I have posted a video on the key differences between genomic DNA (gDNA) and complementary DNA (cDNA) libraries - DNA Libraries: Genomic vs. cDNA - Key Differences and Applications.
DNA libraries are essential tools for researchers to study and manipulate genetic material. If you're working in a lab, you'll likely encounter two main types of DNA libraries: genomic DNA (gDNA) libraries and complementary DNA (cDNA) libraries. Though both serve important roles in research, they are fundamentally different in their composition, creation, and applications.
What Are DNA Libraries?
A DNA library is a collection of DNA sequences cloned into vectors, small pieces of DNA that can carry foreign DNA into a host cell, such as bacteria. These libraries allow scientists to store, access, and manipulate specific DNA sequences for various research purposes.
Genomic DNA (gDNA) Libraries
A genomic DNA library is created from the complete genomic DNA of an organism. This means it contains all the genetic material necessary to build that organism. In eukaryotes, this includes both the coding regions (exons) and the non-coding regions (introns) of genes.
How is a gDNA Library Made?
- DNA Extraction: Genomic DNA is extracted from cells.
- DNA Fragmentation: The extracted DNA is cut into smaller fragments using restriction enzymes.
- Cloning: These fragments are then cloned into plasmids, which are circular DNA molecules.
- Transformation: The plasmids are inserted into bacteria, which replicate the DNA fragments, creating a library.
When to Use a gDNA Library?
gDNA libraries are ideal when studying the full structure of genes, including regulatory elements, or when exploring gene functions across the genome.
However, there are some limitations. Because a gDNA library contains both introns and exons, it can complicate the task of isolating and studying the sequences that actually code for proteins. Additionally, genes in a gDNA library may be fragmented across multiple clones, making it challenging to reconstruct the complete gene sequence through sequence alignment.
Complementary DNA (cDNA) Libraries
cDNA libraries are derived from messenger RNA (mRNA) in a cell. This means that a cDNA library only contains the sequences actively expressed as proteins when the library is made. Therefore, the content of a cDNA library depends on the type of cell, the time of day, and the cell's conditions.
How is a cDNA Library Made?
- mRNA Isolation: Cells are lysed, and the mRNA is purified from the lysate using affinity chromatography, which often involves oligo-dT beads that bind to the poly-A tails of mRNA molecules.
- Reverse Transcription: Using reverse transcriptase, the mRNA is used as a template to synthesise complementary DNA (cDNA). This gives a mRNA/DNA molecule.
- RNA Removal: The original mRNA is removed from the mRNA/DNA molecule using an enzyme (RNase H). The now single-stranded cDNA is converted into a double-stranded DNA molecule using DNA polymerase.
- Cloning: The cDNA is cloned into plasmids and inserted into bacteria.
When to Use a cDNA Library?
cDNA libraries are particularly useful for studying gene expression, identifying specific mRNA sequences, and producing recombinant proteins.
Choosing the Right Library
It is essential to understand the distinctions between gDNA and cDNA libraries. A gDNA library is the right choice if the goal is to study the entire genome or understand gene regulation. However, a cDNA library must be used if you're interested in the proteins a cell produces or need to work with specific mRNA sequences.
