Library (biology)
Encyclopedia
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
(formed from reverse-transcribed RNA
), genomic libraries
(formed from genomic DNA) and randomized mutant libraries (formed by de novo gene synthesis where alternative nucleotides or codons are incorporated). DNA library technology is a mainstay of current molecular biology, 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 (vector + insert). As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated (thus, "cloned").
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 tag
s".
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:
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:
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.
or the genome of a bacteriophage
that has been used to infect bacterial cells.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, 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
Molecular cloning
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
. There are different types of DNA libraries, including cDNA libraries
CDNA library
A cDNA library is a combination of cloned cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an...
(formed from reverse-transcribed RNA
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
), genomic libraries
Gene library
A genomic library is a population of host bacteria, each of which carries a DNA molecule that was inserted into a cloning vector, such that the collection of cloned DNA molecules represents the entire genome of the source organism...
(formed from genomic DNA) and randomized mutant libraries (formed by de novo gene synthesis where alternative nucleotides or codons are incorporated). DNA library technology is a mainstay of current molecular biology, 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 (vector + insert). As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated (thus, "cloned").
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
main article: cDNA libraryCDNA library
A cDNA library is a combination of cloned cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an...
A cDNA library
CDNA library
A cDNA library is a combination of cloned cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an...
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
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
. 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 tag
Expressed sequence tag
An expressed sequence tag or EST is a short sub-sequence of a cDNA sequence. They may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination. The identification of ESTs has proceeded rapidly, with approximately 65.9 million ESTs now available in...
s".
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 splicingAlternative splicingAlternative splicing is a process by which the exons of the RNA produced by transcription of a gene are reconnected in multiple ways during RNA 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 vectorCloning vector
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments...
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 projectGenome projectGenome projects are scientific endeavours that ultimately aim to determine the complete genome sequence of an organism and to annotate protein-coding genes and other important genome-encoded features...
) - Serving as a source of genomic sequence for generation of transgenic animals through genetic engineeringGenetic engineeringGenetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
- Study of the function of regulatory sequences in vitro
- Study of genetic mutations in cancerCancerCancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
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. 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 clonesCloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
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
Directed evolution
thumb|250px|right|An example of a possible round to evolve a protein based fluorescent sensor for a specific analyte using two consecutive FACS sortings...
experiments.
Vectors
The nucleotide sequences of interest are preserved as inserts to a plasmidPlasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
or the genome of a bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
that has been used to infect bacterial cells.