What are you going to learn?
- What are DNA libaries?
- What types of DNA libraries do we have?
- How do we make a DNA library?
- Why are DNA libraries useful?
- terms: genomic DNA library, complementary DNA (cDNA) library, library screening, probe
DNA libraries are collections of cloned DNA fragments. If we isolated all the DNA from human cells, cut it into fragments, insert it into cloning vectors, let it clone in bacterial cells then we would get a DNA library.
There are two main types of DNA libraries, genomic DNA libraries and complementary DNA (cDNA) libraries. A genomic library contains copies of every DNA sequence in the organism. To make a genomic library, we take DNA from cells and cut it with restriction enzymes. The fragments are then inserted into vectors and cloned. The problem with genomic libraries is that because the whole genome is cloned, the library also contains non-coding DNA segments. The cloning vectors are carefully chosen based on the length of DNA that is to be inserted. For example, for the human genome, yeast artificial chromosomes were used, because they can carry large DNA fragments.
1) genomic DNA libraries
2) complementary (cDNA) libraries
Complementary DNA (cDNA) libraries, contain DNA copies made from mRNA molecules. For that reason, cDNA libraries only consist of DNA sequences that are transcribed into mRNA and that were transcriptionally active at the time the library was made.
In order to create a cDNA library, first a short oligo(dT) molecule must anneal to the poly-A tail at the end of an mRNA. This molecule serves as a primer for an enzyme called reverse transcriptase, an enzyme isolated from retroviruses, that uses the mRNA as a template to synthesize a complementary DNA strand (cDNA). The mRNA is then partially digested with the enzyme RNAse H. The 3’ ends of the remaining mRNA serve as primers for DNA polymerase, which synthesizes a second DNA strand. DNA ligase then seals the nicks in the DNA backbone. The result is a double-stranded cDNA molecule, which can now be cloned. It is important to realise that DNA differs from standard DNA because it does not contain noncoding sequences as it was made from mRNA.
1) an oligo(dT) molecule anneals to the poly-A tail at the end of an mRNA
2) reverse transcriptase synthesizes a complementary DNA strand
3) mRNA is partially digested by RNAse H
4) DNA polymerase synthesizes a second DNA strand
5) DNA ligase seals the nicks in the DNA backbone
cDNA libraries are useful for studying genes that are expressed under different conditions or for comparing expressed genes from different tissues.
To find a specific gene in a DNA library, different methods of library screening are used. One method uses a probe, a DNA or RNA sequence that is complementary to some part of the gene of interest that is to be identified. The probe binds to these complementary DNA sequences in the clones and shows the location of the gene. Of course, the probe must be labelled so that it can be identified. Probes used to be labelled with radioactive isotopes, but now they are labelled with compounds that undergo colour reactions for an easy identificaiton. Probes can be obtained from similar genes from other organisms or they can be synthetically produced.
Another method that is used for screening DNA libraries is finding the protein product of the gene. The DNA library is cloned in an expression vector, a vector that ensures that the protein product of the cloned gene is produced. The clones are then tested for the presence of the protein product by using different chemical tests.
Even though DNA libraries are still important today, they have been largely replaced by new genomic techniques.
References:
Klug, W. S., Cummings, M. R., Spencer, C. A., Palladino, M. A., & Killian, D. (2019). Concepts of Genetics. Pearson.
Pierce, B. A. (2019). Genetics: A Conceptual Approach (Seventh ed.). W. H. Freeman.