Library (biology)
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In
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
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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 sequencesin vitro
- Study of genetic mutations in cancertissues
Synthetic mutant libraries
In contrast to the library types described above, a variety of artificial methods exist for making libraries of variant genes.
The
Overview of cDNA library preparation techniques
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DNA extraction
If creating an mRNA library (i.e. 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.
Insert preparation
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.
Using libraries
This involves "screening" for the sequences of interest. There are multiple possible methods to achieve this.