cDNA libraries
A cDNA library is a collection of cloned DNA copies derived from the messenger RNAs (mRNAs) of a specific cell type at a particular time. This library serves multiple purposes, including gene mapping, studying gene expression changes over time, and cloning genes for further research or protein production. The process of constructing a cDNA library involves isolating mRNAs from a cell sample and using the enzyme reverse transcriptase to synthesize complementary DNA (cDNA) strands. This results in double-stranded cDNAs, which can then be cloned into bacterial vectors for maintenance and study.
Citing its advantages, cDNA libraries are smaller and more manageable than genomic libraries because they contain only the sequences of actively expressed genes, without introns. They also facilitate the examination of gene expression patterns among different tissues or during various developmental stages. However, it is important to note that cDNA versions of genes may differ from their original sequences and lack necessary regulatory regions for transcription. Overall, cDNA libraries are valuable tools in genetic research, aiding in the identification of expressed genes and their chromosomal locations, contributing to our understanding of molecular biology and disease.
cDNA libraries
SIGNIFICANCE: A cDNA library is a set of cloned DNA copies of the RNAs found in a specific cell type at a specific time. This library can be used to construct probes for mapping these genes, to study the changing expression of genes over time (during development, for example), or to clone genes into organisms for further study or production of proteins.
Gene Cloning and DNA Libraries
In order to study and map genes, researchers need to take potentially very large sections of DNA (such as a chromosome or whole genome), break them into smaller, manageable fragments, and clone these fragments to construct a DNA library. A genomic or chromosome library may contain many thousands of cloned fragments, many of which will represent stretches of between genes. If the researcher is interested in studying the protein-coding regions, or genes, of the DNA, it is better to start with the messenger RNAs (mRNAs) of the cell, which represent the genes being actively transcribed in the cell at that time. By constructing and cloning copies of these mRNAs, researchers can create a library that contains copies of only the active genes.
cDNA Library Construction
DNA copies of mRNAs are synthesized using the reverse transcriptase. This enzyme was independently discovered by Howard Temin and David Baltimore in 1970 in retroviruses, which “reverse transcribe” their RNA genomes into DNA after infecting their host cells. In the late 1970s, researchers began using the enzyme to make DNA copies of mRNAs, and later to construct cDNA libraries.
To create a from a sample of cells, mRNAs from the cells are isolated and purified. Reverse transcriptase is used to synthesize a complementary DNA strand using each strand as a template, resulting in a collection of double-stranded RNA-DNA hybrids. To obtain double-stranded cDNAs suitable for cloning, the enzyme H is used to digest the RNA strand, and I is used to synthesize the second DNA strand using the first as a template. If desired, “sticky ends” can be added to the cDNAs for cloning into a vector. The set of recombinant vectors are inserted into bacterial cells in the process of transformation, resulting in a cloned cDNA library. The library is maintained as a collection of bacterial colonies, each colony containing a different cloned DNA fragment.
Applications
A cDNA library represents the coding sequences of genes that were actively expressed in the original cell sample at the time the sample was taken. In effect, it can represent a snapshot of active genes in the cells at that time. Comparing the cDNAs of different tissues from the same organism can reveal the differences in of these tissues. Also, comparing cDNAs of cells in the same tissue over time can show how gene expression changes in the same cells. This approach has been especially fruitful in developmental genetic research, because the developmental pattern of an organism can be correlated with the activity of specific genes.
Cloned cDNAs can also be used to find the chromosomal location of an expressed gene. One strand of the cDNA clone is labeled with a fluorescent tag and used as a molecular probe. In the technique of in situ hybridization, the will base pair, or hybridize, to the complementary sequence in a preparation of partially denatured chromosomes, and the chromosomal location of the original gene will be visible because of the fluorescent label. Such a probe can also be used to screen a chromosome or for the cloned fragment containing the target gene. Using the entire cDNA library to probe a genome will generate a cDNA map that suggests the most biologically and medically important parts of the genome, aiding researchers in the search for disease genes.
Genes of eukaryotes (nonbacterial organisms) usually contain introns, noncoding segments that are transcribed but removed from mRNAs before translation, but bacterial genes do not. Often, a eukaryotic gene put into a bacterial cell will not produce a functional because the cell does not have the biochemical machinery for removing introns. If the goal of the research is to have a bacterium make the protein product of a gene, it may be necessary to clone a cDNA version of the gene, which lacks introns, using a special that allows the cell to transcribe the inserted gene and translate it to the proper polypeptide.
Advantages and Disadvantages
Because cDNA libraries contain only DNA of expressed genes, they are much smaller and more easily managed and studied than chromosome or genomic libraries that have all coding and noncoding regions. The cDNA versions of genes have only the protein-coding sequence, without introns, so that cloning them in bacteria allows expression of the protein products of the genes. In contrast to other DNA libraries, cDNA libraries can be used to study variable patterns of gene expression among cell types or over time. In eukaryotes, cDNA copies of genes are not identical to the original sequences of the genes and also lack the region necessary for proper transcription of the gene. However, using cDNA as a molecular probe can lead to the identification of the original gene.
Key Terms
- complementary DNA (cDNA)also known as copy DNA, a form of DNA synthesized by reverse transcribing RNAs (usually messenger RNAs) into DNA
- DNA librarya collection of DNA fragments cloned from a single source, such as a genome, chromosome, or set of mRNAs
- in situ hybridizationa technique that uses a molecular probe to determine the chromosomal location of a gene
- intronsnoncoding segments of DNA within a gene that are removed from mRNA copies of the gene before polypeptide translation
- reverse transcriptasean enzyme, isolated from retroviruses, that synthesizes a DNA strand from an RNA template
Bibliography
Bacher, Rhonda, et al. "Enhancing Biological Signals and Detection Rates in Single-Cell RNA-Seq Experiments with cDNA Library Equalization." Nucleic Acids Research, vol. 50, no. 2, 2022, p. e12, doi.org/10.1093/nar/gkab1071. Accessed 9 Sept. 2024.
Dale, Jeremy, and Malcolm von Schantz. “Genomic and cDNA Libraries.” From Genes to Genomes: Concepts and Applications of DNA Technology. 3rd ed. Hoboken: Wiley, 2012. Print.
Lu, Chaofu, John Browse, and James G. Wallis. CDNA Libraries: Methods and Applications. New York: Humana, 2011. Print.
Sambrook, Joseph, and David Russell. Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor: Cold Spring Harbor Laboratory, 2012. Print.
Watson, James D., et al. Recombinant DNA: Genes and Genomes: A Short Course. 3rd ed. New York: Freeman, 2007. Print.
Ying, Shao-Yao. Generation of cDNA Libraries: Methods and Protocols. Totowa: Humana, 2003. Print.