A standard research tool remains useful despite technology advances
The questions of gene function, interaction, and regulation are central to the science of molecular biology. Despite the myriad of new technologies, products, and techniques produced by the genomics revolution, some old standards remain just as useful as ever. One such cDNA libraries and products that facilitate library the sheer number of companies offering premade and custom cDNA libraries and products that facilitate their use serves as evidence of the tool's sustained power and efficacy.
Assessing which genes are active in a given tissue at a specific time and determining the relative degree of gene activity are asocial steps in understanding how the genome as a whole functions within the tissue or cell under investigation. The term "trascriptome" refers to the set of genes that are expressed or transcribed from genomic DNA in a particular cell or tissue. An understanding of the transcriptome translates into an understanding of the activity and roles of the proteins that these genes encode. For technical reasons however, it is much easier to work with DNA than with RNA. Therefore, researchers synthesize cDNA, which is a DNA copy of mRNA, in vitro. This produces a highly versatile tool that can be used to study gene regulation, expression, splice variation, post-translational modifications, and protein-protein interactions. Changes in patterns of gene expression provide valuable information about the causes and consequences of disease, the body's response to drugs, and the normal operation of cells.
Unfortunately, the preparation of a cDNA library is a challenging, time-consuming, and complex process. The first step, extracting the cellular RNA, is itself a difficult task because of the prevalence of RNA-degrading enzymes (RNAses). Most functional mRNAs have a 3' poly-adenine tract, enabling researchers to purify the mRNA by passing total cellular RNA over a poly-thymine oligonucleotide column. The oligo-dT provides a 3'-hydroxyl group to prime first-strand synthesis with reverse transcriptase. This DNA strand usually turns back on itself to form a hairpin loop before the completion of synthesis, providing a primer for second strand synthesis with DNA polymerase I. An added RNAse H degrades the remaining mRNA, and SI nuclease opens the hairpins, degrading the extension. The resulting cDNA copies can be cloned by blunt-end ligation into a plasmid cloning vector to form a cDNA library, which can then be screened by DNA hybridization or by immunological assays.
Researchers facing this grueling process often turn to commercially prepared libraries. Companies such as BD Biosciences-CLONTECH of Palo Alto, Calif., and Stratagene of La Jolla, Calif., offer researchers a large and diverse array of cDNA libraries, isolated from a striking variety of organisms and tissues. Many other companies offer more limited groups of libraries and all can produce customized libraries for researchers whose needs are not met through existing libraries.
WORKING ON YOUR EXPRESSIONS
cDNA libraries are commonly used in gene expression studies. Expression cloning of cDNAs using mammalian cells was first described more than a decade ago and has been used to isolate a variety of genes. The most common approach is to identify the cDNA clone of interest within the total population of cDNA clones by screening with a nucleic acid probe consisting of a previously cloned cDNA fragment, genomic DNA, or a synthetic oligonucleotide specifying a particular RNA sequence.
Alternatively, cDNA expression libraries can be used to identify sequences encoding polypeptides or proteins of interest. Certain lambda vectors allow the expression of the polypeptide encoded by its DNA insert and can be screened with an antibody raised against the protein of interest. Nucleic acid-binding proteins can be cloned from expression libraries based on the ability to bind a labeled DNA or RNA fragment.
For investigators interested in expression screening in mammalian cells, Carlsbad, Calif.-based Invitrogen's Discovery Line[TM] premade cDNA libraries are available prepared from human normal, fetal, or tumor tissue. The eukaryotic expression vector pcDNA3.1 is used to construct these libraries to facilitate expression in mammalian cells. These libraries are cloned unidirectionally (5' to 3') …