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Functional cloning is a
molecular cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
technique that relies on prior knowledge of the encoded
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
’s
sequence
In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is calle ...
or
function
Function or functionality may refer to:
Computing
* Function key, a type of key on computer keyboards
* Function model, a structured representation of processes in a system
* Function object or functor or functionoid, a concept of object-oriente ...
for
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
identification.
In this assay, a
genomic
Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dim ...
or
cDNA library is screened to identify the genetic sequence of a protein of interest.
Expression
Expression may refer to:
Linguistics
* Expression (linguistics), a word, phrase, or sentence
* Fixed expression, a form of words with a specific meaning
* Idiom, a type of fixed expression
* Metaphorical expression, a particular word, phrase, o ...
cDNA libraries may be screened with
antibodies
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
specific for the protein of interest or may rely on selection via the protein function.
Historically, the amino acid sequence of a protein was used to prepare degenerate oligonucleotides which were then
probed against the library to identify the gene encoding the protein of interest.
Once candidate clones carrying the gene of interest are identified, they are
sequenced
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which suc ...
and their identity is confirmed. This method of cloning allows researchers to screen entire
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
s without prior knowledge of the location of the gene or the genetic sequence.
This technique can be used to identify genes that encode similar proteins from one organism to another. Similarly, this technique can be paired with
metagenomic
Metagenomics is the study of genetic material recovered directly from environmental or clinical samples by a method called sequencing. The broad field may also be referred to as environmental genomics, ecogenomics, community genomics or microb ...
libraries to identify novel genes and proteins that perform similar functions, such as the identification of novel
antibiotics
An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention o ...
by screening for
beta-lactamase
Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem), although carbapenems ...
activity or selecting for growth in the presence of
penicillin
Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...
.
Experimental workflow
![Genomic Library in S](https://upload.wikimedia.org/wikipedia/commons/0/0a/Genomic_Library_in_S._cerevisiae.jpg)
The workflow of a functional cloning experiment varies depending on the source of genetic material, the extent of prior knowledge of the protein or gene of interest and the ability to screen for the protein function. In general, a functional cloning experiment consists of four steps: 1) sample collection, 2) library preparation, 3) screening or selection and 4)
sequencing
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...
.
Sample collection
Genetic material is collected from a particular cell type, organism or environmental sample relevant to the biological question. In functional cloning,
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
is commonly isolated and
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
is prepared from the isolated mRNA (
RNA extraction
RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology t ...
). In certain circumstances
genomic DNA
Genomic deoxyribonucleic acid (abbreviated as gDNA) is chromosomal DNA, in contrast to extra-chromosomal DNAs like plasmids. Most organisms have the same genomic DNA in every cell; however, only certain genes are active in each cell to allow for ...
may be isolated, particularly when environmental samples are used as the source of genetic material.
Library preparation
If the starting material is
genomic DNA
Genomic deoxyribonucleic acid (abbreviated as gDNA) is chromosomal DNA, in contrast to extra-chromosomal DNAs like plasmids. Most organisms have the same genomic DNA in every cell; however, only certain genes are active in each cell to allow for ...
, the DNA is sheared to produce fragments of appropriate length for the
vector
Vector most often refers to:
*Euclidean vector, a quantity with a magnitude and a direction
*Vector (epidemiology), an agent that carries and transmits an infectious pathogen into another living organism
Vector may also refer to:
Mathematic ...
of choice. The DNA fragments or cDNA are then treated with
restriction endonucleases
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
and
ligated to a
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
or chromosomal vectors. In the case of assays that screen for the protein or for its function, an
expression vector
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
is used to ensure that the gene product is expressed. The vector choice will depend on the origin of the DNA or cDNA to ensure proper expression and to ensure that the encoded gene will fall within the limits of the vector’s insert size.
The choice of host is important to ensure that the
codon usage will be similar to the donor organism. The host will also need to guarantee that the proper
post-translational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
s and
protein folding
Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduci ...
will occur to enable proper functioning of the expressed proteins.
Screening or selection
The method of screening the prepared genomic or cDNA libraries for the gene of interest is highly variable depending on the experimental design and biological question. One method of screening is to
probe colonies via
Southern blot
A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detecti ...
ting with degenerate oligonucleotides prepared from the amino acid sequence of the query protein.
In expression libraries, the protein of interest can be identified by screening with an antibody specific for the query protein via
Western blot
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...
ting to identify colonies carrying the gene of interest. In other circumstances, a specific assay can be used to screen or select for the protein’s activity.
For example, genes conferring
antibiotic resistance
Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. ...
can be selected by growing the colonies of the library on media containing a specified
antibiotic
An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of ...
.
Another example is screening for
enzymatic activity
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.
Enzyme units
The quantity or concentration of an enzyme can be expressed in molar amounts, as with an ...
by incubating with a substrate that is catalyzed to a colorimetric compound that can easily be visualized.
Sequencing
The final step of functional cloning is to
sequence
In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is calle ...
the DNA or cDNA from the clones that were successfully identified in the screen or selection step. The sequence can then be annotated and used for downstream applications, such as
protein expression and
purification for industrial applications.
Advantages
The advantages of functional cloning include the ability to screen for novel genes with desired applications in organisms that cannot be cultured, particularly from bacterial or viral specimens.
Additionally, genes encoding proteins with related functions can be identified when there is low sequence similarity due to the ability to screen for the protein function alone. Functional cloning allows for gene identification without prior knowledge of the organism’s genome sequence or position of the gene within the genome.
Limitations
As with other cloning techniques, vector and host choice affect the success of gene identification via functional cloning due to cloning bias. The vector must have an insert size that will accommodate the entire DNA sequence of the expressed protein. Additionally, in expression vectors the
promoters and
terminators must function within the chosen host organism. The host choice may affect
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
and
translation
Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
due to differing
codon usage, transcriptional and translational machinery or
post-translational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
s within the host.
Other limitations include the labour-intensive library preparation and potential screens which can be both expensive and time-consuming.
Alternative approaches
Positional cloning
Positional cloning
A genetic screen or mutagenesis screen is an experimental technique used to identify and select individuals who possess a phenotype of interest in a mutagenized population. Hence a genetic screen is a type of phenotypic screen. Genetic screens c ...
is another molecular cloning technique for identification of a gene of interest. This method uses exact chromosomal location instead of function to guide gene identification.
Because of this, this method focuses on all the genetic material at a chromosomal
locus
Locus (plural loci) is Latin for "place". It may refer to:
Entertainment
* Locus (comics), a Marvel Comics mutant villainess, a member of the Mutant Liberation Front
* ''Locus'' (magazine), science fiction and fantasy magazine
** ''Locus Award' ...
and makes no assumptions about function.
In
model organism
A model organism (often shortened to model) is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workin ...
s such as
mice
A mouse ( : mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus' ...
or
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
, this method is used more frequently as the information about the position of a gene of interest can be obtained from the
sequenced genome. However, this method becomes much more cumbersome when sequence information is not available. In this case,
linkage analysis
Genetic linkage is the tendency of DNA sequences that are close together on a chromosome to be inherited together during the meiosis phase of sexual reproduction. Two genetic markers that are physically near to each other are unlikely to be separ ...
can also be used.
Functional cloning on the other hand is more readily used in organisms such as
bacterial pathogens that are
viable but nonculturable
Viable but nonculturable (VBNC) bacteria refers as to bacteria that are in a state of very low metabolic activity and do not divide, but are alive and have the ability to become culturable once resuscitated.
Bacteria in a VBNC state cannot grow on ...
and where sequence data is not available but gene homology or protein function is still of interest.
A way to differentiate between functional and positional cloning is to visualize genes as words. Functional cloning is like using a thesaurus to look up words and selecting for new words that have the same meanings (or functions).
Positional cloning is more like picking a specific page of a dictionary and then browsing only that page for any words of interest.
Computationally determine homology
With the advent of
sequencing
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...
technology becoming cheaper and cheaper, it is now more feasible to sequence an unknown genome and then computationally determine
homology
Homology may refer to:
Sciences
Biology
*Homology (biology), any characteristic of biological organisms that is derived from a common ancestor
* Sequence homology, biological homology between DNA, RNA, or protein sequences
*Homologous chrom ...
instead of screening. This brings the added benefit of being able to screen for multiple genes of interest at the same time and reduces experimental time. It also allows one to avoid labour-intensive cloning procedures as well. However, if this route is taken, there are other biases and hurdles one must consider. By using sequenced data, one is able to screen based on homology alone.
A function-based approach thus allows for discovery of novel enzymes whose functions would not have been predicted based on DNA sequence alone.
Therefore, while sequencing is less labour-intensive experimentally, it can also lead to missed genes of interest due to differing sequence homology in genes of related function.
Gibson assembly
Gibson assembly Gibson assembly is a molecular cloning method that allows for the joining of multiple DNA fragments in a single, isothermal reaction. It is named after its creator, Daniel G. Gibson, who is the chief technology officer and co-founder of the synthet ...
is a quick cloning method that uses three primary enzymes; 5'
exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is the ...
,
polymerase
A polymerase is an enzyme ( EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by copying a DNA template strand using base- ...
and
ligase
In biochemistry, a ligase is an enzyme that can catalyze the joining (ligation) of two large molecules by forming a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one of the larger molecules or the enzym ...
.
The exonuclease digests the 5' end of DNA fragments leaving a 3' overhang.
If there is significant homology (20-40 bp) on each end of the DNA insert, it can anneal with a complementary backbone.
Afterwards the polymerase can fill in the gaps while ligase fuses the nicks at the end.
This method greatly increases the rate of cloning and success rate of cloning into a vector backbone.
However, it requires the DNA fragment to have significant homology with the plasmid.
For this reason, knowledge of the sequence being cloned must be known beforehand. This is not a requirement with functional cloning.
TOPO cloning
TOPO Cloning TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosi ...
is a cloning method that uses
Taq polymerase
''Taq'' polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism ''Thermus aquaticus,'' from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to ''Taq'' or ''Ta ...
.
This is because Taq leaves a single
adenosine
Adenosine ( symbol A) is an organic compound that occurs widely in nature in the form of diverse derivatives. The molecule consists of an adenine attached to a ribose via a β-N9-glycosidic bond. Adenosine is one of the four nucleoside building ...
overhang on the 3’ end of
PCR reaction products.
Utilizing this knowledge, backbones with a 5’
thymine
Thymine () ( symbol T or Thy) is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nu ...
overhang can be used for cloning purposes.
In this case knowledge of the fragment being cloned must be known to be able to make PCR primers for it and the number of TOPO Cloning compatible vectors is relatively small. However, it provides the advantage that reactions only take about 5 minutes to do.
Gateway recombination cloning
Gateway recombination cloning is a cloning method in which a DNA fragment is moved from one plasmid backbone to another via a single
homologous recombination
Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may ...
event.
However, for this method to work, the DNA fragment of interest must be flanked by recombination sites.
While this method isn't strictly an alternative, it does allow the movement of DNA fragments from one plasmid to another quicker than creating a whole new genomic library. The reason this method may be used in conjunction with functional cloning is to put a library under a different promoter or on a backbone with a different selection marker.
This can come in handy if an individual wants to try functional cloning in a wide range of bacteria to try to combat the issue with
codon bias.
Applications
Determining homology in the environment
Metagenomics
Metagenomics is the study of genetic material recovered directly from environmental or clinical samples by a method called sequencing. The broad field may also be referred to as environmental genomics, ecogenomics, community genomics or microb ...
is one of the largest fields that commonly uses functional cloning. Metagenomics studies all the genetic material from a specific environmental sample, such as the gut microbiome or lake water.
Functional libraries are created that contain DNA fragments from the environment.
As the original bacterium that a DNA sequence originated from cannot be easily detected, creating metagenomic functional libraries possesses advantages. Less than 1% of all bacteria are easily cultured in the lab, leaving a large percentage of bacteria that cannot be grown. By using functional libraries, the gene functions of unculturable bacteria can still be studied.
Furthermore, these uncultured microbes provide a source for the discovery of novel enzymes with biotechnological applications. Some novel proteins that have been discovered from marine environments include enzymes such as proteases, amylases, lipases, chitinases, deoxyribonucleases and phosphatases.
Determining homology in a known species
There are situations in which it is imperative to determine if a gene homolog from one source is present in another organism. For example, identification of novel
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
s for
polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
reactions which synthesize DNA molecules from deoxyribonucleotides.
While human polymerase optimally works at 37 °C (98.6 °F), DNA does not denature until 94–98 °C (201–208 °F).
This poses a problem as at these temperatures the human DNA polymerase would denature during the denaturation step of the PCR reaction resulting in a non-functioning polymerase protein and a failed PCR. To combat this a DNA polymerase from a
thermophile
A thermophile is an organism—a type of extremophile—that thrives at relatively high temperatures, between . Many thermophiles are archaea, though they can be bacteria or fungi. Thermophilic eubacteria are suggested to have been among the earl ...
, or bacteria that grows at high temperatures, could be used. An example is
Taq polymerase
''Taq'' polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism ''Thermus aquaticus,'' from which it was originally isolated by Chien et al. in 1976. Its name is often abbreviated to ''Taq'' or ''Ta ...
which comes from the thermophilic bacterium ''
Thermus aquaticus
''Thermus aquaticus'' is a species of bacteria that can tolerate high temperatures, one of several thermophilic bacteria that belong to the ''Deinococcota'' phylum. It is the source of the heat-resistant enzyme ''Taq'' DNA polymerase, one of th ...
''.
One could set up a functional cloning screen to find homologous polymerases that have the added advantage of being thermostable at high temperatures.
With this in mind, 3173 Polymerase, another polymerase enzyme, now commonly used in
RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
reactions was discovered using the above theory.
In RT-PCR reactions, two separate enzymes are commonly used. The first is a retroviral
reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
to convert
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
to
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
.
The second is a thermostable DNA polymerase to amplify the target sequence.
3173 Polymerase is able to perform both enzymatic functions resulting in a better option for RT-PCR.
The enzyme was discovered using functional cloning from a viral host originally found in Octopus hot springs (93 °C) in Yellowstone National Park.
Human health applications
One of the ongoing challenges of treating bacterial infections is
antibiotic resistance
Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. ...
which commonly arises when patients do not take their full treatment of medication and hence allow bacteria to develop resistance to antibiotics over time.
[{{Cite journal, last1=Sommer, first1=Morten O. A., last2=Dantas, first2=Gautam, last3=Church, first3=George M., date=2009-08-28, title=Functional Characterization of the Antibiotic Resistance Reservoir in the Human Microflora, journal=Science, language=en, volume=325, issue=5944, pages=1128–1131, doi=10.1126/science.1176950, issn=0036-8075, pmid=19713526, pmc=4720503, bibcode=2009Sci...325.1128S] To understand how to combat antibiotic resistance it is important to understand how the bacterial genome is evolving and changing in healthy individuals with no recent usage of antibiotics to provide a baseline.
Using a functional cloning-based technique, DNA isolated from human
microflora
Microbiota are the range of microorganisms that may be commensal, symbiotic, or pathogenic found in and on all multicellular organisms, including plants. Microbiota include bacteria, archaea, protists, fungi, and viruses, and have been found to ...
were cloned into expression vectors in ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
''.
Afterwards, antibiotics were applied as a screen.
If a plasmid contained a gene insert that provided antibiotic resistance the cell survived and was selected on the plate.
If the insert provided no resistance, the cell died and did not form a colony.
Based on selection of cell colonies that survived, a better picture of genetic factors contributing to antibiotic resistance were pieced together. Most of the resistance genes that were identified were previously unknown.
By using a functional cloning-based technique one is able to elucidate genes giving rise to antibiotic resistance to better understand treatment for bacterial infections.
See also
*
cDNA library
*
Genetic screen
A genetic screen or mutagenesis screen is an experimental technique used to identify and select individuals who possess a phenotype of interest in a mutagenized population. Hence a genetic screen is a type of phenotypic screen. Genetic screens c ...
*
Genomic library A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is ex ...
*
Hybridization probe
In molecular biology, a hybridization probe (HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RNA ...
*
Molecular cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
References
External links
Functional Cloning, Sorting, and Expression Profiling of Nucleic Acid-Binding Proteins''Genome Research.com''
A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases''Oxford Journals]
Molecular genetics of Cohen syndrome''Department of Medical Genetics, University of Helsinki''
Genetics techniques
Molecular genetics
Medical genetics