The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific
DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study in detail. PCR was invented in 1983 by the American
biochemist
Biochemists are scientists who are trained in biochemistry. They study chemical processes and chemical transformations in living organisms. Biochemists study DNA, proteins and Cell (biology), cell parts. The word "biochemist" is a portmanteau of ...
Kary Mullis
Kary Banks Mullis (December 28, 1944August 7, 2019) was an American biochemist. In recognition of his role in the invention of the polymerase chain reaction (PCR) technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and wa ...
at
Cetus Corporation
Cetus Corporation was one of the first biotechnology companies. It was established in Berkeley, California, in 1971, but conducted most of its operations in nearby Emeryville. Before merging with Chiron Corporation in 1991 (now a part of Novarti ...
; Mullis and biochemist
Michael Smith, who had developed other essential ways of manipulating DNA,
were jointly awarded the
Nobel Prize in Chemistry
)
, image = Nobel Prize.png
, alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
in 1993.
PCR is fundamental to many of the procedures used in genetic testing and research, including analysis of
ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of
DNA sequences
A nucleic acid sequence is a succession of bases signified by a series of a set of five different letters that indicate the order of nucleotides forming alleles within a DNA (using GACT) or RNA (GACU) molecule. By convention, sequences are usua ...
are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in
medical laboratory
A medical laboratory or clinical laboratory is a laboratory where tests are conducted out on clinical specimens to obtain information about the health of a patient to aid in diagnosis, treatment, and prevention of disease. Clinical Medical labor ...
research for a broad variety of applications including
biomedical research
Medical research (or biomedical research), also known as experimental medicine, encompasses a wide array of research, extending from "basic research" (also called ''bench science'' or ''bench research''), – involving fundamental scientif ...
and
criminal forensics.
The majority of PCR methods rely on
thermal cycling
Thermal analysis is a branch of materials science where the properties of materials are studied as they change with temperature. Several methods are commonly used – these are distinguished from one another by the property which is measured:
* ...
. Thermal cycling exposes reactants to repeated cycles of heating and cooling to permit different temperature-dependent reactions—specifically,
DNA melting
Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of double-stranded DNA (dsDNA). The melting temperature (''Tm'') is defined as the temperature at which half of the DNA strands are in the random coil o ...
and
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
-driven
DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
. PCR employs two main reagents—
primers (which are short single strand DNA fragments known as
oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids c ...
s that are a
complementary
A complement is something that completes something else.
Complement may refer specifically to:
The arts
* Complement (music), an interval that, when added to another, spans an octave
** Aggregate complementation, the separation of pitch-class ...
sequence to the target DNA region) and a
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
. 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
Template may refer to:
Tools
* Die (manufacturing), used to cut or shape material
* Mold, in a molding process
* Stencil, a pattern or overlay used in graphic arts (drawing, painting, etc.) and sewing to replicate letters, shapes or designs
Co ...
for DNA polymerase to
enzymatically
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
assemble a new DNA strand from free
nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...
s, 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
A chain reaction is a sequence of reactions where a reactive product or by-product causes additional reactions to take place. In a chain reaction, positive feedback leads to a self-amplifying chain of events.
Chain reactions are one way that syst ...
in which the original DNA template is
exponentially
Exponential may refer to any of several mathematical topics related to exponentiation, including:
*Exponential function, also:
**Matrix exponential, the matrix analogue to the above
* Exponential decay, decrease at a rate proportional to value
*Exp ...
amplified.
Almost all PCR applications employ a heat-stable DNA polymerase, such as
''Taq'' polymerase, an enzyme originally isolated from the
thermophilic
A thermophile is an organism—a type of extremophile—that thrives at relatively high temperatures, between . Many thermophiles are archaea, though they can be bacteria or fungi. Thermophilic eubacteria are suggested to have been among the earl ...
bacterium ''
Thermus aquaticus
''Thermus aquaticus'' is a species of bacteria that can tolerate high temperatures, one of several thermophilic bacteria that belong to the ''Deinococcota'' phylum. It is the source of the heat-resistant enzyme ''Taq'' DNA polymerase, one of th ...
''. 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.
Applications of the technique include
DNA cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
for
sequencing
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...
, gene cloning and manipulation, gene mutagenesis; construction of DNA-based
phylogenies
A phylogenetic tree (also phylogeny or evolutionary tree Felsenstein J. (2004). ''Inferring Phylogenies'' Sinauer Associates: Sunderland, MA.) is a branching diagram or a tree showing the evolutionary relationships among various biological spec ...
, or functional analysis of
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s;
diagnosis
Diagnosis is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in many different disciplines, with variations in the use of logic, analytics, and experience, to determine " cause and effect". In systems engin ...
and
monitoring
Monitoring may refer to:
Science and technology Biology and healthcare
* Monitoring (medicine), the observation of a disease, condition or one or several medical parameters over time
* Baby monitoring
* Biomonitoring, of toxic chemical compounds, ...
of
genetic disorder
A genetic disorder is a health problem caused by one or more abnormalities in the genome. It can be caused by a mutation in a single gene (monogenic) or multiple genes (polygenic) or by a chromosomal abnormality. Although polygenic disorders ...
s; amplification of ancient DNA;
analysis of genetic fingerprints for
DNA profiling
DNA profiling (also called DNA fingerprinting) is the process of determining an individual's DNA characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding.
DNA profiling is a forensic tec ...
(for example, in
forensic science
Forensic science, also known as criminalistics, is the application of science to criminal and civil laws, mainly—on the criminal side—during criminal investigation, as governed by the legal standards of admissible evidence and criminal ...
and
parentage testing
DNA paternity testing is the use of DNA profiles to determine whether an individual is the biological parent of another individual. Paternity testing can be especially important when the rights and duties of the father are in issue and a child' ...
); and detection of
pathogen
In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ ...
s in
nucleic acid test
A nucleic acid test (NAT) is a technique used to detect a particular nucleic acid sequence and thus usually to detect and identify a particular species or subspecies of organism, often a virus or bacterium that acts as a pathogen in blood, tissu ...
s for the diagnosis of
infectious disease
An infection is the invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce. An infectious disease, also known as a transmissible disease or communicable dise ...
s.
Principles
![Primitive PCR machine for scrap](https://upload.wikimedia.org/wikipedia/commons/2/2f/Primitive_PCR_machine_for_scrap.JPG)
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 pair
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DN ...
s (kbp) in length, although some techniques allow for amplification of fragments up to 40 kbp. The amount of amplified product is determined by the available substrates in the reaction, which becomes limiting as the reaction progresses.
A basic PCR set-up requires several components and reagents,
[ Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction] including:
* a ''DNA template'' that contains the DNA target region to amplify
* a ''
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
''; an enzyme that
polymerizes
In polymer chemistry, polymerization (American English), or polymerisation (British English), is a process of reacting monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks. There are many for ...
new DNA strands; heat-resistant
''Taq'' polymerase is especially common, as it is more likely to remain intact during the high-temperature DNA denaturation process
* two DNA ''
primers'' that are
complementary
A complement is something that completes something else.
Complement may refer specifically to:
The arts
* Complement (music), an interval that, when added to another, spans an octave
** Aggregate complementation, the separation of pitch-class ...
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);
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";
nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...
s containing triphosphate groups), the building blocks from which the DNA polymerase synthesizes a new DNA strand
* a ''
buffer solution
A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is ...
'' providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
* ''
bivalent Bivalent may refer to:
* Bivalent (chemistry), a molecule formed from two or more atoms bound together
*Bivalent (engine), an engine that can operate on two different types of fuel
*Bivalent (genetics), a pair of homologous chromosomes
*Bivalent log ...
cations
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
'', typically
magnesium
Magnesium is a chemical element with the symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 of the periodic ta ...
(Mg) or
manganese
Manganese is a chemical element with the symbol Mn and atomic number 25. It is a hard, brittle, silvery metal, often found in minerals in combination with iron. Manganese is a transition metal with a multifaceted array of industrial alloy use ...
(Mn) ions; Mg
2+ is the most common, but Mn
2+ can be used for
PCR-mediated DNA mutagenesis, as a higher Mn
2+ concentration increases the error rate during DNA synthesis; and ''monovalent cations'', typically
potassium
Potassium is the chemical element with the symbol K (from Neo-Latin ''kalium'') and atomic number19. Potassium is a silvery-white metal that is soft enough to be cut with a knife with little force. Potassium metal reacts rapidly with atmosphe ...
(K) ions
The reaction is commonly carried out in a volume of 10–200
μL
The litre (international spelling) or liter (American English spelling) (SI symbols L and l, other symbol used: ℓ) is a metric unit of volume. It is equal to 1 cubic decimetre (dm3), 1000 cubic centimetres (cm3) or 0.001 cubic metre (m3). ...
in small reaction tubes (0.2–0.5 mL volumes) in a
thermal cycler
The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). Thermal cyclers may also be used in laboratories to fa ...
. 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
The thermoelectric effect is the direct conversion of temperature differences to electric voltage and vice versa via a thermocouple. A thermoelectric device creates a voltage when there is a different temperature on each side. Conversely, when ...
, 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
The thermal conductivity of a material is a measure of its ability to conduct heat. It is commonly denoted by k, \lambda, or \kappa.
Heat transfer occurs at a lower rate in materials of low thermal conductivity than in materials of high thermal ...
to allow for rapid thermal equilibrium. Most thermal cyclers have heated lids to prevent
condensation
Condensation is the change of the state of matter from the gas phase into the liquid phase, and is the reverse of vaporization. The word most often refers to the water cycle. It can also be defined as the change in the state of water vapor to ...
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 (>), 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 (''T
m'') of the primers. 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.
It consists of heating the reaction chamber to a temperature of , or 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 for 20–30 seconds. This causes
DNA melting
Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of double-stranded DNA (dsDNA). The melting temperature (''Tm'') is defined as the temperature at which half of the DNA strands are in the random coil o ...
, or denaturation, of the double-stranded DNA template by breaking the
hydrogen bond
In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a ...
s between complementary bases, yielding two single-stranded DNA molecules.
* ''
Annealing'': In the next step, the reaction temperature is lowered to 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
Hybridization (or hybridisation) may refer to:
*Hybridization (biology), the process of combining different varieties of organisms to create a hybrid
*Orbital hybridization, in chemistry, the mixing of atomic orbitals into new hybrid orbitals
*Nu ...
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 ''T
m'' 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'' polymerase is approximately ,
though a temperature of 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 is complementary to the template in the
5'-to-3' direction,
condensing the 5'-
phosphate group
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosphoric acid .
The phosphate or orthophosphate ion is derived from phosphor ...
of the dNTPs with the 3'-
hydroxy group
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
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 2
n, where ''n'' is the number of cycles. Thus, a reaction set for 30 cycles results in 2
30, or , copies of the original double-stranded DNA target region.
* ''Final elongation'': This single step is optional, but is performed at a temperature of (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 for an indefinite time, and may be employed for short-term storage of the PCR products.
![Polymerase chain reaction-en](https://upload.wikimedia.org/wikipedia/commons/a/ab/Polymerase_chain_reaction-en.svg)
To check whether the PCR successfully generated the anticipated DNA target region (also sometimes referred to as the amplimer or
amplicon
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification (molecular biology), amplification or DNA replication, replication events. It can be formed artificially, using various methods including ...
),
agarose gel electrophoresis
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the ...
may be employed for size separation of the PCR products. The size of the PCR products is determined by comparison with a
DNA ladder
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle th ...
, a molecular weight marker which contains DNA fragments of known sizes, which runs on the gel alongside the PCR products.
Stages
![Exponential Amplification](https://upload.wikimedia.org/wikipedia/commons/1/1a/Exponential_Amplification.svg)
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.
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
In practice, PCR can fail for various reasons, such as sensitivity or contamination. Contamination with extraneous DNA can lead to spurious products and is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.
For instance, if DNA from a crime scene is analyzed, a single DNA molecule from lab personnel could be amplified and misguide the investigation. Hence the PCR-setup areas is separated from the analysis or purification of other PCR products, disposable plasticware used, and the work surface between reaction setups needs to be thoroughly cleaned.
Specificity can be adjusted by experimental conditions so that no spurious products are generated. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of unspecific products. The usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. For instance, Q5 polymerase is said to be ~280 times less error-prone than Taq polymerase. Both the running parameters (e.g. temperature and duration of cycles), or the addition of reagents, such as
formamide
Formamide is an amide derived from formic acid. It is a colorless liquid which is miscible with water and has an ammonia-like odor. It is chemical feedstock for the manufacture of sulfa drugs and other pharmaceuticals, herbicides and pesticide ...
, may increase the specificity and yield of PCR. Computer simulations of theoretical PCR results (
Electronic PCR) may be performed to assist in primer design.
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 probe
In molecular biology, a hybridization probe (HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RN ...
s for
Southern
Southern may refer to:
Businesses
* China Southern Airlines, airline based in Guangzhou, China
* Southern Airways, defunct US airline
* Southern Air, air cargo transportation company based in Norwalk, Connecticut, US
* Southern Airways Express, M ...
or
northern
Northern may refer to the following:
Geography
* North, a point in direction
* Northern Europe, the northern part or region of Europe
* Northern Highland, a region of Wisconsin, United States
* Northern Province, Sri Lanka
* Northern Range, a ra ...
hybridization and
DNA cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
, 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
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in
Sanger sequencing
Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederi ...
, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
,
phage
A bacteriophage (), also known informally as a ''phage'' (), is a duplodnaviria virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν ('), meaning "to devour". Bacterio ...
, or
cosmid
A cosmid is a type of hybrid plasmid that contains a Lambda phage ''cos'' sequence. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in ...
(depending on size) or the genetic material of another organism. Bacterial colonies ''(such as
E. coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
)'' can be rapidly screened by PCR for correct DNA
vector
Vector most often refers to:
*Euclidean vector, a quantity with a magnitude and a direction
*Vector (epidemiology), an agent that carries and transmits an infectious pathogen into another living organism
Vector may also refer to:
Mathematic ...
constructs.
PCR may also be used for
genetic fingerprinting
DNA profiling (also called DNA fingerprinting) is the process of determining an individual's DNA characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding.
DNA profiling is a forensic tec ...
; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.
![Pcr fingerprint](https://upload.wikimedia.org/wikipedia/commons/0/02/Pcr_fingerprint.png)
Some PCR fingerprint 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 clock
The molecular clock is a figurative term for a technique that uses the mutation rate of biomolecules to deduce the time in prehistory when two or more life forms diverged. The biomolecular data used for such calculations are usually nucleoti ...
s are used (i.e. the
16S rRNA 16S rRNA may refer to:
* 16S ribosomal RNA
16 S ribosomal RNA (or 16 S rRNA) is the RNA component of the 30S subunit of a prokaryotic ribosome ( SSU rRNA). It binds to the Shine-Dalgarno sequence and provides most of the SSU structure.
The g ...
and recA genes of microorganisms).
Amplification and quantification of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for
forensic analysis
Forensic science, also known as criminalistics, is the application of science to criminal and civil laws, mainly—on the criminal side—during criminal investigation, as governed by the legal standards of admissible evidence and criminal p ...
, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of
ancient DNA
Ancient DNA (aDNA) is DNA isolated from ancient specimens. Due to degradation processes (including cross-linking, deamination and fragmentation) ancient DNA is more degraded in comparison with contemporary genetic material. Even under the bes ...
that is tens of thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old
mammoth
A mammoth is any species of the extinct elephantid genus ''Mammuthus'', one of the many genera that make up the order of trunked mammals called proboscideans. The various species of mammoth were commonly equipped with long, curved tusks and, ...
, and also on human DNA, in applications ranging from the analysis of Egyptian
mummies
A mummy is a dead human or an animal whose soft tissues and organs have been preserved by either intentional or accidental exposure to chemicals, extreme cold, very low humidity, or lack of air, so that the recovered body does not decay furth ...
to the identification of a Russian
tsar
Tsar ( or ), also spelled ''czar'', ''tzar'', or ''csar'', is a title used by East Slavs, East and South Slavs, South Slavic monarchs. The term is derived from the Latin word ''Caesar (title), caesar'', which was intended to mean "emperor" i ...
and the body of English king
Richard III
Richard III (2 October 145222 August 1485) was King of England and Lord of Ireland from 26 June 1483 until his death in 1485. He was the last king of the House of York and the last of the Plantagenet dynasty. His defeat and death at the Battl ...
.
Quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
or Real Time PCR (qPCR, not to be confused with
RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
) methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of
gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.
qPCR allows the quantification and detection of a specific DNA sequence in real time since it measures concentration while the synthesis process is taking place. There are two methods for simultaneous detection and quantification. The first method consists of using
fluorescent
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its
complementary DNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
(cDNA) takes place. An interesting technique combination is real-time PCR and reverse transcription. This sophisticated technique, called RT-qPCR, allows for the quantification of a small quantity of RNA. Through this combined technique, mRNA is converted to cDNA, which is further quantified using qPCR. This technique lowers the possibility of error at the end point of PCR,
increasing chances for detection of genes associated with genetic diseases such as cancer.
Laboratories use RT-qPCR for the purpose of sensitively measuring gene regulation. The mathematical foundations for the reliable quantification of the PCR and RT-qPCR facilitate the implementation of accurate fitting procedures of experimental data in research, medical, diagnostic and infectious disease applications.
Medical and diagnostic applications
Prospective parents can be tested for being
genetic carrier
A hereditary carrier (genetic carrier or just carrier), is a person or other organism that has inherited a recessive allele for a genetic trait or mutation but usually does not display that trait or show symptoms of the disease. Carriers are, ho ...
s, or their children might be tested for actually being affected by a
disease
A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that a ...
.
DNA samples for
prenatal testing
Prenatal testing consists of prenatal screening and prenatal diagnosis, which are aspects of prenatal care that focus on detecting problems with the pregnancy as early as possible. These may be anatomic and physiologic problems with the health of ...
can be obtained by
amniocentesis
Amniocentesis is a medical procedure used primarily in the prenatal diagnosis of genetic conditions. It has other uses such as in the assessment of infection and fetal lung maturity. Prenatal diagnostic testing, which includes amniocentesis, is ne ...
,
chorionic villus sampling
Chorionic villus sampling (CVS), sometimes called "chorionic ''villous'' sampling" (as "villous" is the adjectival form of the word "villus"), is a form of prenatal diagnosis done to determine chromosomal or genetic disorders in the fetus. It ent ...
, or even by the analysis of rare fetal cells circulating in the mother's bloodstream. PCR analysis is also essential to
preimplantation genetic diagnosis
Preimplantation genetic diagnosis (PGD or PIGD) is the genetic profiling of embryos prior to implantation (as a form of embryo profiling), and sometimes even of oocytes prior to fertilization. PGD is considered in a similar fashion to prenatal ...
, 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 Tissue typing is a procedure in which the tissues of a prospective donor and recipient are tested for compatibility prior to transplantation. Mismatched donor and recipient tissues can lead to rejection of the tissues. There are multiple methods of ...
'', vital to
organ transplantation
Organ transplantation is a medical procedure in which an organ (anatomy), organ is removed from one body and placed in the body of a recipient, to replace a damaged or missing organ. The donor and recipient may be at the same location, or organ ...
. there is even a proposal to replace the traditional antibody-based tests for
blood type
A blood type (also known as a blood group) is a classification of blood, based on the presence and absence of antibodies and inherited antigenic substances on the surface of red blood cells (RBCs). These antigens may be proteins, carbohydrate ...
with PCR-based tests.
* Many forms of cancer involve alterations to ''
oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels. s''. 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
Malignancy () is the tendency of a medical condition to become progressively worse.
Malignancy is most familiar as a characterization of cancer. A ''malignant'' tumor contrasts with a non-cancerous ''benign'' tumor in that a malignancy is not s ...
diseases such as
leukemia
Leukemia ( also spelled leukaemia and pronounced ) is a group of blood cancers that usually begin in the bone marrow and result in high numbers of abnormal blood cells. These blood cells are not fully developed and are called ''blasts'' or ' ...
and
lymphoma
Lymphoma is a group of blood and lymph tumors that develop from lymphocytes (a type of white blood cell). In current usage the name usually refers to just the cancerous versions rather than all such tumours. Signs and symptoms may include enlar ...
s, 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. 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.
Infectious disease applications
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses.
PCR also permits identification of non-cultivatable or slow-growing microorganisms such as
mycobacteria
''Mycobacterium'' is a genus of over 190 species in the phylum Actinomycetota, assigned its own family, Mycobacteriaceae. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis ('' M. tuberculosis'') and ...
,
anaerobic bacteria
An anaerobic organism or anaerobe is any organism that does not require molecular oxygen for growth. It may react negatively or even die if free oxygen is present. In contrast, an aerobic organism (aerobe) is an organism that requires an oxygenate ...
, or
virus
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's 1 ...
es from
tissue culture
Tissue culture is the growth of tissues or cells in an artificial medium separate from the parent organism. This technique is also called micropropagation. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, su ...
assays and
animal model
An animal model (short for animal disease model) is a living, non-human, often genetic-engineered animal used during the research and investigation of human disease, for the purpose of better understanding the disease process without the risk of ha ...
s. 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.
Characterization and detection of infectious disease organisms have been revolutionized by PCR in the following ways:
* The ''human immunodeficiency virus'' (or ''
HIV
The human immunodeficiency viruses (HIV) are two species of ''Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immune ...
''), 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
Test(s), testing, or TEST may refer to:
* Test (assessment), an educational assessment intended to measure the respondents' knowledge or other abilities
Arts and entertainment
* ''Test'' (2013 film), an American film
* ''Test'' (2014 film), ...
have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells. 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
Tuberculosis (TB) is an infectious disease usually caused by '' Mycobacterium tuberculosis'' (MTB) bacteria. Tuberculosis generally affects the lungs, but it can also affect other parts of the body. Most infections show no symptoms, in ...
'', 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
Domestic may refer to:
In the home
* Anything relating to the human home or family
** A domestic animal, one that has undergone domestication
** A domestic appliance, or home appliance
** A domestic partnership
** Domestic science, sometimes c ...
or
wild
Wild, wild, wilds or wild may refer to:
Common meanings
* Wild animal
* Wilderness, a wild natural environment
* Wildness, the quality of being wild or untamed
Art, media and entertainment Film and television
* ''Wild'' (2014 film), a 2014 A ...
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.
Such early detection may give physicians a significant lead time in treatment. The amount of virus ("
viral load
Viral load, also known as viral burden, is a numerical expression of the quantity of virus in a given volume of fluid, including biological and environmental specimens. It is not to be confused with viral titre or viral titer, which depends on the ...
") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below). A variant of PCR (
RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
) is used for detecting viral RNA rather than DNA: in this test the enzyme reverse transcriptase is used to generate a DNA sequence which matches the viral RNA; this DNA is then amplified as per the usual PCR method. RT-PCR is widely used to detect the SARS-CoV-2 viral genome.
* Diseases such as pertussis (or
whooping cough
Whooping cough, also known as pertussis or the 100-day cough, is a highly contagious bacterial disease. Initial symptoms are usually similar to those of the common cold with a runny nose, fever, and mild cough, but these are followed by two or ...
) are caused by the bacteria ''
Bordetella pertussis
''Bordetella pertussis'' is a Gram-negative, aerobic, pathogenic, encapsulated coccobacillus of the genus ''Bordetella'', and the causative agent of pertussis or whooping cough. Like '' B. bronchiseptica'', ''B. pertussis'' is motile and expres ...
''. 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 play a role in cell immunity. PCR is an important testing tool that can detect sequences within the gene for the pertussis toxin. Because PCR has a high sensitivity for the toxin and a rapid turnaround time, it is very efficient for diagnosing pertussis when compared to culture.
Forensic applications
The development of PCR-based
genetic (or
DNA) fingerprinting protocols has seen widespread application in
forensics
Forensic science, also known as criminalistics, is the application of science to criminal and civil laws, mainly—on the criminal side—during criminal investigation, as governed by the legal standards of admissible evidence and crimina ...
:
*
![US Army CID agents at crime scene](https://upload.wikimedia.org/wikipedia/commons/f/f1/US_Army_CID_agents_at_crime_scene.jpg)
In its most discriminating form, ''
genetic fingerprinting
DNA profiling (also called DNA fingerprinting) is the process of determining an individual's DNA characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding.
DNA profiling is a forensic tec ...
'' can uniquely discriminate any one person from the entire population of the
world
In its most general sense, the term "world" refers to the totality of entities, to the whole of reality or to everything that is. The nature of the world has been conceptualized differently in different fields. Some conceptions see the worl ...
. Minute samples of DNA can be isolated from a
crime scene
A crime scene is any location that may be associated with a committed crime. Crime scenes contain physical evidence that is pertinent to a criminal investigation. This evidence is collected by crime scene investigators (CSI) and law enforcement ...
, and
compared to that from suspects, or from a
DNA database
A DNA database or DNA databank is a database of DNA profiles which can be used in the analysis of genetic diseases, genetic fingerprinting for criminology, or genetic genealogy. DNA databases may be public or private, the largest ones being na ...
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
Exoneration occurs when the conviction for a crime is reversed, either through demonstration of innocence, a flaw in the conviction, or otherwise. Attempts to exonerate convicts are particularly controversial in death penalty cases, especially wh ...
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.
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.
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.
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.
* Less discriminating forms of
DNA fingerprinting
DNA profiling (also called DNA fingerprinting) is the process of determining an individual's DNA characteristics. DNA analysis intended to identify a species, rather than an individual, is called DNA barcoding.
DNA profiling is a forensic tec ...
can help in ''
DNA paternity testing
DNA paternity testing is the use of DNA profiles to determine whether an individual is the biological parent of another individual. Paternity testing can be especially important when the rights and duties of the father are in issue and a child ...
'', 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
In Christian denominations that practice infant baptism, confirmation is seen as the sealing of the covenant created in baptism. Those being confirmed are known as confirmands. For adults, it is an affirmation of belief. It involves laying on ...
(or ruled out).
* The PCR AMGX/AMGY design facilitate 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 a powerful and effective way to determine gender in forensic cases and ancient specimens.
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
Hybridization (or hybridisation) may refer to:
*Hybridization (biology), the process of combining different varieties of organisms to create a hybrid
*Orbital hybridization, in chemistry, the mixing of atomic orbitals into new hybrid orbitals
*Nu ...
probes'' for
Southern
Southern may refer to:
Businesses
* China Southern Airlines, airline based in Guangzhou, China
* Southern Airways, defunct US airline
* Southern Air, air cargo transportation company based in Norwalk, Connecticut, US
* Southern Airways Express, M ...
or
northern blot
The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.K ...
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
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
'' 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
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
''. 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 site A sequence-tagged site (or STS) is a short (200 to 500 base pair) DNA sequence that has a single occurrence in the genome and whose location and base sequence are known.
Usage
STSs can be easily detected by the polymerase chain reaction (PCR) usin ...
s'' 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
The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a ...
found this application vital to mapping the cosmid clones they were sequencing, and to coordinating the results from different laboratories.
* An application of PCR is the
phylogenic
In biology, phylogenetics (; from Greek φυλή/ φῦλον [] "tribe, clan, race", and wikt:γενετικός, γενετικός [] "origin, source, birth") is the study of the evolutionary history and relationships among or within groups o ...
analysis of DNA from ''
ancient sources'', such as that found in the recovered bones of
Neanderthal
Neanderthals (, also ''Homo neanderthalensis'' and erroneously ''Homo sapiens neanderthalensis''), also written as Neandertals, are an extinct species or subspecies of archaic humans who lived in Eurasia until about 40,000 years ago. While th ...
s, from frozen tissues of
mammoth
A mammoth is any species of the extinct elephantid genus ''Mammuthus'', one of the many genera that make up the order of trunked mammals called proboscideans. The various species of mammoth were commonly equipped with long, curved tusks and, ...
s, or from the brain of Egyptian mummies.
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
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
''. 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
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
to quantitate the actual levels of expression
* The ability of PCR to simultaneously amplify several loci from individual sperm has greatly enhanced the more traditional task of ''
genetic mapping
Genetic linkage is the tendency of DNA sequences that are close together on a chromosome to be inherited together during the meiosis phase of sexual reproduction. Two genetic markers that are physically near to each other are unlikely to be separ ...
'' by studying
chromosomal crossover
Chromosomal crossover, or crossing over, is the exchange of genetic material during sexual reproduction between two homologous chromosomes' non-sister chromatids that results in recombinant chromosomes. It is one of the final phases of geneti ...
s after
meiosis
Meiosis (; , since it is a reductional division) is a special type of cell division of germ cells in sexually-reproducing organisms that produces the gametes, such as sperm or egg cells. It involves two rounds of division that ultimately resu ...
. 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
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesi ...
: 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.
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.
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.
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.
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. Pipettors with disposable plungers and extra-long pipette tips should be routinely used.
It is moreover recommended to ensure that the lab set-up follows a unidirectional workflow. No materials or reagents used in the PCR and analysis rooms should ever be taken into the PCR preparation room without thorough decontamination.
Environmental samples that contain humic acids may inhibit PCR amplification and lead to inaccurate results.
Variations
* ''Allele-specific PCR'': a diagnostic or cloning technique based on single-nucleotide variations (SNVs not to be confused with
SNPs
In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...
) (single-base differences in a patient). It requires prior knowledge of a DNA sequence, including differences between
allele
An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution.
::"The chro ...
s, 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. See
SNP genotyping
SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common ...
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.
* ''
Asymmetric PCR'': preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in
sequencing
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...
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
Arithmetic () is an elementary part of mathematics that consists of the study of the properties of the traditional operations on numbers— addition, subtraction, multiplication, division, exponentiation, and extraction of roots. In the 19th ...
) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.
A recent modification on this process, known as ''L''inear-''A''fter-''T''he-''E''xponential-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.
* ''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. 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. 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.
* ''
Digital PCR
Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a biotechnology, biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids ...
(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 Helicase-dependent amplification (HDA) is a method for ''in vitro'' DNA amplification (like the polymerase chain reaction) that takes place at a constant temperature.
Introduction
The polymerase chain reaction is the most widely used method for '' ...
'': similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles.
DNA helicase
Helicases are a class of enzymes thought to be vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separatin ...
, an enzyme that unwinds DNA, is used in place of thermal denaturation.
* ''
Hot start PCR Hot start PCR is a modified form of conventional polymerase chain reaction (PCR) that reduces the presence of undesired products and primer dimers due to non-specific DNA amplification at room (or colder) temperatures. Many variations and modificat ...
'': 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.
Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an
antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
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
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
or
transcriptome
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The t ...
. In silico PCR was proposed as an educational tool for molecular biology.
* ''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.
* ''
Inverse PCR'': is commonly used to identify the flanking sequences around
genomic
Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dim ...
inserts. It involves a series of
DNA digestions and
self ligation, resulting in known sequences at either end of the unknown sequence.
* ''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
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
,
genome walking, and
DNA footprinting DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and within cells.
The regulation of transcription has been studied ...
.
* ''
Methylation-specific PCR'' (MSP): developed by
Stephen Baylin and
James G. Herman
James Gordon Herman is an American oncologist.
Herman studied chemistry at Hope College and earned a medical degree from the Johns Hopkins School of Medicine. He completed his residency in internal medicine at Duke University in 1992, and a fello ...
at the Johns Hopkins School of Medicine, 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.
* ''
Multiplex ligation-dependent probe amplification
Multiplex ligation-dependent probe amplification (MLPA) is a variation of the multiplex polymerase chain reaction that permits amplification of multiple targets with only a single primer pair. It detects copy number changes at the molecular level, ...
'' (''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
amplicon
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification (molecular biology), amplification or DNA replication, replication events. It can be formed artificially, using various methods including ...
s 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
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
.
* ''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
Zinc oxide is an inorganic compound with the formula . It is a white powder that is insoluble in water. ZnO is used as an additive in numerous materials and products including cosmetics, food supplements, rubbers, plastics, ceramics, glass, cemen ...
,
titanium dioxide
Titanium dioxide, also known as titanium(IV) oxide or titania , is the inorganic compound with the chemical formula . When used as a pigment, it is called titanium white, Pigment White 6 (PW6), or CI 77891. It is a white solid that is insolubl ...
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.
* ''
Nested PCR
Nested polymerase chain reaction (nested PCR) is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites.
Polymerase chain reaction
Polymerase chai ...
'': 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
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
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.
* ''PAN-AC'': uses isothermal conditions for amplification, and may be used in living cells.
* ''
Quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
'' (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
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
-containing DNA probes, such as
TaqMan TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-L ...
, to measure the amount of amplified product in real time. It is also sometimes abbreviated to
RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
(''real-time'' PCR) but this abbreviation should be used only for
reverse transcription PCR. qPCR is the appropriate contractions for
quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
(real-time PCR).
* ''
Reverse Complement PCR'' (RC-PCR): Allows the addition of functional domains or sequences of choice to be appended independently to either end of the generated amplicon in a single closed tube reaction. This method generates target specific primers within the reaction by the interaction of universal primers (which contain the desired sequences or domains to be appended) and RC probes.
* ''Reverse Transcription PCR (
RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase cha ...
)'': for amplifying DNA from RNA.
Reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
reverse transcribes
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
into
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
, which is then amplified by PCR. RT-PCR is widely used in
expression profiling
In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between c ...
, 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
An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequence ...
and
introns
An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene. ...
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
RNase H-dependent PCR (rhPCR) is a modification of the standard PCR technique. In rhPCR, the primers are designed with a removable amplification block on the 3’ end. Amplification of the blocked primer is dependent on the cleavage activity o ...
'' (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.
* ''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.
* ''Solid Phase PCR'': encompasses multiple meanings, including
Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (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 (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 Paleogenetics is the study of the past through the examination of preserved genetic material from the remains of ancient organisms. Emile Zuckerkandl and Linus Pauling introduced the term in 1963, long before the sequencing of DNA, in reference to ...
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
''Yersinia pestis'' (''Y. pestis''; formerly '' Pasteurella pestis'') is a gram-negative, non-motile, coccobacillus bacterium without spores that is related to both ''Yersinia pseudotuberculosis'' and ''Yersinia enterocolitica''. It is a facult ...
in dental samples obtained from 14th Century graves of people supposedly killed by the plague during the medieval
Black Death
The Black Death (also known as the Pestilence, the Great Mortality or the Plague) was a bubonic plague pandemic occurring in Western Eurasia and North Africa from 1346 to 1353. It is the most fatal pandemic recorded in human history, causi ...
epidemic. 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.
* ''
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 T
m of the primers used, while at the later cycles, it is a few degrees (3–5 °C) below the primer T
m. 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.
* ''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')
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),
5'RACE LaNe
and 3'RACE LaNe.
History
![Primers RevComp](https://upload.wikimedia.org/wikipedia/commons/9/91/Primers_RevComp.svg)
The heat-resistant enzymes that are a key component in polymerase chain reaction were discovered in the 1960s as a product of a microbial life form that lived in the superheated waters of
Yellowstone
Yellowstone National Park is an American national park located in the western United States, largely in the northwest corner of Wyoming and extending into Montana and Idaho. It was established by the 42nd U.S. Congress with the Yellowston ...
's Mushroom Spring.
A 1971 paper in the ''
Journal of Molecular Biology
The ''Journal of Molecular Biology'' is a biweekly peer-reviewed scientific journal covering all aspects of molecular biology. It was established in 1959 and is published by Elsevier. The editor-in-chief is Peter Wright ( The Scripps Research In ...
'' by
Kjell Kleppe
Kjell Kleppe (1934-1988) was a Norwegian biochemist and molecular biologist who was a pioneer in the PCR technique and built the first laboratory in the country for bio and gene technology.
Kjell Kleppe earned a B.S. degree from the University of ...
and co-workers in the laboratory of
H. Gobind Khorana first described a method of using an enzymatic assay to replicate a short DNA template with primers ''in vitro''. However, this early manifestation of the basic PCR principle did not receive much attention at the time and the invention of the polymerase chain reaction in 1983 is generally credited to
Kary Mullis
Kary Banks Mullis (December 28, 1944August 7, 2019) was an American biochemist. In recognition of his role in the invention of the polymerase chain reaction (PCR) technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and wa ...
.
When Mullis developed the PCR in 1983, he was working in
Emeryville Emeryville may refer to:
* Emeryville, California
Emeryville is a city located in northwest Alameda County, California, in the United States. It lies in a corridor between the cities of Berkeley and Oakland, with a border on the shore of San ...
, California for
Cetus Corporation
Cetus Corporation was one of the first biotechnology companies. It was established in Berkeley, California, in 1971, but conducted most of its operations in nearby Emeryville. Before merging with Chiron Corporation in 1991 (now a part of Novarti ...
, one of the first
biotechnology
Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used b ...
companies, where he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived the idea for PCR while cruising along the
Pacific Coast Highway one night in his car.
He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase. In ''
Scientific American
''Scientific American'', informally abbreviated ''SciAm'' or sometimes ''SA'', is an American popular science magazine. Many famous scientists, including Albert Einstein and Nikola Tesla, have contributed articles to it. In print since 1845, it i ...
'', Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat." DNA fingerprinting was first used for
paternity testing
DNA paternity testing is the use of DNA profiles to determine whether an individual is the biological parent of another individual. Paternity testing can be especially important when the rights and duties of the father are in issue and a child ...
in 1988.
Mullis has credited his use of
LSD
Lysergic acid diethylamide (LSD), also known colloquially as acid, is a potent psychedelic drug. Effects typically include intensified thoughts, emotions, and sensory perception. At sufficiently high dosages LSD manifests primarily mental, vi ...
as integral to his development of PCR: "Would I have invented PCR if I hadn't taken LSD? I seriously doubt it. I could sit on a DNA molecule and watch the polymers go by. I learnt that partly on psychedelic drugs."
Mullis and biochemist
Michael Smith, who had developed other essential ways of manipulating DNA,
were jointly awarded the
Nobel Prize in Chemistry
)
, image = Nobel Prize.png
, alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
in 1993, seven years after Mullis and his colleagues at Cetus first put his proposal to practice.
Mullis's 1985 paper with R. K. Saiki and H. A. Erlich, "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia"—the polymerase chain reaction invention (PCR)—was honored by a Citation for Chemical Breakthrough Award from the Division of History of Chemistry of the American Chemical Society in 2017.
At the core of the PCR method is the use of a suitable
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
able to withstand the high temperatures of > required for separation of the two DNA strands in the
DNA double helix after each
replication cycle. The DNA polymerases initially employed for
in vitro
''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
experiments presaging PCR were unable to withstand these high temperatures.
So the early procedures for DNA replication were very inefficient and time-consuming, and required large amounts of DNA polymerase and continuous handling throughout the process.
The discovery in 1976 of
''Taq'' polymerase—a DNA polymerase purified from the
thermophilic bacterium, ''
Thermus aquaticus
''Thermus aquaticus'' is a species of bacteria that can tolerate high temperatures, one of several thermophilic bacteria that belong to the ''Deinococcota'' phylum. It is the source of the heat-resistant enzyme ''Taq'' DNA polymerase, one of th ...
'', which naturally lives in hot () environments
such as hot springs—paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from ''T. aquaticus'' is stable at high temperatures remaining active even after DNA denaturation,
thus obviating the need to add new DNA polymerase after each cycle.
This allowed an automated thermocycler-based process for DNA amplification.
Patent disputes
The PCR technique was patented by
Kary Mullis
Kary Banks Mullis (December 28, 1944August 7, 2019) was an American biochemist. In recognition of his role in the invention of the polymerase chain reaction (PCR) technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and wa ...
and assigned to
Cetus Corporation
Cetus Corporation was one of the first biotechnology companies. It was established in Berkeley, California, in 1971, but conducted most of its operations in nearby Emeryville. Before merging with Chiron Corporation in 1991 (now a part of Novarti ...
, where Mullis worked when he invented the technique in 1983. The ''Taq'' polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by
DuPont
DuPont de Nemours, Inc., commonly shortened to DuPont, is an American multinational chemical company first formed in 1802 by French-American chemist and industrialist Éleuthère Irénée du Pont de Nemours. The company played a major role in ...
. The Swiss pharmaceutical company
Hoffmann-La Roche
F. Hoffmann-La Roche AG, commonly known as Roche, is a Swiss multinational healthcare company that operates worldwide under two divisions: Pharmaceuticals and Diagnostics. Its holding company, Roche Holding AG, has shares listed on the SIX ...
purchased the rights to the patents in 1992. The last of the commercial PCR patents expired in 2017.
A related patent battle over the ''Taq'' polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and
Promega
Promega Corporation is a Madison, Wisconsin-based manufacturer of enzymes and other products for biotechnology and molecular biology with a portfolio covering the fields of genomics, protein analysis and expression, cellular analysis, drug disc ...
. The legal arguments have extended beyond the lives of the original PCR and ''Taq'' polymerase patents, which expired on 28 March 2005.
See also
*
COVID-19 testing
COVID-19 testing involves analyzing samples to assess the current or past presence of SARS-CoV-2. The two main types of tests detect either the presence of the virus or antibodies produced in response to infection. Molecular tests for viral ...
*
DNA spiking
*
Loop-mediated isothermal amplification
Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA and a low-cost alternative to detect certain diseases. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with ...
*
Selector technique
*
Thermus thermophilus
''Thermus thermophilus'' is a Gram-negative bacterium used in a range of biotechnological applications, including as a model organism for genetic manipulation, structural genomics, and systems biology. The bacterium is extremely thermophilic, w ...
*
Pfu DNA polymerase
''Pfu'' DNA polymerase is an enzyme found in the hyperthermophilic archaeon ''Pyrococcus furiosus'', where it functions to copy the organism's DNA during cell division. In the laboratory setting, ''Pfu'' is used to amplify DNA in the polymeras ...
References
External links
US Patent for PCRWhat is PCR plateau effect?YouTube tutorial video
History of the Polymerase Chain Reactionfrom the
Smithsonian Institution Archives
Smithsonian Libraries and Archives is an institutional archives and library system comprising 21 branch libraries serving the various Smithsonian Institution museums and research centers. The Libraries and Archives serve Smithsonian Institution ...
{{DEFAULTSORT:Polymerase Chain Reaction
Molecular biology
Laboratory techniques
DNA profiling techniques
Amplifiers
Roche
Biotechnology
Molecular biology techniques
American inventions