Polymerase chain reaction (PCR) is a method widely used in molecular biology to rapidly

make millions to billions of copies of a specific DNA sample allowing scientists to take a very
small sample of DNA and amplify it to a large enough amount to study in detail. PCR was
invented in 1983 by Kary Mullis. It is fundamental to much of genetic testing including
analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies
of very small amounts of DNA sequences are exponentially amplified in a series or cycles of
temperature changes. PCR is now a common and often indispensable technique used in
medical laboratory and clinical laboratory research for a broad variety of applications
including biomedical research and criminal forensics.[1][2] The vast majority of PCR
methods rely on thermal cycling. Thermal cycling exposes reactants to repeated cycles of
heating and cooling to permit different temperature-dependent reactions – specifically,
DNA melting and enzyme-driven DNA replication. PCR employs two main reagents – primers
(which are short single strand DNA fragments known as oligonucleotides that are a
complementary sequence to the target DNA region) and a DNA polymerase. In the first step
of PCR, the two strands of the DNA double helix are physically separated at a high
temperature in a process called Nucleic acid denaturation. In the second step, the
temperature is lowered and the primers bind to the complementary sequences of DNA. The
two DNA strands then become templates for DNA polymerase to enzymatically assemble a
new DNA strand from free nucleotides, the building blocks of DNA. As PCR progresses, the
DNA generated is itself used as a template for replication, setting in motion a chain reaction
in which the original DNA template is exponentially amplified.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase,
an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the
polymerase used was heat-susceptible, it would denature under the high temperatures of
the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be
manually added every cycle, which was a tedious and costly process.[3]

Applications of the technique include DNA cloning for sequencing, gene cloning and
manipulation, gene mutagenesis; construction of DNA-based phylogenies, or functional
analysis of genes; diagnosis and monitoring of hereditary diseases; amplification of ancient
DNA;[4] analysis of genetic fingerprints for DNA profiling (for example, in forensic science
and parentage testing); and detection of pathogens in nucleic acid tests for the diagnosis of
infectious diseases.
Principles

A thermal cycler for PCR

An older, three-temperature thermal cycler for PCR

PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify
DNA fragments of between 0.1 and 10 kilo base pairs (kbp) in length, although some
techniques allow for amplification of fragments up to 40 kbp.[5] The amount of amplified
product is determined by the available substrates in the reaction, which become limiting as
the reaction progresses.[6]

A basic PCR set-up requires several components and reagents,[7] including a DNA template
that contains the DNA target region to amplify; a DNA polymerase; an enzyme that
polymerizes new DNA strands; heat-resistant Taq polymerase is especially common,[8] as it
is more likely to remain intact during the high-temperature DNA denaturation process; two
DNA primers that are complementary to the 3' (three prime) ends of each of the sense and
anti-sense strands of the DNA target (DNA polymerase can only bind to and elongate from a
double-stranded region of DNA; without primers there is no double-stranded initiation site
at which the polymerase can bind);[9] specific primers that are complementary to the DNA
target region are selected beforehand, and are often custom-made in a laboratory or
purchased from commercial biochemical suppliers; deoxynucleoside triphosphates, or
dNTPs (sometimes called "deoxynucleotide triphosphates"; nucleotides containing
triphosphate groups), the building blocks from which the DNA polymerase synthesizes a
new DNA strand; a buffer solution providing a suitable chemical environment for optimum
activity and stability of the DNA polymerase; bivalent cations, typically magnesium (Mg) or
manganese (Mn) ions; Mg2+ is the most common, but Mn2+ can be used for PCR-mediated
DNA mutagenesis, as a higher Mn2+ concentration increases the error rate during DNA
synthesis;[10] and monovalent cations, typically potassium (K) ions

The reaction is commonly carried out in a volume of 10–200 μL in small reaction tubes (0.2–
0.5 mL volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes
to achieve the temperatures required at each step of the reaction (see below). Many
modern thermal cyclers make use of the Peltier effect, which permits both heating and
cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-
walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal
equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of
the reaction tube. Older thermal cyclers lacking a heated lid require a layer of oil on top of
the reaction mixture or a ball of wax inside the tube.
Procedure
Typically, PCR consists of a series of 20–40 repeated temperature changes, called thermal
cycles, with each cycle commonly consisting of two or three discrete temperature steps (see
figure below). The cycling is often preceded by a single temperature step at a very high
temperature (>90 °C (194 °F)), and followed by one hold at the end for final product
extension or brief storage. The temperatures used and the length of time they are applied in
each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis,
the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature
(Tm) of the primers.[11] The individual steps common to most PCR methods are as follows:
Initialization: This step is only required for DNA polymerases that require heat activation by
hot-start PCR.[12] It consists of heating the reaction chamber to a temperature of 94–96 °C
(201–205 °F), or 98 °C (208 °F) if extremely thermostable polymerases are used, which is
then held for 1–10 minutes.
Denaturation: This step is the first regular cycling event and consists of heating the reaction
chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA melting, or
denaturation, of the double-stranded DNA template by breaking the hydrogen bonds
between complementary bases, yielding two single-stranded DNA molecules.
Annealing: In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F)
for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA
templates. Two different primers are typically included in the reaction mixture: one for each
of the two single-stranded complements containing the target region. The primers are
single-stranded sequences themselves, but are much shorter than the length of the target
region, complementing only very short sequences at the 3' end of each strand.
It is critical to determine a proper temperature for the annealing step because efficiency
and specificity are strongly affected by the annealing temperature. This temperature must
be low enough to allow for hybridization of the primer to the strand, but high enough for
the hybridization to be specific, i.e., the primer should bind only to a perfectly
complementary part of the strand, and nowhere else. If the temperature is too low, the
primer may bind imperfectly. If it is too high, the primer may not bind at all. A typical
annealing temperature is about 3–5 °C below the Tm of the primers used. Stable hydrogen
bonds between complementary bases are formed only when the primer sequence very
closely matches the template sequence. During this step, the polymerase binds to the
primer-template hybrid and begins DNA formation.
Extension/elongation: The temperature at this step depends on the DNA polymerase used;
the optimum activity temperature for the thermostable DNA polymerase of Taq (Thermus
aquaticus) polymerase is approximately 75–80 °C (167–176 °F),[13][14] though a
temperature of 72 °C (162 °F) is commonly used with this enzyme. In this step, the DNA
polymerase synthesizes a new DNA strand complementary to the DNA template strand by
adding free dNTPs from the reaction mixture that are complementary to the template in the
5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy
group at the end of the nascent (elongating) DNA strand. The precise time required for
elongation depends both on the DNA polymerase used and on the length of the DNA target
region to amplify. As a rule of thumb, at their optimal temperature, most DNA polymerases
polymerize a thousand bases per minute. Under optimal conditions (i.e., if there are no
limitations due to limiting substrates or reagents), at each extension/elongation step, the
number of DNA target sequences is doubled. With each successive cycle, the original
template strands plus all newly generated strands become template strands for the next
round of elongation, leading to exponential (geometric) amplification of the specific DNA
target region.
The processes of denaturation, annealing and elongation constitute a single cycle. Multiple
cycles are required to amplify the DNA target to millions of copies. The formula used to
calculate the number of DNA copies formed after a given number of cycles is 2n, where n is
the number of cycles. Thus, a reaction set for 30 cycles results in 230, or 1073741824, copies
of the original double-stranded DNA target region.
Final elongation: This single step is optional, but is performed at a temperature of 70–74 °C
(158–165 °F) (the temperature range required for optimal activity of most polymerases used
in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-
stranded DNA is fully elongated.
Final hold: The final step cools the reaction chamber to 4–15 °C (39–59 °F) for an indefinite
time, and may be employed for short-term storage of the PCR products.
To check whether the PCR successfully generated the anticipated DNA target region (also
sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis may be
employed for size separation of the PCR products. The size(s) of PCR products is determined
by comparison with a DNA ladder, a molecular weight marker which contains DNA
fragments of known size run on the gel alongside the PCR products.
Stages
As with other chemical reactions, the reaction rate and efficiency of PCR are affected by
limiting factors. Thus, the entire PCR process can further be divided into three stages based
on reaction progress:

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100%
reaction efficiency). After 30 cycles, a single copy of DNA can be increased up to
1,000,000,000 (one billion) copies. In a sense, then, the replication of a discrete strand of
DNA is being manipulated in a tube under controlled conditions.[15] The reaction is very
sensitive: only minute quantities of DNA must be present.
Leveling off stage: The reaction slows as the DNA polymerase loses activity and as
consumption of reagents, such as dNTPs and primers, causes them to become more limited.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.
Optimization
Main article: PCR optimization
In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination
causing amplification of spurious DNA products. Because of this, a number of techniques
and procedures have been developed for optimizing PCR conditions.[16][17] Contamination
with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR
mixtures from potential DNA contaminants.[7] This usually involves spatial separation of
PCR-setup areas from areas for analysis or purification of PCR products, use of disposable
plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-
design techniques are important in improving PCR product yield and in avoiding the
formation of spurious products, and the usage of alternate buffer components or
polymerase enzymes can help with amplification of long or otherwise problematic regions
of DNA. Addition of reagents, such as formamide, in buffer systems may increase the
specificity and yield of PCR.[18] Computer simulations of theoretical PCR results (Electronic
PCR) may be performed to assist in primer design.[19]

Applications
Selective DNA isolation
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a
specific region of DNA. This use of PCR augments many ways, such as generating
hybridization probes for Southern or northern hybridization and DNA cloning, which require
larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques
with high amounts of pure DNA, enabling analysis of DNA samples even from very small
amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified

sequences in which one of the amplification primers may be used in Sanger sequencing,
isolation of a DNA sequence to expedite recombinant DNA technologies involving the
insertion of a DNA sequence into a plasmid, phage, or cosmid (depending on size) or the
genetic material of another organism. Bacterial colonies (such as E. coli) can be rapidly
screened by PCR for correct DNA vector constructs.[20] PCR may also be used for genetic
fingerprinting; a forensic technique used to identify a person or organism by comparing
experimental DNAs through different PCR-based methods.

Some PCR 'fingerprints' methods have high discriminative power and can be used to identify
genetic relationships between individuals, such as parent-child or between siblings, and are
used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary
relationships among organisms when certain molecular clocks are used (i.e., the 16S rRNA
and recA genes of microorganisms).[21]
Medical and diagnostic applications
Prospective parents can be tested for being genetic carriers, or their children might be
tested for actually being affected by a disease.[1] DNA samples for prenatal testing can be
obtained by amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal
cells circulating in the mother's bloodstream. PCR analysis is also essential to
preimplantation genetic diagnosis, where individual cells of a developing embryo are tested
for mutations.

PCR can also be used as part of a sensitive test for tissue typing, vital to organ
transplantation. As of 2008, there is even a proposal to replace the traditional antibody-
based tests for blood type with PCR-based tests.[25]
Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study
these mutations, therapy regimens can sometimes be individually customized to a patient.
PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas, which
is currently the highest-developed in cancer research and is already being used routinely.
PCR assays can be performed directly on genomic DNA samples to detect translocation-
specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other
methods.[26] PCR is very useful in the medical field since it allows for the isolation and
amplification of tumor suppressors. Quantitative PCR for example, can be used to quantify
and analyze single cells, as well as recognize DNA, mRNA and protein confirmations and
combinations.[24]
Infectious disease applications
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those
caused by bacteria or viruses.[27] PCR also permits identification of non-cultivatable or
slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from
tissue culture assays and animal models. The basis for PCR diagnostic applications in
microbiology is the detection of infectious agents and the discrimination of non-pathogenic
from pathogenic strains by virtue of specific genes.[27][28]

Characterization and detection of infectious disease organisms have been revolutionized by

PCR in the following ways:

The human immunodeficiency virus (or HIV), is a difficult target to find and eradicate. The
earliest tests for infection relied on the presence of antibodies to the virus circulating in the
bloodstream. However, antibodies don't appear until many weeks after infection, maternal
antibodies mask the infection of a newborn, and therapeutic agents to fight the infection
don't affect the antibodies. PCR tests have been developed that can detect as little as one
viral genome among the DNA of over 50,000 host cells.[29] Infections can be detected
earlier, donated blood can be screened directly for the virus, newborns can be immediately
tested for infection, and the effects of antiviral treatments can be quantified.
Some disease organisms, such as that for tuberculosis, are difficult to sample from patients
and slow to be grown in the laboratory. PCR-based tests have allowed detection of small
numbers of disease organisms (both live or dead), in convenient samples. Detailed genetic
analysis can also be used to detect antibiotic resistance, allowing immediate and effective
therapy. The effects of therapy can also be immediately evaluated.
The spread of a disease organism through populations of domestic or wild animals can be
monitored by PCR testing. In many cases, the appearance of new virulent sub-types can be
detected and monitored. The sub-types of an organism that were responsible for earlier
epidemics can also be determined by PCR analysis.
Viral DNA can be detected by PCR. The primers used must be specific to the targeted
sequences in the DNA of a virus, and PCR can be used for diagnostic analyses or DNA
sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon
after infection and even before the onset of disease.[27] Such early detection may give
physicians a significant lead time in treatment. The amount of virus ("viral load") in a patient
can also be quantified by PCR-based DNA quantitation techniques (see below).
Diseases such as pertussis (or whooping cough) are cause by the bacteria Bordetella
pertussis. This bacteria is marked by a serious acute respiratory infection that affects various
animals and humans and has led to the deaths of many young children. The pertussis toxin
is a protein exotoxin that binds to cell receptors by two dimers and reacts with different cell
types such as T lymphocytes which plays a role in cell immunity.[30] PCR is an important
testing tool that can detect the sequences that are within the pertussis toxin gene. This is
because PCR has a high sensitivity for the toxin and has demonstrated a rapid turnaround
time. PCR is very efficient for diagnosing pertussis when compared to culture.[31]
Forensic applications
The development of PCR-based genetic (or DNA) fingerprinting protocols has seen
widespread application in forensics:

In its most discriminating form, genetic fingerprinting can uniquely discriminate any one
person from the entire population of the world. Minute samples of DNA can be isolated
from a crime scene, and compared to that from suspects, or from a DNA database of earlier
evidence or convicts. Simpler versions of these tests are often used to rapidly rule out
suspects during a criminal investigation. Evidence from decades-old crimes can be tested,
confirming or exonerating the people originally convicted.
Forensic DNA typing has been an effective way of identifying or exonerating criminal
suspects due to analysis of evidence discovered at a crime scene. The human genome has
many repetitive regions that can be found within gene sequences or in non-coding regions
of the genome. Specifically, up to 40% of human DNA is repetitive.[4] There are two distinct
categories for these repetitive, non-coding regions in the genome. The first category is
called variable number tandem repeats (VNTR), which are 10–100 base pairs long and the
second category is called short tandem repeats (STR) and these consist of repeated 2–10
base pair sections. PCR is used to amplify several well-known VNTRs and STRs using primers
that flank each of the repetitive regions. The sizes of the fragments obtained from any
individual for each of the STRs will indicate which alleles are present. By analyzing several
STRs for an individual, a set of alleles for each person will be found that statistically is likely
to be unique.[4] Researchers have identified the complete sequence of the human genome.
This sequence can be easily accessed through the NCBI website and is used in many real-life
applications. For example, the FBI has compiled a set of DNA marker sites used for
identification, and these are called the Combined DNA Index System (CODIS) DNA
database.[4] Using this database enables statistical analysis to be used to determine the
probability that a DNA sample will match. PCR is a very powerful and significant analytical
tool to use for forensic DNA typing because researchers only need a very small amount of
the target DNA to be used for analysis. For example, a single human hair with attached hair
follicle has enough DNA to conduct the analysis. Similarly, a few sperm, skin samples from
under the fingernails, or a small amount of blood can provide enough DNA for conclusive
analysis.[4]
Less discriminating forms of DNA fingerprinting can help in DNA paternity testing, where an
individual is matched with their close relatives. DNA from unidentified human remains can
be tested, and compared with that from possible parents, siblings, or children. Similar
testing can be used to confirm the biological parents of an adopted (or kidnapped) child.
The actual biological father of a newborn can also be confirmed (or ruled out).
The PCR AMGX/AMGY design has been shown to not only facilitating in amplifying DNA
sequences from a very minuscule amount of genome. However it can also be used for real
time sex determination from forensic bone samples. This provides us with a powerful and
effective way to determine the sex of not only ancient specimens but also current suspects
in crimes.[32]
Research applications
PCR has been applied to many areas of research in molecular genetics:

PCR allows rapid production of short pieces of DNA, even when not more than the sequence
of the two primers is known. This ability of PCR augments many methods, such as
generating hybridization probes for Southern or northern blot hybridization. PCR supplies
these techniques with large amounts of pure DNA, sometimes as a single strand, enabling
analysis even from very small amounts of starting material.
The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can easily
be produced from a patient with a genetic disease mutation. Modifications to the
amplification technique can extract segments from a completely unknown genome, or can
generate just a single strand of an area of interest.
PCR has numerous applications to the more traditional process of DNA cloning. It can
extract segments for insertion into a vector from a larger genome, which may be only
available in small quantities. Using a single set of 'vector primers', it can also analyze or
extract fragments that have already been inserted into vectors. Some alterations to the PCR
protocol can generate mutations (general or site-directed) of an inserted fragment.
Sequence-tagged sites is a process where PCR is used as an indicator that a particular
segment of a genome is present in a particular clone. The Human Genome Project found this
application vital to mapping the cosmid clones they were sequencing, and to coordinating
the results from different laboratories.
An exciting application of PCR is the phylogenic analysis of DNA from ancient sources, such
as that found in the recovered bones of Neanderthals, from frozen tissues of mammoths, or
from the brain of Egyptian mummies. Have been amplified and sequenced.[15] In some
cases the highly degraded DNA from these sources might be reassembled during the early
stages of amplification.
A common application of PCR is the study of patterns of gene expression. Tissues (or even
individual cells) can be analyzed at different stages to see which genes have become active,
or which have been switched off. This application can also use quantitative PCR to
quantitate the actual levels of expression
The ability of PCR to simultaneously amplify several loci from individual sperm[33] has
greatly enhanced the more traditional task of genetic mapping by studying chromosomal
crossovers after meiosis. Rare crossover events between very close loci have been directly
observed by analyzing thousands of individual sperms. Similarly, unusual deletions,
insertions, translocations, or inversions can be analyzed, all without having to wait (or pay)
for the long and laborious processes of fertilization, embryogenesis, etc.
Site-directed mutagenesis: PCR can be used to create mutant genes with mutations chosen
by scientists at will. These mutations can be chosen in order to understand how proteins
accomplish their functions, and to change or improve protein function.
Advantages
PCR has a number of advantages. It is fairly simple to understand and to use, and produces
results rapidly. The technique is highly sensitive with the potential to produce millions to
billions of copies of a specific product for sequencing, cloning, and analysis. qRT-PCR shares
the same advantages as the PCR, with an added advantage of quantification of the
synthesized product. Therefore, it has its uses to analyze alterations of gene expression
levels in tumors, microbes, or other disease states.[34]

PCR is a very powerful and practical research tool. The sequencing of unknown etiologies of
many diseases are being figured out by the PCR. The technique can help identify the
sequence of previously unknown viruses related to those already known and thus give us a
better understanding of the disease itself. If the procedure can be further simplified and
sensitive non radiometric detection systems can be developed, the PCR will assume a
prominent place in the clinical laboratory for years to come.[15]

Limitations
One major limitation of PCR is that prior information about the target sequence is necessary
in order to generate the primers that will allow its selective amplification.[35] This means
that, typically, PCR users must know the precise sequence(s) upstream of the target region
on each of the two single-stranded templates in order to ensure that the DNA polymerase
properly binds to the primer-template hybrids and subsequently generates the entire target
region during DNA synthesis.

Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations
in the PCR fragments that are generated.[36]

Another limitation of PCR is that even the smallest amount of contaminating DNA can be
amplified, resulting in misleading or ambiguous results. To minimize the chance of
contamination, investigators should reserve separate rooms for reagent preparation, the
PCR, and analysis of product. Reagents should be dispensed into single-use aliquots.
Pipetters with disposable plungers and extra-long pipette tips should be routinely used.[15]
Variations
Main article: Variants of PCR
Allele-specific PCR: a diagnostic or cloning technique based on single-nucleotide variations
(SNVs not to be confused with SNPs) (single-base differences in a patient). It requires prior
knowledge of a DNA sequence, including differences between alleles, and uses primers
whose 3' ends encompass the SNV (base pair buffer around SNV usually incorporated). PCR
amplification under stringent conditions is much less efficient in the presence of a mismatch
between template and primer, so successful amplification with an SNP-specific primer
signals presence of the specific SNP in a sequence.[37] See SNP genotyping for more
information.
Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA
sequences by performing PCR on a pool of long oligonucleotides with short overlapping
segments. The oligonucleotides alternate between sense and antisense directions, and the
overlapping segments determine the order of the PCR fragments, thereby selectively
producing the final long DNA product.[38]
Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA
template. It is used in sequencing and hybridization probing where amplification of only one
of the two complementary strands is required. PCR is carried out as usual, but with a great
excess of the primer for the strand targeted for amplification. Because of the slow
(arithmetic) amplification later in the reaction after the limiting primer has been used up,
extra cycles of PCR are required.[39] A recent modification on this process, known as Linear-
After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting
temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting
primer concentration decreases mid-reaction.[40]
Convective PCR: a pseudo-isothermal way of performing PCR. Instead of repeatedly heating
and cooling the PCR mixture, the solution is subjected to a thermal gradient. The resulting
thermal instability driven convective flow automatically shuffles the PCR reagents from the
hot and cold regions repeatedly enabling PCR.[41] Parameters such as thermal boundary
conditions and geometry of the PCR enclosure can be optimized to yield robust and rapid
PCR by harnessing the emergence of chaotic flow fields.[42] Such convective flow PCR setup
significantly reduces device power requirement and operation time.
Dial-out PCR: a highly parallel method for retrieving accurate DNA molecules for gene
synthesis. A complex library of DNA molecules is modified with unique flanking tags before
massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules
with desired sequences by PCR.[43]
Digital PCR (dPCR): used to measure the quantity of a target DNA sequence in a DNA
sample. The DNA sample is highly diluted so that after running many PCRs in parallel, some
of them do not receive a single molecule of the target DNA. The target DNA concentration is
calculated using the proportion of negative outcomes. Hence the name 'digital PCR'.
Helicase-dependent amplification: similar to traditional PCR, but uses a constant
temperature rather than cycling through denaturation and annealing/extension cycles. DNA
helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.[44]
Hot start PCR: a technique that reduces non-specific amplification during the initial set up
stages of the PCR. It may be performed manually by heating the reaction components to the
denaturation temperature (e.g., 95 °C) before adding the polymerase.[45] Specialized
enzyme systems have been developed that inhibit the polymerase's activity at ambient
temperature, either by the binding of an antibody[12][46] or by the presence of covalently
bound inhibitors that dissociate only after a high-temperature activation step. Hot-
start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient
temperature and are instantly activated at elongation temperature.
In silico PCR (digital PCR, virtual PCR, electronic PCR, e-PCR) refers to computational tools
used to calculate theoretical polymerase chain reaction results using a given set of primers
(probes) to amplify DNA sequences from a sequenced genome or transcriptome. In silico
PCR was proposed as an educational tool for molecular biology.[47]
Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies
regions between simple sequence repeats to produce a unique fingerprint of amplified
fragment lengths.[48]
Inverse PCR: is commonly used to identify the flanking sequences around genomic inserts. It
involves a series of DNA digestions and self ligation, resulting in known sequences at either
end of the unknown sequence.[49]
Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple
primers annealing to the DNA linkers; it has been used for DNA sequencing, genome
walking, and DNA footprinting.[50]
Methylation-specific PCR (MSP): developed by Stephen Baylin and James G. Herman at the
Johns Hopkins School of Medicine,[51] and is used to detect methylation of CpG islands in
genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated
cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then
carried out on the modified DNA, using primer sets identical except at any CpG islands
within the primer sequences. At these points, one primer set recognizes DNA with cytosines
to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify
unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather
than qualitative information about methylation.
Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers
("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller
primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S
(or eukaryotic 18S) rRNA gene.[52]
Multiplex ligation-dependent probe amplification (MLPA): permits amplifying multiple
targets with a single primer pair, thus avoiding the resolution limitations of multiplex PCR
(see below).
Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce
amplicons of varying sizes that are specific to different DNA sequences. By targeting
multiple genes at once, additional information may be gained from a single test-run that
otherwise would require several times the reagents and more time to perform. Annealing
temperatures for each of the primer sets must be optimized to work correctly within a
single reaction, and amplicon sizes. That is, their base pair length should be different
enough to form distinct bands when visualized by gel electrophoresis.
Nanoparticle-Assisted PCR (nanoPCR): some nanoparticles (NPs) can enhance the efficiency
of PCR (thus being called nanoPCR), and some can even outperform the original PCR
enhancers. It was reported that quantum dots (QDs) can improve PCR specificity and
efficiency. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes
(MWCNTs) are efficient in enhancing the amplification of long PCR. Carbon nanopowder
(CNP) can improve the efficiency of repeated PCR and long PCR, while zinc oxide, titanium
dioxide and Ag NPs were found to increase the PCR yield. Previous data indicated that non-
metallic NPs retained acceptable amplification fidelity. Given that many NPs are capable of
enhancing PCR efficiency, it is clear that there is likely to be great potential for nanoPCR
technology improvements and product development.[53][54]
Nested PCR: increases the specificity of DNA amplification, by reducing background due to
non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In
the first reaction, one pair of primers is used to generate DNA products, which besides the
intended target, may still consist of non-specifically amplified DNA fragments. The
product(s) are then used in a second PCR with a set of primers whose binding sites are
completely or partially different from and located 3' of each of the primers used in the first
reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments
than conventional PCR, but it requires more detailed knowledge of the target sequences.
Overlap-extension PCR or Splicing by overlap extension (SOEing) : a genetic engineering
technique that is used to splice together two or more DNA fragments that contain
complementary sequences. It is used to join DNA pieces containing genes, regulatory
sequences, or mutations; the technique enables creation of specific and long DNA
constructs. It can also introduce deletions, insertions or point mutations into a DNA
sequence.[55][56]
PAN-AC: uses isothermal conditions for amplification, and may be used in living
cells.[57][58]
quantitative PCR (qPCR): used to measure the quantity of a target sequence (commonly in
real-time). It quantitatively measures starting amounts of DNA, cDNA, or RNA. quantitative
PCR is commonly used to determine whether a DNA sequence is present in a sample and the
number of its copies in the sample. Quantitative PCR has a very high degree of precision.
Quantitative PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or
fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified
product in real time. It is also sometimes abbreviated to RT-PCR (real-time PCR) but this
abbreviation should be used only for reverse transcription PCR. qPCR is the appropriate
contractions for quantitative PCR (real-time PCR).
Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase
reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in
expression profiling, to determine the expression of a gene or to identify the sequence of an
RNA transcript, including transcription start and termination sites. If the genomic DNA
sequence of a gene is known, RT-PCR can be used to map the location of exons and introns
in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically
identified by RACE-PCR (Rapid Amplification of cDNA Ends).
RNase H-dependent PCR (rhPCR): a modification of PCR that utilizes primers with a 3’
extension block that can be removed by a thermostable RNase HII enzyme. This system
reduces primer-dimers and allows for multiplexed reactions to be performed with higher
numbers of primers.[59]
Single Specific Primer-PCR (SSP-PCR): allows the amplification of double-stranded DNA even
when the sequence information is available at one end only. This method permits
amplification of genes for which only a partial sequence information is available, and allows
unidirectional genome walking from known into unknown regions of the chromosome.[60]
Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where
PCR colonies are derived in a gel matrix, for example), Bridge PCR[61] (primers are
covalently linked to a solid-support surface), conventional Solid Phase PCR (where
Asymmetric PCR is applied in the presence of solid support bearing primer with sequence
matching one of the aqueous primers) and Enhanced Solid Phase PCR[62] (where
conventional Solid Phase PCR can be improved by employing high Tm and nested solid
support primer with optional application of a thermal 'step' to favour solid support priming).
Suicide PCR: typically used in paleogenetics or other studies where avoiding false positives
and ensuring the specificity of the amplified fragment is the highest priority. It was originally
described in a study to verify the presence of the microbe Yersinia pestis in dental samples
obtained from 14th Century graves of people supposedly killed by plague during the
medieval Black Death epidemic.[63] The method prescribes the use of any primer
combination only once in a PCR (hence the term "suicide"), which should never have been
used in any positive control PCR reaction, and the primers should always target a genomic
region never amplified before in the lab using this or any other set of primers. This ensures
that no contaminating DNA from previous PCR reactions is present in the lab, which could
otherwise generate false positives.
Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence
flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of
primers with differing annealing temperatures; a degenerate primer is used to amplify in the
other direction from the unknown sequence.[64]
Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific
background by gradually lowering the annealing temperature as PCR cycling progresses. The
annealing temperature at the initial cycles is usually a few degrees (3–5 °C) above the Tm of
the primers used, while at the later cycles, it is a few degrees (3–5 °C) below the primer Tm.
The higher temperatures give greater specificity for primer binding, and the lower
temperatures permit more efficient amplification from the specific products formed during
the initial cycles.[65]
Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific
'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific
primer and one general primer—which can lead to artefactual 'noise')[66] by virtue of a
mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe
RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends),[67]
5'RACE LaNe[68] and 3'RACE LaNe.

SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a variant of

polyacrylamide gel electrophoresis, an analytical method in biochemistry for the separation
of charged molecules in mixtures by their molecular masses in an electric field. It uses
sodium dodecyl sulfate (SDS) molecules to help identify and isolate protein molecules.

SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is

commonly used as a method to separate proteins with molecular masses between 5 and
250 KDa.[1] The publication describing it is the most frequently cited paper by a single
author, and the second most cited overall.[2]
Properties
Unfolding of a protein with SDS
Unfolding of a protein with heat
SDS-PAGE is an electrophoresis method that allows protein separation by mass. The
medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel. In
addition, SDS (sodium dodecyl sulfate) is used. About 1.4 grams of SDS bind to a gram of
protein,[3][4][5] corresponding to one SDS molecule per two amino acids. SDS acts as a
surfactant, covering the proteins' intrinsic charge and conferring them very similar charge-
to-mass ratios. The intrinsic charges of the proteins are negligible in comparison to the SDS
loading, and the positive charges are also greatly reduced in the basic pH range of a
separating gel. Upon application of a constant electric field, the protein migrate towards the
anode, each with a different speed, depending on its mass. This simple procedure allows
precise protein separation by mass.

SDS tends to form spherical micelles in aqueous solutions above a certain concentration
called the critical micellar concentration (CMC). Above the critical micellar concentration of
7 to 10 millimolar in solutions, the SDS simultaneously occurs as single molecules
(monomer) and as micelles, below the CMC SDS occurs only as monomers in aqueous
solutions. At the critical micellar concentration, a micelle consists of about 62 SDS
molecules.[6] However, only SDS monomers bind to proteins via hydrophobic interactions,
whereas the SDS micelles are anionic on the outside and do not adsorb any protein.[3] SDS
is amphipathic in nature, which allows it to unfold both polar and nonpolar sections of
protein structure.[7] In SDS concentrations above 0.1 millimolar, the unfolding of proteins
begins,[3] and above 1 mM, most proteins are denatured.[3] Due to the strong denaturing
effect of SDS and the subsequent dissociation of protein complexes, quaternary structures
can generally not be determined with SDS. Exceptions are e.g. proteins that were previously
stabilised by covalent cross-linking and the SDS-resistant protein complexes, which are
stable even in the presence of SDS (the latter, however, only at room temperature). To
denature the SDS-resistant complexes a high activation energy is required, which is achieved
by heating. SDS resistance is based on a metastability of the protein fold. Although the
native, fully folded, SDS-resistant protein does not have sufficient stability in the presence of
SDS, the chemical equilibrium of denaturation at room temperature occurs slowly. Stable
protein complexes are characterised not only by SDS resistance but also by stability against
proteases and an increased biological half-life.[8]

Alternatively, polyacrylamide gel electrophoresis can also be performed with the cationic
surfactants CTAB in a CTAB-PAGE,[9][10][11] or 16-BAC in a BAC-PAGE.[12]

Procedure
The SDS-PAGE method is composed of gel preparation, sample preparation, electrophoresis,
protein staining or western blotting and analysis of the generated banding pattern.

Gel production

Sample combs with different numbers of pockets, each prong leaves a pocket in the gel
when pulled out

Polymerised separating and stacking gel before removing the sample comb (white) between
the spacers (black), in the stacking gel are small amounts of bromophenol blue for improved
visibility, the separating gel is unstained
When using different buffers in the gel (discontinuous gel electrophoresis), the gels are
made up to one day prior to electrophoresis, so that the diffusion does not lead to a mixing
of the buffers. The gel is produced by radical polymerisation in a mold consisting of two
sealed glass plates with spacers between the glass plates. In a typical mini-gel setting, the
spacers have a thickness of 0.75 mm or 1.5 mm, which determines the loading capacity of
the gel. For pouring the gel solution, the plates are usually clamped in a stand which
temporarily seals the otherwise open underside of the glass plates with the two spacers. For
the gel solution, acrylamide is mixed as gel-former (usually 4% V/V in the stacking gel and
10-12 % in the separating gel), methylenebisacrylamide as a cross-linker, stacking or
separating gel buffer, water and SDS. By adding the catalyst TEMED and the radical initiator
ammonium persulfate (APS) the polymerisation is started. The solution is then poured
between the glass plates without creating bubbles. Depending on the amount of catalyst
and radical starter and depending on the temperature, the polymerisation lasts between a
quarter of an hour and several hours. The lower gel (separating gel) is poured first and
covered with a few drops of a barely water-soluble alcohol (usually buffer-saturated butanol
or isopropanol), which eliminates bubbles from the meniscus and protects the gel solution
of the radical scavenger oxygen. After the polymerisation of the separating gel, the alcohol
is discarded and the residual alcohol is removed with filter paper. After addition of APS and
TEMED to the stacking gel solution, it is poured on top of the solid separation gel.
Afterwards, a suitable sample comb is inserted between the glass plates without creating
bubbles. The sample comb is carefully pulled out after polymerisation, leaving pockets for
the sample application. For later use of proteins for protein sequencing, the gels are often
prepared the day before electrophoresis to reduce reactions of unpolymerised acrylamide
with cysteines in proteins.

By using a gradient mixer, gradient gels with a gradient of acrylamide (usually from 4 to
12%) can be cast, which have a larger separation range of the molecular masses.[13]
Commercial gel systems (so-called pre-cast gels) usually use the buffer substance Bis-tris
methane with a pH value between 6.4 and 7.2 both in the stacking gel and in the separating
gel.[14][15] These gels are delivered cast and ready-to-use. Since they use only one buffer
(continuous gel electrophoresis) and have a nearly neutral pH, they can be stored for several
weeks. The more neutral pH slows the hydrolysis and thus the decomposition of the
polyacrylamide. Furthermore, there are fewer acrylamide-modified cysteines in the
proteins.[14] Due to the constant pH in collecting and separating gel there is no stacking
effect. Proteins in BisTris gels can not be stained with ruthenium complexes.[16] This gel
system has a comparatively large separation range, which can be varied by using MES or
MOPS in the running buffer.[14]

Sample preparation

Disulfide reduction by DTT

During sample preparation, the sample buffer, and thus SDS, is added in excess to the
proteins, and the sample is then heated to 95 °C for five minutes, or alternatively 70 °C for
ten minutes. Heating disrupts the secondary and tertiary structures of the protein by
disrupting hydrogen bonds and stretching the molecules. Optionally, disulfide bridges can
be cleaved by reduction. For this purpose, reducing thiols such as β-mercaptoethanol (β-ME,
5% by volume), dithiothreitol (DTT, 10 millimolar) or dithioerythritol (DTE, 10 millimolar) are
added to the sample buffer. After cooling to room temperature, each sample is pipetted
into its own well in the gel, which was previously immersed in electrophoresis buffer in the
electrophoresis apparatus.

In addition to the samples, a molecular-weight size marker is usually loaded onto the gel.
This consists of proteins of known sizes and thereby allows the estimation (with an error of
± 10%) of the sizes of the proteins in the actual samples, which migrate in parallel in
different tracks of the gel.[17] The size marker is often pipetted into the first or last pocket
of a gel.

Electrophoresis

Electrophoresis chamber after a few minutes of electrophoresis. In the first pocket a size
marker was applied with bromophenol blue, in the other pockets, the samples were added
bromocresol green
For separation, the denatured samples are loaded onto a gel of polyacrylamide, which is
placed in an electrophoresis buffer with suitable electrolytes. Thereafter, a voltage (usually
around 100 V, 10-20 V per cm gel length) is applied, which causes a migration of negatively
charged molecules through the gel in the direction of the positively charged anode. The gel
acts like a sieve. Small proteins migrate relatively easily through the mesh of the gel, while
larger proteins are more likely to be retained and thereby migrate more slowly through the
gel, thereby allowing proteins to be separated by molecular size. The electrophoresis lasts
between half an hour to several hours depending on the voltage and length of gel used.

The fastest-migrating proteins (with a molecular weight of less than 5 KDa) form the buffer
front together with the anionic components of the electrophoresis buffer, which also
migrate through the gel. The area of the buffer front is made visible by adding the
comparatively small, anionic dye bromophenol blue to the sample buffer. Due to the
relatively small molecule size of bromophenol blue, it migrates faster than proteins. By
optical control of the migrating colored band, the electrophoresis can be stopped before the
dye and also the samples have completely migrated through the gel and leave it.

The most commonly used method is the discontinuous SDS-PAGE. In this method, the
proteins migrate first into a collecting gel with neutral pH, in which they are concentrated
and then they migrate into a separating gel with basic pH, in which the actual separation
takes place. Stacking and separating gels differ by different pore size (4-6 % T and 10-20 %
T), ionic strength and pH values (pH 6.8 or pH 8.8). The electrolyte most frequently used is
an SDS-containing Tris-glycine-chloride buffer system. At neutral pH, glycine predominantly
forms the zwitterionic form, at high pH the glycines lose positive charges and become
predominantly anionic. In the collection gel, the smaller, negatively charged chloride ions
migrate in front of the proteins (as leading ions) and the slightly larger, negatively and
partially positively charged glycinate ions migrate behind the proteins (as initial trailing
ions), whereas in the comparatively basic separating gel both ions migrate in front of the
proteins. The pH gradient between the stacking and separation gel buffers leads to a
stacking effect at the border of the stacking gel to the separation gel, since the glycinate
partially loses its slowing positive charges as the pH increases and then, as the former
trailing ion, overtakes the proteins and becomes a leading ion, which causes the bands of
the different proteins (visible after a staining) to become narrower and sharper - the
stacking effect. For the separation of smaller proteins and peptides, the TRIS-Tricine buffer
system of Schägger and von Jagow is used due to the higher spread of the proteins in the
range of 0.5 to 50 KDa.[18]

Gel staining

Coomassie-stained 10% Tris/Tricine gel. In the left lane, a molecular weight size marker was
used to estimate the size (from top to bottom: 66, 45, 35, 24, 18 and 9 kDa). In the
remaining lanes purified yeast proteins were separated.
At the end of the electrophoretic separation, all proteins are sorted by size and can then be
analyzed by other methods, e. g. protein staining such as Coomassie staining (most common
and easy to use),[19][20] silver staining (highest sensitivity),[21][22][23][24][25][26] stains
all staining, Amido black 10B staining,[20] Fast green FCF staining,[20] fluorescent stains
such as epicocconone stain[27] and SYPRO orange stain,[28] and immunological detection
such as the Western Blot.[29][30] The fluorescent dyes have a comparatively higher linearity
between protein quantity and color intensity of about three orders of magnitude above the
detection limit, i. e. the amount of protein can be estimated by color intensity. When using
the fluorescent protein dye trichloroethanol, a subsequent protein staining is omitted if it
was added to the gel solution and the gel was irradiated with UV light after
electrophoresis.[31][32]

In Coomassie Staining, Gel is Fixed in a 50% ethanol 10% glacial acetic acid solution for 1 hr.
Then the solution is changed for fresh one and after 1 to 12 hrs Gel is changed to a Staining
solution (50% methanol, 10% Glacial acetic Acid, 0.1% Coomassie Brilliant Blue) followed by
destaining changing several times a destaining solution of 40% methanol, 10% glacial acetic
acid.

Analysis
Protein staining in the gel creates a documentable banding pattern of the various proteins.
Glycoproteins have differential levels of glycosylations and adsorb SDS more unevenly at the
glycosylations, resulting in broader and blurred bands.[33] Membrane proteins, because of
their transmembrane domain, are often composed of the more hydrophobic amino acids,
have lower solubility in aqueous solutions, tend to bind lipids, and tend to precipitate in
aqueous solutions due to hydrophobic effects when sufficient amounts of detergent are not
present. This precipitation manifests itself for membrane proteins in a SDS-PAGE in "tailing"
above the band of the transmembrane protein. In this case, more SDS can be used (by using
more or more concentrated sample buffer) and the amount of protein in the sample
application can be reduced. An overloading of the gel with a soluble protein creates a
semicircular band of this protein (e. g. in the marker lane of the image at 66 KDa), allowing
other proteins with similar molecular weights to be covered. A low contrast (as in the
marker lane of the image) between bands within a lane indicates either the presence of
many proteins (low purity) or, if using purified proteins and a low contrast occurs only below
one band, it indicates a proteolytic degradation of the protein, which first causes
degradation bands, and after further degradation produces a homogeneous color ("smear")
below a band.[34] The documentation of the banding pattern is usually done by
photographing or scanning. For a subsequent recovery of the molecules in individual bands,
a gel extraction can be performed.

Archiving

Two SDS gels after completed separation of the samples and staining in a drying frame
After protein staining and documentation of the banding pattern, the polyacrylamide gel
can be dried for archival storage. Proteins can be extracted from it at a later date. The gel is
either placed in a drying frame (with or without the use of heat) or in a vacuum dryer. The
drying frame consists of two parts, one of which serves as a base for a wet cellophane film
to which the gel and a one percent glycerol solution are added. Then a second wet
cellophane film is applied bubble-free, the second frame part is put on top and the frame is
sealed with clips. The removal of the air bubbles avoids a fragmentation of the gel during
drying. The water evaporates through the cellophane film. In contrast to the drying frame, a
vacuum dryer generates a vacuum and heats the gel to about 50 °C.

Molecular mass determination

The proteins of the size marker (black X) show an approximately straight line in the
representation of log M over Rf. The molecular weight of the unknown protein (red X) can
be determined on the y-axis.
For a more accurate determination of the molecular weight, the relative migration distances
of the individual protein bands are measured in the separating gel.[35][36] The
measurements are usually performed in triplicate for increased accuracy. The relative
mobility (called Rf value or Rm value) is the quotient of the distance of the band of the
protein and the distance of the buffer front. The distances of the bands and the buffer front
are each measured from the beginning of the separation gel. The distance of the buffer
front roughly corresponds to the distance of the bromophenol blue contained in the sample
buffer. The relative distances of the proteins of the size marker are plotted semi-
logarithmically against their known molecular weights. By comparison with the linear part of
the generated graph or by a regression analysis, the molecular weight of an unknown
protein can be determined by its relative mobility. Bands of proteins with glycosylations can
be blurred.[33] Proteins with many basic amino acids (e. g. histones)[37] can lead to an
overestimation of the molecular weight or even not migrate into the gel at all, because they
move slower in the electrophoresis due to the positive charges or even to the opposite
direction. Accordingly, many acidic amino acids can lead to accelerated migration of a
protein and an underestimation of its molecular mass.[38]

Applications
The SDS-PAGE in combination with a protein stain is widely used in biochemistry for the
quick and exact separation and subsequent analysis of proteins. It has comparatively low
instrument and reagent costs and is an easy-to-use method. Because of its low scalability, it
is mostly used for analytical purposes and less for preparative purposes, especially when
larger amounts of a protein are to be isolated.

Additionally, SDS-PAGE is used in combination with the western blot for the determination
of the presence of a specific protein in a mixture of proteins - or for the analysis of post-
translational modifications. Post-translational modifications of proteins can lead to a
different relative mobility (i.e. a band shift) or to a change in the binding of a detection
antibody used in the western blot (i.e. a band disappears or appears).

In mass spectrometry of proteins, SDS-PAGE is a widely used method for sample preparation
prior to spectrometry, mostly using in-gel digestion. In regards to determining the molecular
mass of a protein, the SDS-PAGE is a bit more exact than an analytical ultracentrifugation,
but less exact than a mass spectrometry or - ignoring post-translational modifications - a
calculation of the protein molecular mass from the DNA sequence.

In medical diagnostics, SDS-PAGE is used as part of the HIV test and to evaluate proteinuria.
In the HIV test, HIV proteins are separated by SDS-PAGE and subsequently detected by
Western Blot with HIV-specific antibodies of the patient, if they are present in his blood
serum. SDS-PAGE for proteinuria evaluates the levels of various serum proteins in the urine,
e.g. Albumin, Alpha-2-macroglobulin and IgG.

Variants
SDS-PAGE is the most widely used method for gel electrophoretic separation of proteins.
Two-dimensional gel electrophoresis sequentially combines isoelectric focusing or BAC-
PAGE with a SDS-PAGE. Native PAGE is used if native protein folding is to be maintained. For
separation of membrane proteins, BAC-PAGE or CTAB-PAGE may be used as an alternative
to SDS-PAGE. For electrophoretic separation of larger protein complexes, agarose gel
electrophoresis can be used, e.g. the SDD-AGE. Some enzymes can be detected via their
enzyme activity by zymography.

Alternatives
While being one of the more precise and low-cost protein separation and analysis methods,
the SDS-PAGE denatures proteins. Where non-denaturing conditions are necessary, proteins
are separated by a native PAGE or different chromatographic methods with subsequent
photometric quantification, for example affinity chromatography (or even tandem affinity
purification), size exclusion chromatography, ion exchange chromatography.[39] Proteins
can also be separated by size in a tangential flow filtration[40] or an ultrafiltration.[41]
Single proteins can be isolated from a mixture by affinity chromatography or by a pull-down
assay. Some historically early and cost effective but crude separation methods usually based
upon a series of extractions and precipitations using kosmotropic molecules, for example
the ammonium sulfate precipitation and the polyethyleneglycol precipitation.

Immunoelectrophoresis is a general name for a number of biochemical methods for

separation and characterization of proteins based on electrophoresis and reaction with
antibodies. All variants of immunoelectrophoresis require immunoglobulins, also known as
antibodies, reacting with the proteins to be separated or characterized. The methods were
developed and used extensively during the second half of the 20th century. In somewhat
chronological order: Immunoelectrophoretic analysis (one-dimensional
immunoelectrophoresis ad modum Grabar), crossed immunoelectrophoresis (two-
dimensional quantitative immunoelectrophoresis ad modum Clarke and Freeman or ad
modum Laurell), rocket-immunoelectrophoresis (one-dimensional quantitative
immunoelectrophoresis ad modum Laurell), fused rocket immunoelectrophoresis ad
modum Svendsen and Harboe, affinity immunoelectrophoresis ad modum Bøg-Hansen.

Agarose as 1% gel slabs of about 1 mm thickness buffered at high pH (around 8.6) is

traditionally preferred for the electrophoresis as well as the reaction with antibodies. The
agarose was chosen as the gel matrix because it has large pores allowing free passage and
separation of proteins, but provides an anchor for the immunoprecipitates of protein and
specific antibodies. The high pH was chosen because antibodies are practically immobile at
high pH. An electrophoresis equipment with a horizontal cooling plate was normally
recommended for the electrophoresis.

Immunoprecipitates may be seen in the wet agarose gel, but are stained with protein stains
like Coomassie Brilliant Blue in the dried gel. In contrast to SDS-gel electrophoresis, the
electrophoresis in agarose allows native conditions, preserving the native structure and
activities of the proteins under investigation, therefore immunoelectrophoresis allows
characterization of enzyme activities and ligand binding etc. in addition to electrophoretic
separation.

The immunoelectrophoretic analysis ad modum Grabar is the classical method of

immunoelectrophoresis. Proteins are separated by electrophoresis, then antibodies are
applied in a trough next to the separated proteins and immunoprecipitates are formed after
a period of diffusion of the separated proteins and antibodies against each other. The
introduction of the immunoelectrophoretic analysis gave a great boost to protein chemistry,
some of the very first results were the resolution of proteins in biological fluids and
biological extracts. Among the important observations made were the great number of
different proteins in serum, the existence of several immunoglobulin classes and their
electrophoretic heterogeneity.

Sex change is a process by which a person or animal changes sex – that is, by which
female sexual characteristics are substituted for male ones or vice versa. Sex change may
occur naturally, as in the case of the sequential hermaphroditism observed in some species.
Most commonly, however, the term is used for sex reassignment therapy, including sex
reassignment surgery, carried out on humans. It is also sometimes used for the medical
procedures applied to intersex people. The term may also be applied to the broader process
of changing gender role ("living as a woman" instead of living as a man, or vice versa),
including but not necessarily limited to medical procedures.
In animals

A clownfish – a species in which sex change from male to female is a normal process in
biology
Some species exhibit sequential hermaphroditism. In these species, such as many species of
coral reef fishes, sex change is a normal anatomical process.[1] Clownfish, wrasses, moray
eels, gobies[2] and other fish species are known to change sex, including reproductive
functions. A school of clownfish is always built into a hierarchy with a female fish at the top.
When she dies, the most dominant male changes sex and takes her place.[3] In the wrasses
(the family Labridae), sex change is from female to male, with the largest female of the
harem changing into a male and taking over the harem upon the disappearance of the
previous dominant male.

Natural sex change, in both directions, has also been reported in mushroom corals. This is
posited to take place in response to environmental or energetic constraints, and to improve
the organism's evolutionary fitness; similar phenomena are observed in some dioecious
plants.[4]

Chickens can sometimes undergo natural sex changes. Normally, female chickens have just
one functional ovary, on their left side. Although two sex organs are present during the
embryonic stages of all birds, once a chicken's female hormones come into effect, it typically
develops only the left ovary. The right gonad, which has yet to be defined as an ovary,
testes, or both (called an ovotestis), typically remains dormant. Certain medical conditions
can cause a chicken's left ovary to regress. In the absence of a functional left ovary, the
dormant right sex organ may begin to grow, if the activated right gonad is an ovotestis or
testes, it will begin secreting androgens. The hen does not completely change into a rooster,
however. This transition is limited to making the bird phenotypically male.[5] The condition
could also be caused by mycotoxins that can develop when animal feed is stored, and these
have the same effect as synthetic hormones.[6] In about 10 percent of cases, if eggs
fertilized with male chromosomes are cooled by a few degrees for three days after laying,
the relative activity of the sex hormones will favour development of female characteristics.
The sex chromosomes work by coding for enzymes that affect the bird’s development in the
egg and during its life. This cooling will produce a chicken with a fully functioning and
reproductively fertile female body-type; even though the chicken is genetically male.

Sequential hermaphroditism (called dichogamy in botany) is a type of

hermaphroditism that occurs in many fish, gastropods, and plants. Sequential
hermaphroditism occurs when the individual changes its sex at some point in its life.[1] In
particular, a sequential hermaphrodite produces eggs (female gametes) and sperm (male
gametes) at different stages in life.[2] Species that can undergo these changes from one sex
to another do so as a normal event within their reproductive cycle that is usually cued by
either social structure or the achievement of a certain age or size.[3]

In animals, the different types of change are male to female (protandry) (is this opposite?
See below), female to male (protogyny),[4] female to hermaphrodite (protogynous
hermaphroditism), and male to hermaphrodite (protandrous hermaphroditism). Both
protogynous and protandrous hermaphroditism allow the organism to switch between
functional male and functional female.[5] These various types of sequential
hermaphroditism may indicate that there is no advantage based on the original sex of an
individual organism.[5] Those that change gonadal sex can have both female and male germ
cells in the gonads or can change from one complete gonadal type to the other during their
last life stage.[6]

In plants, individual flowers are called dichogamous if their function has the two sexes
separated in time, although the plant as a whole may have functionally male and
functionally female flowers open at any one moment. A flower is protogynous if its function
is first female, then male, and protandrous if its function is male then female. It used to be
thought that this reduced inbreeding,[7] but it may be a more general mechanism for
reducing pollen-pistil interference.
Protandry

Ocellaris clownfish, Amphiprion ocellaris, a protandrous animal species

In general, protandrous hermaphrodites are animals that develop as males, but can later
reproduce as females.[9] However, protandry features a spectrum of different forms, which
are characterized by the overlap between male and female reproductive function
throughout an organism's lifetime:

Protandrous sequential hermaphroditism: Early reproduction as a pure male and later

reproduction as a pure female.
Protandrous hermaphroditism with overlap: Early reproduction as a pure male and later
reproduction as a pure female with an intervening overlap between both male and female
reproduction.
Protandrous simultaneous hermaphroditism: Early pure male reproduction and later
reproduction in both sexes.[10]
Furthermore, there are also species that reproduce as both sexes throughout their lifespans
(i.e simultaneous hermaphrodites), but shift their reproductive resources from male to
female over time.[11]

Protandrous examples
Protandry is uncommon, but does occur in a widespread range of animal phyla.[12] In fact,
protandrous hermaphroditism occurs in many fish,[13] mollusks,[10] and crustaceans,[14]
but is completely absent in terrestrial vertebrates.[9]

Protandrous fishes include teleost species in the Pomacentridae, Sparidae, and Gobiidae
families.[15] A common example of a protandrous species are clownfish, which have a very
structured society. In the Amphiprion percula species, there are zero to four individuals
excluded from breeding and a breeding pair living in a sea anemone. Dominance is based on
size, the female being the largest and the reproductive male being the second largest. The
rest of the group is made up of progressively smaller males that do not breed and have no
functioning gonads.[16] If the female dies, in many cases, the reproductive male gains
weight and becomes the female for that group. The largest non-breeding male then sexually
matures and becomes the reproductive male for the group.[17]

Other protandrous fishes can be found in the classes clupeiformes, siluriformes,

stomiiformes. Since these groups are distantly related and have many intermediate relatives
that are not protandrous, it strongly suggests that protandry evolved multiple times.[18]

Phylogenies support this assumption because ancestral states differ for each family. For
example, the ancestral state of the Pomacentridae family was gonochoristic (single-sexed),
indicating that protandry evolved within the family.[15] Therefore, because other families
also contain protandrous species, protandry likely has evolved multiple times.

Other examples of protandrous animals include:

The Platyctenida order of comb jellies. Unlike most ctenophores, which are simultaneous
hermaphrodites, Platyctenida are primarily protandrous, but asexual reproduction has also
been observed in some species.[19]
The flatworms Hymanella retenuova.[20]
Laevapex fuscus, a gastropod, is described as being functionally protandric. The sperm
matures in late winter and early spring, the eggs mature in early summer, and copulation
occurs only in June. This shows that males cannot reproduce until the females appear, thus
why they are considered to be functionally protandric.[21][22]
Speyeria mormonia, or the Mormon Fritillary, is a butterfly species exhibiting protandry. In
its case, functional protandry refers to the emergence of male adults 2–3 weeks before
female adults.[23]
The shrimp genus Lysmata perform protandric simultaneous hermaphroditism where they
become true hermaphrodites instead of females.[14] During the "female phase," they have
both male and female tissues in their gonads and produce both gametes.[24]

Lysmata, a genus of shrimp that performs protandric simultaneous hermaphroditism.

Protogyny

Moon wrasse, Thalassoma lunare, a protogynous animal species

Protogynous hermaphrodites are animals that are born female and at some point in their
lifespan change sex to male.[25] Protogyny is a more common form of sequential
hermaphroditism, especially when compared to protandry.[26] As the animal ages, it shifts
sex to become a male animal due to internal or external triggers. Unlike females, male
fecundity increases greatly with age, and it is hypothesized that it is more selectively
advantageous to be a male when an organism's body is larger.[25] This advantage may
cause certain species to be protogynous hermaphrodites as the sex change to male leads to
an increased reproductive fitness advantage.[15]

Protogynous examples
Protogyny is the most common form of hermaphroditism in fish in nature.[27] About 75% of
the 500 known sequentially hermaphroditic fish species are protogynous and often have
polygynous mating systems.[28][29] In these systems, large males use aggressive territorial
defense to dominate female mating. This causes small males to have a severe reproductive
disadvantage, which promotes strong selection of size-based protogyny.[30] Therefore, if an
individual is small, it is more reproductively advantageous to be female because they will
still be able to reproduce, unlike small males.

Common model organisms for this type of sequential hermaphroditism are wrasses. They
are one of the largest families of coral reef fish and belong to the family Labridae. Wrasses
are found around the world in all marine habitats and tend to bury themselves in sand at
night or when they feel threatened.[31] In wrasses, the larger of a mating pair is the male,
while the smaller is the female. In most cases, females and immature males have a uniform
color while the male has the terminal bicolored phase.[32] Large males hold territories and
try to pair spawn, while small to mid-size initial-phase males live with females and group
spawn.[33] In other words, both the initial- and terminal-phase males can breed, but they
differ in the way they do it.
In the California sheephead (Semicossyphus pulcher), a type of wrasse, when the female
changes to male, the ovaries degenerate and spermatogenic crypts appear in the
gonads.[34] The general structure of the gonads remains ovarian after the transformation
and the sperm is transported through a series of ducts on the periphery of the gonad and
oviduct. Here, sex change is age-dependent. For example, the California sheephead stays a
female for four to six years before changing sex[32] since all California sheephead are born
female.[35]

A terminal-phase male bluehead wrasse

Bluehead wrasses begin life as males or females, but females can change sex and function as
males. Young females and males start with a dull initial-phase coloration before progressing
into a brilliant terminal-phase coloration, which has a change in intensity of color, stripes,
and bars. Terminal-phase coloration occurs when males become large enough to defend
territory.[36] Initial-phase males have larger testes than larger, terminal phase males, which
enables the initial-phase males to produce a large amount of sperm. This strategy allows
these males to compete with the larger territorial male.[37]

Botryllus schlosseri, a colonial tunicate, is a protogynous hermaphrodite. In a colony, eggs

are released about two days before the peak of sperm emission.[38] Although self-
fertilization is avoided and cross-fertilization favored by this strategy, self-fertilization is still
possible. Self-fertilized eggs develop with a substantially higher frequency of anomalies
during cleavage than cross-fertilized eggs (23% vs. 1.6%).[38] Also a significantly lower
percentage of larvae derived from self-fertilized eggs metamorphose, and the growth of the
colonies derived from their metamorphosis is significantly lower. These findings suggest that
self-fertilization gives rise to inbreeding depression associated with developmental deficits
that are likely caused by expression of deleterious recessive mutations.[39]

Other examples of protogynous organisms include:

In the following fish families: Serranidae (groupers),[40][41][42] Sparidae (porgies),[43]

Synbranchidae (swamp eels),[44] Labridae (wrasses),[36] Scaridae (parrotfishes),[45]
Pomacanthidae (angelfishes),[46] Gobiidae (gobies),[47] Lethrinidae (emperors),[48] and
possibly others.[49]
The intertidal isopod Gnorimosphaeroma oregonense.[50]
Protogyny sometimes occurs in the frog Rana temporaria, where older females will
sometimes switch to being males.[21]
Ultimate causes
Size-advantage model
The size-advantage model states that individuals of a given sex reproduce more effectively if
they are a certain size or age. To create selection for sequential hermaphroditism, small
individuals must have higher reproductive fitness as one sex and larger individuals must
have higher reproductive fitness as the opposite sex. For example, eggs are larger than
sperm, thus larger individuals are able to make more eggs, so individuals could maximize
their reproductive potential by beginning life as male and then turning female upon
achieving a certain size.[51]

In most ectotherms, body size and female fecundity are positively correlated.[4] This
supports the size-advantage model. Kazancioglu and Alonzo (2010) performed the first
comparative analysis of sex change in Labridae. Their analysis supports the size-advantage
model and suggest that sequential hermaphroditism is correlated to the size-advantage.
They determined that dioecy was less likely to occur when the size advantage is stronger
than other advantages.[52] Warner suggests that selection for protandry may occur in
populations where female fecundity is augmented with age and individuals mate randomly.
Selection for protogyny may occur where there are traits in the population that depress
male fecundity at early ages (territoriality, mate selection or inexperience) and when female
fecundity is decreased with age, the latter seems to be rare in the field.[4] An example of
territoriality favoring protogyny occurs when there is a need to protect their habitat and
being a large male is advantageous for this purpose. In the mating aspect, a large male has a
higher chance of mating, while this has no effect on the female mating fitness.[52] Thus, he
suggests that female fecundity has more impact on sequential hermaphroditism than the
age structures of the population.[4]

The size-advantage model predicts that sex change would only be absent if the relationship
between size/age with reproductive potential is identical in both sexes. With this prediction
one would assume that hermaphroditism is very common, but this is not the case.
Sequential hermaphroditism is very rare and according to scientists this is due to some cost
that decreases fitness in sex changers as opposed to those who don't change sex. Some of
the hypotheses proposed for the dearth of hermaphrodites are the energetic cost of sex
change, genetic and/or physiological barriers to sex change, and sex-specific mortality
rates.[4][53][54]

In 2009, Kazanciglu and Alonzo found that dioecy was only favored when the cost of
changing sex was very large. This indicates that the cost of sex change does not explain the
rarity of sequential hermaphroditism by itself.[55]

Protection against inbreeding

Sequential hermaphroditism can also protect against inbreeding in populations of organisms
that have low enough motility and/or are sparsely distributed enough that there is a
considerable risk of siblings encountering each other after reaching sexual maturity, and
interbreeding. If siblings are all the same or similar ages, and if they all begin life as one sex
and then transition to the other sex at about the same age, then siblings are highly likely to
be the same sex at any given time. This should dramatically reduce the likelihood of
inbreeding. Both protandry and protogyny are known to help prevent inbreeding in
plants,[2] and many examples of sequential hermaphroditism attributable to inbreeding
prevention have been identified in a wide variety of animals.[51]

Proximate causes
The proximate cause of a biological event concerns the molecular and physiological
mechanisms that produce the event. Many studies have focused on the proximate causes of
sequential hermaphroditism, which may be caused by various hormonal and enzyme
changes in organisms.

The role of aromatase has been widely studied in this area. Aromatase is an enzyme that
controls the androgen/estrogen ratio in animals by catalyzing the conversion of
testosterone into oestradiol, which is irreversible. It has been discovered that the aromatase
pathway mediates sex change in both directions in organisms.[56] Many studies also involve
understanding the effect of aromatase inhibitors on sex change. One such study was
performed by Kobayashi et al. In their study they tested the role of estrogens in male three-
spot wrasses (Halichoeres trimaculatus). They discovered that fish treated with aromatase
inhibitors showed decreased gonodal weight, plasma estrogen level and spermatogonial
proliferation in the testis as well as increased androgen levels. Their results suggest that
estrogens are important in the regulation of spermatogenesis in this protogynous
hermaphrodite.[57]

Previous studies have also investigated sex reversal mechanisms in teleost fish. During sex
reversal, their whole gonads including the germinal epithelium undergoes significant
changes, remodeling, and reformation. One study on the teleost Synbranchus marmoratus
found that metalloproteinases (MMPs) were involved in gonadal remodeling. In this
process, the ovaries degenerated and were slowly replaced by the germinal male tissue. In
particular, the action of MMPs induced significant changes in the interstitial gonadal tissue,
allowing for reorganization of germinal epithelial tissue. The study also found that sex
steroids help in the sex reversal process by being synthesized as Leydig cells replicate and
differentiate. Thus, the synthesis of sex steroids coincides with gonadal remodeling, which is
triggered by MMPs produced by germinal epithelial tissue. These results suggests that
MMPs and changes in steroid levels play a large role in sequential hermaphroditism in
teleosts.[58]

Genetic consequences
Sequential hermaphrodites almost always have a sex ratio biased towards the birth sex, and
consequently experience significantly more reproductive success after switching sexes.
According to the population genetics theory, this should decrease genetic diversity and
effective population size (Ne). However, a study of two ecologically similar santer sea bream
(gonochoric) and slinger sea bream (protogynous) in South African waters found that
genetic diversities were similar in the two species, and while Ne was lower in the instant for
the sex-changer, they were similar over a relatively short time horizon.[59] The ability of
these organisms to change biological sex has allowed for better reproductive success based
on the ability for certain genes to pass down more easily from generation to generation. The
change in sex also allows for organisms to reproduce if no individuals of the opposite sex are
already present.

Bioluminescence is the production and emission of light by a living organism. It is a form

of chemiluminescence. Bioluminescence occurs widely in marine vertebrates and
invertebrates, as well as in some fungi, microorganisms including some bioluminescent
bacteria and terrestrial arthropod such as fireflies. In some animals, the light is
bacteriogenic, produced by symbiotic organisms such as Vibrio bacteria; in others, it is
autogenic, produced by the animals themselves.

In a general sense, the principal chemical reaction in bioluminescence involves some light-
emitting molecule and an enzyme, generally called the luciferin and the luciferase,
respectively. Because these are generic names, the luciferins and luciferases are often
distinguished by including the species or group, i.e. Firefly luciferin. In all characterized
cases, the enzyme catalyzes the oxidation of the luciferin.

In some species, the luciferase requires other cofactors, such as calcium or magnesium ions,
and sometimes also the energy-carrying molecule adenosine triphosphate (ATP). In
evolution, luciferins vary little: one in particular, coelenterazine, is found in eleven different
animal (phyla), though in some of these, the animals obtain it through their diet. Conversely,
luciferases vary widely between different species, and consequently bioluminescence has
arisen over forty times in evolutionary history.

Both Aristotle and Pliny the Elder mentioned that damp wood sometimes gives off a glow
and many centuries later Robert Boyle showed that oxygen was involved in the process,
both in wood and in glow-worms. It was not until the late nineteenth century that
bioluminescence was properly investigated. The phenomenon is widely distributed among
animal groups, especially in marine environments where dinoflagellates cause
phosphorescence in the surface layers of water. On land it occurs in fungi, bacteria and
some groups of invertebrates, including insects.

The uses of bioluminescence by animals include counter-illumination camouflage, mimicry

of other animals, for example to lure prey, and signalling to other individuals of the same
species, such as to attract mates. In the laboratory, luciferase-based systems are used in
genetic engineering and for biomedical research. Other researchers are investigating the
possibility of using bioluminescent systems for street and decorative lighting, and a
bioluminescent plant has been created.[1]
Chemical mechanism
Main article: luciferase

Protein structure of the luciferase of the firefly Photinus pyralis. The enzyme is a much
larger molecule than luciferin.
Bioluminescence is a form of chemiluminescence where light energy is released by a
chemical reaction. This reaction involves a light-emitting pigment, the luciferin, and a
luciferase, the enzyme component.[31] Because of the diversity of luciferin/luciferase
combinations, there are very few commonalities in the chemical mechanism. From currently
studied systems, the only unifying mechanism is the role of molecular oxygen, though many
examples have a concurrent release of carbon dioxide. For example, the firefly
luciferin/luciferase reaction requires magnesium and ATP and produces carbon dioxide
(CO2), adenosine monophosphate (AMP) and pyrophosphate (PP) as waste products. Other
cofactors may be required for the reaction, such as calcium (Ca2+) for the photoprotein
aequorin, or magnesium (Mg2+) ions and ATP for the firefly luciferase.[32] Generically, this
reaction could be described as:

{\displaystyle {\ce {L+O2->[{\text{Luciferase}}][{\text{other cofactors}}]oxy-

L+lightenergy}}}{\displaystyle {\ce {L+O2->[{\text{Luciferase}}][{\text{other cofactors}}]oxy-
L+lightenergy}}}

Coelenterazine is a luciferin found in many different marine phyla from comb jellies to
vertebrates. Like all luciferins, it is oxidised to produce light.
Instead of a luciferase, the jellyfish Aequorea victoria makes use of another type of protein
called a photoprotein, in this case specifically aequorin.[33] When calcium ions are added,
the rapid catalysis creates a brief flash quite unlike the prolonged glow produced by
luciferase. In a second, much slower, step luciferin is regenerated from the oxidised
(oxyluciferin) form, allowing it to recombine with aequorin, in readiness for a subsequent
flash. Photoproteins are thus enzymes, but with unusual reaction kinetics.[34] Furthermore,
some of the blue light released by aequorin in contact with calcium ions is absorbed by a
green fluorescent protein, which in turn releases green light in a process called resonant
energy transfer.[35]

Overall, bioluminescence has arisen over forty times in evolutionary history.[31] In

evolution, luciferins tend to vary little: one in particular, coelenterazine, is the light emitting
pigment for nine phyla (groups of very different organisms), including polycystine radiolaria,
Cercozoa (Phaeodaria), protozoa, comb jellies, cnidaria including jellyfish and corals,
crustaceans, molluscs, arrow worms and vertebrates (ray-finned fish). Not all these
organisms synthesize coelenterazine: some of them obtain it through their diet.[31]
Conversely, luciferase enzymes vary widely and tend to be different in each species.[31]

A fungus gnat from New Zealand, Arachnocampa luminosa, lives in the predator-free
environment of caves and its larvae emit bluish-green light.[42] They dangle silken threads
that glow and attract flying insects, and wind in their fishing-lines when prey becomes
entangled.[43] The bioluminescence of the larvae of another fungus gnat from North
America which lives on streambanks and under overhangs has a similar function. Orfelia
fultoni builds sticky little webs and emits light of a deep blue colour. It has an inbuilt
biological clock and, even when kept in total darkness, turns its light on and off in a
circadian rhythm.[44]

Fireflies use light to attract mates. Two systems are involved according to species; in one,
females emit light from their abdomens to attract males; in the other, flying males emit
signals to which the sometimes sedentary females respond.[39][45] Click beetles emit an
orange light from the abdomen when flying and a green light from the thorax when they are
disturbed or moving about on the ground. The former is probably a sexual attractant but the
latter may be defensive.[39] Larvae of the click beetle Pyrophorus nyctophanus live in the
surface layers of termite mounds in Brazil. They light up the mounds by emitting a bright
greenish glow which attracts the flying insects on which they feed.[39]

In the marine environment, use of luminescence for mate attraction is chiefly known among
ostracods, small shrimplike crustaceans, especially in the family Cyprididae. Pheromones
may be used for long-distance communication, with bioluminescence used at close range to
enable mates to "home in".[31] A polychaete worm, the Bermuda fireworm creates a brief
display, a few nights after the full moon, when the female lights up to attract males.

Communication in the form of quorum sensing plays a role in the regulation of

luminescence in many species of bacteria. Small extracellularly secreted molecules stimulate
the bacteria to turn on genes for light production when cell density, measured by
concentration of the secreted molecules, is high.[31]

Pyrosomes are colonial tunicates and each zooid has a pair of luminescent organs on either
side of the inlet siphon. When stimulated by light, these turn on and off, causing rhythmic
flashing. No neural pathway runs between the zooids, but each responds to the light
produced by other individuals, and even to light from other nearby colonies.[52]
Communication by light emission between the zooids enables coordination of colony effort,
for example in swimming where each zooid provides part of the propulsive force.[53]

Some bioluminous bacteria infect nematodes that parasitize Lepidoptera larvae. When
these caterpillars die, their luminosity may attract predators to the dead insect thus
assisting in the dispersal of both bacteria and nematodes.[39] A similar reason may account
for the many species of fungi that emit light. Species in the genera Armillaria, Mycena,
Omphalotus, Panellus, Pleurotus and others do this, emitting usually greenish light from the
mycelium, cap and gills. This may attract night-flying insects and aid in spore dispersal, but
other functions may also be involved.[39]

Quantula striata is the only known bioluminescent terrestrial mollusc. Pulses of light are
emitted from a gland near the front of the foot and may have a communicative function,
although the adaptive significance is not fully understood

Bioluminescence is used by a variety of animals to mimic other species. Many species of

deep sea fish such as the anglerfish and dragonfish make use of aggressive mimicry to
attract prey. They have an appendage on their heads called an esca that contains
bioluminescent bacteria able to produce a long-lasting glow which the fish can control. The
glowing esca is dangled or waved about to lure small animals to within striking distance of
the fish.[31][55]

The cookiecutter shark uses bioluminescence to camouflage its underside by

counterillumination, but a small patch near its pectoral fins remains dark, appearing as a
small fish to large predatory fish like tuna and mackerel swimming beneath it. When such
fish approach the lure, they are bitten by the shark.[56][57]

Female Photuris fireflies sometimes mimic the light pattern of another firefly, Photinus, to
attract its males as prey. In this way they obtain both food and the defensive chemicals
named lucibufagins, which Photuris cannot synthesize.[58]

South American giant cockroaches of the genus Lucihormetica were believed to be the first
known example of defensive mimicry, emitting light in imitation of bioluminescent,
poisonous click beetles.[59] However, doubt has been cast on this assertion, and there is no
conclusive evidence that the cockroaches are bioluminescent.

Bioluminescent organisms are a target for many areas of research. Luciferase systems are
widely used in genetic engineering as reporter genes, each producing a different colour by
fluorescence,[64][65] and for biomedical research using bioluminescence
imaging.[66][67][68] For example, the firefly luciferase gene was used as early as 1986 for
research using transgenic tobacco plants.[69] Vibrio bacteria symbiose with marine
invertebrates such as the Hawaiian bobtail squid (Euprymna scolopes), are key experimental
models for bioluminescence.[70][71] Bioluminescent activated destruction is an
experimental cancer treatment.[72] See also optogenetics which involves the use of light to
control cells in living tissue, typically neurons, that have been genetically modified to
express light-sensitive ion channels, and also see biophoton, a photon of non-thermal origin
in the visible and ultraviolet spectrum emitted from a biological system.

The structures of photophores, the light producing organs in bioluminescent organisms, are
being investigated by industrial designers. Engineered bioluminescence could perhaps one
day be used to reduce the need for street lighting, or for decorative purposes if it becomes
possible to produce light that is both bright enough and can be sustained for long periods at
a workable price.[11][73][74] The gene that makes the tails of fireflies glow has been added
to mustard plants. The plants glow faintly for an hour when touched, but a sensitive camera
is needed to see the glow.[75] University of Wisconsin–Madison is researching the use of
genetically engineered bioluminescent E. coli bacteria, for use as bioluminescent bacteria in
a light bulb.[76] In 2011, Philips launched a microbial system for ambience lighting in the
home.[77][78] An iGEM team from Cambridge (England) has started to address the problem
that luciferin is consumed in the light-producing reaction by developing a genetic
biotechnology part that codes for a luciferin regenerating enzyme from the North American
firefly; this enzyme "helps to strengthen and sustain light output".[79] In 2016, Glowee, a
French company started selling bioluminescent lights, targeting shop fronts and municipal
street signs as their main markets.[80] France has a law that forbids retailers and offices
from illuminating their windows between 1 and 7 in the morning in order to minimise
energy consumption and pollution.[81][82] Glowee hoped their product would get around
this ban. They used bacteria called Aliivibrio fischeri which glow in the dark, but the
maximum lifetime of their product was three days.