Library (biology)

cDNA libraries

A cDNA library represents a sample of the mRNA purified from a particular source (either a collection of cells, a particular tissue, or an entire organism), which has been converted back to a DNA template by the use of the enzyme reverse transcriptase. It thus represents the genes that were being actively transcribed in that particular source under the physiological, developmental, or environmental conditions that existed when the mRNA was purified. cDNA libraries can be generated using techniques that promote "full-length" clones or under conditions that generate shorter fragments used for the identification of "expressed sequence tags".

cDNA libraries are useful in reverse genetics, but they only represent a very small (less than 1%) portion of the overall genome in a given organism.

Applications of cDNA libraries include:

• Discovery of novel genes
• Cloning of full-length cDNA molecules for in vitro study of gene function
• Study of the repertoire of mRNAs expressed in different cells or tissues
• Study of alternative splicing in different cells or tissues

Genomic libraries

A genomic library is a set of clones that together represents the entire genome of a given organism. The number of clones that constitute a genomic library depends on (1) the size of the genome in question and (2) the insert size tolerated by the particular cloning vector system. For most practical purposes, the tissue source of the genomic DNA is unimportant because each cell of the body contains virtually identical DNA (with some exceptions).

Applications of genomic libraries include:

• Determining the complete genome sequence of a given organism (see genome project)
• Serving as a source of genomic sequence for generation of transgenic animals through genetic engineering
• Study of the function of regulatory sequences in vitro
• Study of genetic mutations in cancer tissues

Randomized mutant libraries

In contrast to the library types described above, a randomized mutant library is created by de novo synthesis of a gene.^[1] During synthesis, alternative nucleotides or codons are incorporated into the DNA sequence at specific positions. This results in a mixture of double stranded DNA molecules which represent variants of the original gene. These variants can then be ligated into an expression vector, individual clones can be created, and the encoded protein variants can be expressed.

The expressed proteins can then be screened for variants which exhibit favourable properties. Typically the properties that are to be improved by screening a randomized mutant library are the binding affinity of antibodies or other protein-protein interactions, the activity of enzymes, or the stability of a protein. Multiple cycles of creating gene variants and screening the expression products are typically involved in directed evolution experiments.

DNA Extraction

If creating an mRNA library (ie with cDNA clones), there are several possible protocols for isolating full length mRNA. To extract DNA for genomic DNA (also known as gDNA) libraries, a DNA mini-prep may be useful.

Prepare Inserts

cDNA libraries require care to ensure that full length clones of mRNA are captured as cDNA (which will later be inserted into vectors). Several protocols have been designed to optimise the synthesis of the 1st cDNA strand and the 2nd cDNA strand for this reason, and also to make directional cloning into the vector more likely.

gDNA fragments are generated from the extracted gDNA by using non-specific frequent cutter restriction enzymes.

Vectors

The nucleotide sequences of interest are preserved as inserts to a plasmid or the genome of a bacteriophage that has been used to infect bacterial cells.

Vectors are propagated most commonly in bacterial cells, but if using a YAC (Yeast Artificial Chromosome) then yeast cells may be used. Vectors could also be propagated in viruses, but this can be time consuming and tedious. However, the high transfection efficiency achieved by using viruses (often phages) makes them useful for packaging the vector (with the ligated insert) and then introducing them into the bacterial (or yeast) cell.

Additionally, for cDNA libraries, a system using the Lambda Zap II phage, ExAssist, and 2 E. coli species has been developed. A Cre-Lox system using loxP sites and the in vivo expression of the recombinase enzyme can also be used instead. These are examples of in vivo excision systems. In vitro excision involves subcloning often using traditional restriction enzymes and cloning strategies. In vitro excision can be more time-consuming and may require more "hands-on" work than in vivo excision systems. In either case, the systems allow the movement of the vector from the phage into a live cell, where the vector can replicate and propagate until the library is to be used.