Calif. Clontech's extensive collection of libraries includes a genomic human YAC library, human eDNA libraries from different brain regions, and libraries of the chicken gizzard, liver, lung, spleen, eye, and brain. If a researcher's needs aren't met by these, or standard offerings from organisms as diverse as mink and monkey, kangaroo and fungi, custom libraries can be delivered in two or three weeks. Prefab libraries benefit the evolutionary work of Anders Blomquist and colleagues at Sweden's Uppsala University and the Research Institute of Scripps Clinic in La Jolla, Calif. They deduced an evolutionary tree for neuropeptide Y using a chicken brain eDNA library from Clontech; an optic lobe ray library from Stratagene Inc. in La Jolla, Calif.; and genomic chicken and goldfish retina libraries from colleagues. Purchasing libraries from a catalog is not expensive. "You can buy different products to carry out the various stages in constructing a library, and the final cost is about $1,500. A library offered in our catalog is, at the most, $600. You can call and get it the next day," says Clontech's San Mateo. But a special order library is pricier. "We make customized eDNA libraries, as a service, for $2,500 and up, and genomic libraries for $2,000 and up," says Chip MacElroy of Ambion Inc. of Austin, Texas. Lofstrand Laboratories Ltd. in Gaithersburg, Md., provides similar services. If a researcher wants a more hands-on involvement in library construction, prepackaged kits may be the way to go. "A lot of people are not molecular biologists, but work with DNA. They do not have the time to develop protocols for making libraries," says Geoffrey Smith, field application specialist at Pharmacia LKB Biotechnology Inc., Piscataway. N.J. The company's TimeSaver eDNA synthesis kit guides the user to produce a library in just 24 hours. Another kit is eDNA Clonstruct, from U.S. Biochemical Corp. of Cleveland. The eDNA is synthesized directly onto the vector, saving steps and time. And for the truly ambitious who would indeed make their own bagels and pizza, eDNA protocols can be found in conventional libraries (Molecular and Cellular Biology, 2:161- 70, 1982 and 3:280-9, 1983). —R.L. DNA LIBRARY PRODUCT SUPPLIERS AND THEIR CURRENT OFFERINGS: LIBRARIES American Type Culture Collection 12301 Parklawn Dr. Rockville, Md. 20852 (800) 638-6597 Fax: (301) 231-5826 Products: Human chromosome-specific libraries: $100 non-profit, $150 others. Clontech laboratories Inc. 4030 Fabian Way Palo Alto, Calif. 94303 (800) 662-CLON Fax: (415)424-1352 Products: 5'-Stretch cDNA libraries, human YAC libraries: $600. Stratagene Inc. 11099 N. Torrey Pines Rd. La Jolla, Calif. 92037-9963 (619) 535.5400 Fax: (619) 535-S430 Products: Mouse genomic library: in lambda $685; in cosmid $670. KITS U.S. Biochemical Corp. P.O. Box 22400 Cleveland, Ohio 44122 (800) 321-9322 Fax: (800) 535-0898 Products: cDNA CLONSTRUCT: $495. Pharmacia LKB Biotechnology Inc. 800 Centennial Ave. Piscataway, N.J. 08854-9932 (800) 526-3618 Fax: (201) 457-0557 Products: TimeSaver cDNA synthesis kit: $450. DNA libraries—collections of bits of an organism's genome carried in bacteria—are descended from the 1970s recombinant DNA era, made possible by the ability to shuttle foreign genes into bacterial hosts. This vital tool of molecular biology traces its roots to 1950s research into bacterial mating, and will have future repercussions as the backbone of genome research. The tradition of sharing these resources has made many scientists bristle at the National Institutes of Health's recent attempt to patent human DNA libraries (The Scientist, April 27, 1992, page 1). A DNA library provides a way to access an individual gene without the constraints of working with the natural host. Overlapping the sequences of library entries allows gene mappers to piece together chromosomes. Genomics, emerging from this technology, focuses on characterizing organisms' DNA. Molecular evolution, a new subfield of genomics, compares entire genomes among species. Accordingly, a library may contain an entire genome, a single chromosome, or only those genes expressed in a particular cell type at a particular time. Shotgunning Whole Genomes A genomic library is made by cutting a whole genome with restriction enzymes ("shotgunning") and inserting each piece into a bacterium, which is then cloned. A bacterium might require 1,500 cloned fragments to represent its genome, a yeast 4,600, a Drosophila fruit fly 48,000, and a mammal 800,000, using a vector (a structure that carries foreign DNA into a host cell) that can accommodate small inserts of foreign DNA. Vectors with greater capacities yield libraries with fewer, but larger, pieces. These include plasmids (holding 5 to 10 kilobases [kb] of DNA), phage (up to 20 kb), and yeast artificial chromosomes (YACs, 500 to 1,000 kb). A small library may be stored as a frozen vial or spotted onto a single piece of filter paper. A larger DNA library consists of microtiter plates assembled into a grid, such as the genomic library for the mustard weed Arabidopsis thaliana. It consists of 225 96-well microtiter plates, arranged in a 15-by-15 grid. A library can contain a single chromosome type. Researchers at Los Alamos and Lawrence Livermore national laboratories began collaborating on such libraries in 1983. "Both national labs had extensive histories of flow sorting human chromosomes," says Peter DeYoung, who is in charge of the National Laboratory Gene Library Project at Lawrence Livermore. "We did not have a history of recombinant DNA work, and that cost us some delays, but not too much." The project is supported by the Department of Energy, and the libraries can be obtained from the American Type Culture Collection (ATCC) at low cost, thanks to support from NIH's division of research resources. Human chromosome 21, for example, is in a library of 10,000 clones, taking up 100 96-well microtiter plates. More than 8,000 copies of the chromosome libraries have been Circulated all over the world, says DeYoung. A second, larger collection of libraries from Livermore is distributed from 12 laboratories worldwide, DeYoung adds. At times, a genomic library can offer too much information. "When you do the whole genome, you get a lot of other stuff in there. So people prefer to use cDNA libraries," says Christopher Wills, a professor of biology at the University of California, San Diego, and author of The Wisdom of the Genes (New York, Basic Books, 1989) and Exons, Introns and Talking Genes (Basic Books, 1992). cDNA Expression Libraries A cDNA library uses the retroviral enzyme reverse transcriptase to make DNA copies, or complementary DNA (cDNA), from the messenger RNA (mRNA) in a particular cell type. Because the mRNA reflects the genes being expressed, a cDNA library is a functional subset of the whole genome. Different cell types yield different cDNA libraries. This ability to tap into differential gene expression is vital for nailing down disease-related genes. When researchers have a "candidate gene" for a disease, the suspected sequence is screened against cDNA libraries from affected tissues. The gene for cystic fibrosis, for example, binds to its complementary sequence in cDNA libraries made from the human sweat gland, lung, and pancreas, because corresponding mRNAs are abundant in these tissues, in which symptoms arise. A dynamic new twist on the ability of cDNA libraries to capture gene action is a subtraction library. Explains Wills, "Take a cDNA library. Now, stimulate the tissue, by adding a hormone or stimulating an immune response in the organism. Make a new cDNA library, after the stimulation, and hybridize it to the old one." Sequences that do not hybridize are expressed only when the tissue is stimulated. "It is a terrific technique to home in on a very specific subset of genes," Wills adds. A subtraction library helped define the functions of the T cell receptor, the protein on cells to which the immune systems' T cells bind. But for people interested in genomics, cDNA libraries tell only a partial tale. A cDNA compared with a genomic library, like reading a novel condensed in a magazine, gives major points, but misses nuances and connections, evolution, and interactions. Another limitation is that in cDNA libraries, not all genes are represented equally. "There is tremendous bias toward genes that are turned on a lot. You miss those that are not made a lot, and those that are not turned on at all except under special circumstances," says Wills. The recent sequencing of the entire third chromosome of baker's yeast underscores the fact that much information can be overlooked in cDNA libraries. The massive effort by 147 researchers from 35 European labs (Nature, 357:38-46,1992) expected to uncover 34 genes--and found 182. Similar unanticipated genes were found in the roundworm Caenorhabditis elegans (J. Sulston et al., Nature, 356:37,40, 1992). Yeast megateam member Peter Slonimski of the Centre de Génétique Moléculaire du CNRS in France found why so many genes were previously unknown-they are expressed only under stress (which, in yeast, may be altered pH or temperature). "What that tells us," says Wills, "is that you can get a library of cDNA that would certainly not be any kind of representative sampling of genes that are quite important to the organism." DNA libraries have enabled investigators to single out the voices of individual genes from the orchestra of a genome. As genetic research evolves to the next plane-revealing gene interactions-we can be certain that DNA libraries will continue to help decipher how those voices blend to build the organism. Ricki Lewis is the author of Life, a college biology text (Dubuque, Iowa, Wm. C. Brown Publishing, 1992).

DNA Libraries: Offering Researchers A Genome At A Glance

Interested in reading more?

Become a Member of