Gene library

A term used to describe a collection of DNA fragments derived from the genome of an organism
and cloned randomly into suitable cloning vectors (plasmids, phages). If plasmid cloning vectors
are used for the establishment of the library, this library essentially is a collection of host cells,
each of which contains a plasmid with an inserted DNA fragment. If phages have been used to
clone the DNA fragments, the library consists of a phage lysate with each phage containing an
inserted fragment of DNA. Together the individual fragments in the collection of inserted
molecules represent the entire genetic information contained in an organism and in this case the
library is said to be a representative library.

If the library has been established by using fragmented cellular DNA of an organism the library
is said to be a genomic library. The term genomic DNA clone or chromosomal DNA clone
then refers to an individual cell carrying a cloning vector with one of the cellular DNA fragments
or to a phage isolate with a specific DNA insert. Subgenomic libraries are obtained if only
selected portions of the genome, for example fragments from distinct sorted chromosomes, are
represented in the library. A characteristic of genomic libraries is the fact that the individual
inserts still contain non-coding intron sequences. Such libraries are used, therefore, for
determining the genomic structures of genes.
Principle of gene cloning and preparation of gene libraries.

A suitable cloning vector (in this example a circular plasmid) is linearized by cleavage with a
suitable restriction enzyme (1). The cellular DNA containing the gene of interest (red) is also
cleaved into fragments with this enzyme (2). For various reasons the cellular DNA is cleaved in
a way yielding N overlapping fragments with a uniform length of

20 kb. Linearized vector DNA and genomic DNA fragments are then ligated in vitro, yielding a
population of N recircularized cloning vector molecules, each of which contains an insert of
cellular genomic DNA (3). These molecules are introduced into suitable host cells (4).

The collection of cloning vectors with inserts obtained at step 3 or the collection of cells obtained
after step 4 are called a gene library. This library is called a representative library if the sum of
the genomic DNA inserts found in the cloning vectors of all cells represents the entire cellular
DNA. Only a representative library, i.e., only the presence of all DNA fragments generated
originally, guarantees a reasonable chance of finding the desired gene. Statistical analysis shows
that for human DNA more than 600000 cell clones must be screened in order to find a particular
DNA fragment containing the desired gene with a probability of 99 %.

In principle libraries can be made also with cellular RNA as starting material. The resulting
collection of host cells containing recombinant vectors is then called a cDNA library.

The art of cloning is to find the one particular cell (marked red) which contains the cloning
vector with the gene of interest. This process is called library screening. If this goal has been
achieved the gene in question is said to have been cloned.

Many different techniques are available for screening a library. The most important approaches
involve screening by nucleic acid hybridization and screening by functional analysis. Nucleic
acid hybridization requires some prior knowledge of the DNA sequence either of the gene to be
cloned or of stretches of DNA in the vicinity of the gene to be cloned. Functional screening
involves the use of expression vectors that allow cells containing the vector with the desired gene
to express the corresponding protein. Under these circumstances, cells containing a vector with
the desired gene can be identified by means of antibodies directed against the protein. Cells
producing the desired gene product can also be identified in bioassays detecting protein
activities, if these are available.

Principle of gene cloning and preparation of gene libraries.

A suitable cloning vector (in this example a circular plasmid) is linearized by cleavage with a
suitable restriction enzyme (1). The cellular DNA containing the gene of interest (red) is also
cleaved into fragments with this enzyme (2). For various reasons the cellular DNA is cleaved in
a way yielding N overlapping fragments with a uniform length of

20 kb. Linearized vector DNA and genomic DNA fragments are then ligated in vitro, yielding a
population of N recircularized cloning vector molecules, each of which contains an insert of
cellular genomic DNA (3). These molecules are introduced into suitable host cells (4).
The collection of cloning vectors with inserts obtained at step 3 or the collection of cells obtained
after step 4 are called a gene library. This library is called a representative library if the sum of
the genomic DNA inserts found in the cloning vectors of all cells represents the entire cellular
DNA. Only a representative library, i.e., only the presence of all DNA fragments generated
originally, guarantees a reasonable chance of finding the desired gene. Statistical analysis shows
that for human DNA more than 600000 cell clones must be screened in order to find a particular
DNA fragment containing the desired gene with a probability of 99 %.

In principle libraries can be made also with cellular RNA as starting material. The resulting
collection of host cells containing recombinant vectors is then called a cDNA library.

The art of cloning is to find the one particular cell (marked red) which contains the cloning
vector with the gene of interest. This process is called library screening. If this goal has been
achieved the gene in question is said to have been cloned.

Many different techniques are available for screening a library. The most important approaches
involve screening by nucleic acid hybridization and screening by functional analysis. Nucleic
acid hybridization requires some prior knowledge of the DNA sequence either of the gene to be
cloned or of stretches of DNA in the vicinity of the gene to be cloned. Functional screening
involves the use of expression vectors that allow cells containing the vector with the desired gene
to express the corresponding protein. Under these circumstances, cells containing a vector with
the desired gene can be identified by means of antibodies directed against the protein. Cells
producing the desired gene product can also be identified in bioassays detecting protein
activities, if these are available.

A special form a gene library that is gaining importance is the subtractive library or
differential library. Terms such as subtractive hybridization, subtraction cloning,
differential gene screening, differential hybridization, or genomic difference cloning
refer to the screening of such a library with the aim to isolate a specific gene.
General strategy for subtractive library screening.

Consider two cell types, designated A and B. These cells may be completely different (for
example brain and liver cells) or identical cells that differ from each other, for example, in their
activation state or pretreatment with cytokines.

In the initial step (step 1) polyadenylated mRNA (jagged lines ending in AAAAA) is isolated
from both types of cells. The RNA shown in green is expressed in cell type A only while that
shown in blue is expressed in cell type B only. RNAs expressed in either cell type are shown in
black.

RNA isolated from cell type A is used to generate single-stranded cDNA by reverse transcription
(step 2). This cDNA is then hybridized with RNA from cell type B (step 3). Those RNAs (black)
expressed in both cell types will form DNA/RNA hybrids while cDNA single strands derived
from type A-specific mRNA not present in type B-cells will remain single stranded. Likewise,
RNA that is expressed in type B but not in type A cells will remain single stranded. Single and
double stranded molecules are separated by hydroxyapatite chromatography (step 4). This yields
fractions containing only single stranded DNA or RNA molecules. Removal of RNA by
treatment with alkali yields a population of cDNA molecules derived from those RNAs
expressed specifically in type A cells only. These can be converted into double stranded DNA
which can then be cloned.

If the aim is to investigate those RNAs expressed specifically in type B-cells only type B-
specific RNA is converted into single stranded DNA and hybridized with RNA from type A
cells.