Library (biology)


In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depends on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.

Terminology

The term "library" can refer to a population of organisms, each of which carries a DNA molecule inserted into a cloning vector, or alternatively to the collection of all of the cloned vector molecules.

cDNA libraries

A cDNA library represents a sample of the mRNA purified from a particular source, 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 portion of the overall genome in a given organism.
Applications of cDNA libraries include:
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 the size of the genome in question and 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.
Applications of genomic libraries include:
In contrast to the library types described above, a variety of artificial methods exist for making libraries of variant genes. Variation throughout the gene can be introduced randomly by either error-prone PCR, DNA shuffling to recombine parts of similar genes together, or transposon-based methods to introduce indels.
Alternatively, mutations can be targeted to specific codons during de novo synthesis or saturation mutagenesis to construct one or more point mutants of a gene in a controlled way. This results in a mixture of double stranded DNA molecules which represent variants of the original gene.
The expressed proteins from these libraries can then be screened for variants which exhibit favorable properties. This can be repeated in cycles of creating gene variants and screening the expression products in a directed evolution process.

Overview of cDNA library preparation techniques

DNA Extraction

If creating an mRNA library, there are several possible protocols for isolating full length mRNA. To extract DNA for genomic DNA 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. 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 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 makes them useful for packaging the vector and then introducing them into the bacterial 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.

Using libraries

This involves "screening" for the sequences of interest. There are multiple possible methods to achieve this.