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POLYMERASE CHAIN REACTION 243

POLYMERASE CHAIN REACTION


J M Wages Jr., Palmetto Consulting & Research, Tupelo, molecule has been amplified to two molecules. These
MS, USA in turn are denatured in the next cycle and amplified
& 2005, Elsevier Ltd. All Rights Reserved. to produce four molecules. The four molecules are
amplified to eight in the third cycle, and so on. Each
successive cycle effectively doubles the amount of
Introduction DNA product. The three-stage cycle of denaturation,
Polymerase chain reaction (PCR) is a technology for annealing, and primer extension is repeated 25–40
exponential amplification of a fragment of DNA. times in a typical PCR procedure. The product
(The PCR is covered by patents owned by Hoffman- of such a reaction is a large quantity (10  9 to
La Roche. A license is required to use the PCR proc- 10  8 mol l  1 in a 10–100 ml volume) of a double-
ess.) The limit of its sensitivity is a single molecule, stranded DNA molecule whose length is determined
making PCR a superb qualitative tool for the specific by the distance between the primer sites on the
detection of rare DNA sequences. Under proper con- original template molecule. Typically, PCR products
ditions, the yield of amplified DNA is proportional to or amplicons are 100–3000 bp in length, although
the initial number of target molecules, rendering it a much longer (up to B50 000 bp) amplifications are
quantitative analytical tool as well. Since its original possible under specific conditions. PCR products are
description in 1985, PCR has evolved into an assem- visualized by electrophoresis followed by staining
blage of varied methodologies almost universally with fluorescent dyes or by hybridization to labeled
used in basic biological research, biotechnology, clini- oligonucleotide probes.
cal research, clinical diagnostics, forensics, food
technology, environmental testing, archaeology and Technical Aspects of PCR
anthropology, and other fields. Even though other
One of the advantages of PCR technology is the speed
nucleic acid amplification technologies have been
and simplicity with which the technique can be per-
described, PCR remains by far the most widely used.
formed. A PCR reaction can be set up in a short time,
and multiple samples can be handled easily. Compo-
Biochemical Basis of PCR nents of the amplification reaction (template DNA,
PCR involves the enzymatic synthesis of millions of DNA polymerase, oligonucleotide primers, buffers)
copies of a specific DNA segment. The exponential are added to the amplification vessel. The most com-
amplification of a very small amount of template mon containers for PCR are microcentrifuge tubes
DNA is achieved using a heat-stable DNA poly- (0.5–0.2 ml) with thin walls to facilitate rapid heat
merase and an automated heat block that is capable transfer or multiwell plates (96- or 384-well plates
of rapid changes of temperature. The template DNA being the most common). Some commercially avail-
molecule is initially denatured to two single strands able systems utilize specially designed vessels such as
by heating to high temperature (typically 90–951C) capillary tubes. The reaction tubes are then placed
in the first stage of the PCR cycle (Figure 1A). Two into an automated programmable thermal cycler for
small oligonucleotides that are complementary to se- controlled heating and cooling for denaturation, an-
quences on opposite strands of the template molecule nealing, and extension reactions. Following amplifi-
and that flank the DNA segment to be amplified are cation, some type of DNA sequence analysis is
used as primers for the DNA polymerase. The second typically performed, although technology for real-
stage of the PCR cycle consists of cooling the reac- time monitoring of amplification obviates the need
tion, which permits the annealing of the single- even to open the PCR tube after cycling is complete.
stranded oligonucleotide primers to the denatured
Template DNA
template molecule (Figure 1B). The third stage of the
reaction is the extension of the new strand from the One of the advantages of PCR is that DNA from
annealed primer in a 50 to 30 direction by the heat- relatively clean samples need not be highly purified to
stable polymerase (Figure 1C). This is performed be used as a template for amplification. It is possible
at the optimum temperature for the polymerase to simply heat samples to near boiling to release
(68–721C). The most commonly used polymerase is DNA, then to add the resulting lysate to the PCR
the enzyme isolated from Thermus aquaticus (Taq reaction tube. Such simplicity is the hallmark of PCR.
DNA polymerase). After the first cycle, each template With some types of biological samples, inhibitors of
244 POLYMERASE CHAIN REACTION

GATCTTAAAACAATACAGCCTGCCGCTATCGACAGGGAAAGGAATTAAAGAAAA
CTAGAATTTTGTTATGTCGGACGGCGATAGCTGTCCCTTTCCTTAATTTCTTTT

GATCTTAAAACAATACAGCCTGCCGCTATCGACAGGGAAAGGAATTAAAGAAAA
+
CTAGAATTTTGTTATGTCGGACGGCGATAGCTGTCCCTTTCCTTAATTTCTTTT
(A)

GATCTTAAAACAATACAGCCTGCCGCTATCGACAGGGAAAGGAATTAAAGAAAA
+
CTAGAATTTTGTTATGTCGGACGGCGATAGCTGTCCCTTTCCTTAATTTCTTTT

GATCTTAAAACAATACAGCCTGCCGCTATCGACAGGGAAAGGAATTAAAGAAAA
CTTTCCTTAATTTCTTTT
+
GATCTTAAAACAATACAG
CTAGAATTTTGTTATGTCGGACGGCGATAGCTGTCCCTTTCCTTAATTTCTTTT
(B)

GATCTTAAAACAATACAGCCTGCCGCTATCGACAGGGAAAGGAATTAAAGAAAA
CTTTCCTTAATTTCTTTT
+
GATCTTAAAACAATACAG
CTAGAATTTTGTTATGTCGGACGGCGATAGCTGTCCCTTTCCTTAATTTCTTTT

GATCTTAAAACAATACAGCCTGCCGCTATCGACAGGGAAAGGAATTAAAGAAAA
CTAGAATTTTGTTATGTCGGACGGCGATAGCTGTCCCTTTCCTTAATTTCTTTT
+
GATCTTAAAACAATACAGCCTGCCGCTATCGACAGGGAAAGGAATTAAAGAAAA
CTAGAATTTTGTTATGTCGGACGGCGATAGCTGTCCCTTTCCTTAATTTCTTTT
(C)
Figure 1 One cycle of a PCR. (A) The template is shown as a double-stranded DNA molecule, which is denatured to two single
strands. (B) The short oligonucleotide primers anneal to the complementary regions of the single strands. (C) The polymerase
generates a new strand from each single-stranded molecule, creating two double-stranded molecules.

PCR such as heme or polyanionic mucopolysaccha- Often the concentration of template DNA is un-
rides must be removed by DNA purification. known and does not need to be determined. If nece-
Virtually any form of DNA can be used as a tem- ssary, a series of control reactions, each containing a
plate in a PCR reaction. Plasmids, cosmids, phage- different amount of template, can be performed.
mids, lambda phage, M13 phage, genomic DNA,
and many other sources of DNA have all been used
Biochemical Components
successfully. A PCR can be performed directly on
colonies or plaques. Although it is possible to am- DNA polymerase A wide range of DNA poly-
plify a single DNA molecule, a typical PCR reaction merases is available for PCR. A cloned heat-stable
uses 105–106 copies of the target DNA as template. DNA polymerase from Thermus aquaticus (Taq DNA
In practice, this means adding B1 mg of eukaryotic polymerase) is the most commonly used. Other en-
genomic DNA, 10 ng of yeast DNA, or 1 ng of bac- zymes such as DNA polymerase from Pyrococcus
terial DNA. The concentration of DNA is not critical furiosus (Pfu DNA polymerase) or Thermoccocus
for most applications, however, and a PCR reaction litoralis (VentTM DNA polymerase, New England Bio-
will work with a wide range of template concentrations. labs) have been reported to have a lower error rate
POLYMERASE CHAIN REACTION 245

than Taq polymerase and may be advantageous for stringent temperature. A simple technique for Hot
some applications. Typically, 0.5–2.5 units of enzyme Start is manual addition of a small volume (typically
are used per 50 ml reaction. 5–10 ml) of reaction buffer containing the enzyme to
the other components maintained in the thermal cy-
Oligonucleotide primers Oligonucleotide primers cler at B801C, then continuing with standard PCR
are usually used at a concentration of 1 mmol l  1 in amplification. Another method involves sealing a
PCR reactions (50 pmol per 50 ml reaction). Concen- portion of the amplification reaction under wax, then
trations between 0.1 and 1 mmol l  1 can be used. adding the remaining reactants above the wax bar-
Higher concentrations may increase nonspecific an- rier. Enzymatic methods to accomplish the same goal
nealing of primers and thus to nonspecific amplifica- include the use of a modified DNA polymerase that is
tion products. PCR primers are normally between 18 blocked with a thermolabile group or the use of
and 30 nucleotides in length and should preferably uracil DNA glycosylase (uracil-N-glycosylase, UNG),
have a guanine þ cytosine (G þ C) content of B50%. deoxyuridine triphosphate (dUTP), and a brief initial
The temperature at which half the DNA molecules incubation at 501C. Typically, dUTP is substituted on
will be double-stranded, Tm, in degree celsius, for a an equimolar basis for deoxythymidine triphosphate
primer is estimated by the rule-of-thumb equation: (dTTP) (200 mmol l  1), but higher concentrations of
2  (number of As and Ts) þ 4  number of Gs and dUTP (125–300 mmol l  1) may be beneficial in some
Cs) (A ¼ adenine, T ¼ thymine). However, more assays. Uracil DNA glycosylase is used at 1–2 Units
accurate calculation of oligonucleotide Tm requires per reaction. This method ensures that any nonspe-
consideration of the effects of ionic strength and cific product or primer oligomer that is generated
neighboring bases in the DNA strand (nearest neigh- during the initial low-stringency conditions will con-
bor Tm calculation methods). Software is available tain uracil (U) and therefore be susceptible to
for performing these calculations. The Tm values for cleavage by UNG. Uracil is excised during the 501C
the two primers in a reaction should be similar, and incubation, and strand breakage occurs at these
the annealing temperature used is normally B51C abasic sites during the initial denaturation step. In
below the Tm. As annealing temperature approaches addition to inactivating any contaminating ampli-
Tm, more specific amplifications are achieved, but cons, this method reduces nonspecific products and
overall yields may decrease. Despite careful calcula- primer oligomer. Because residual UNG can degrade
tions, empirical testing of annealing temperatures is the U-containing amplicons, PCR products must be
essential for a well-optimized PCR assay. Comple- analyzed immediately or stored at 41C for short pe-
mentarity at the 30 ends of primer pairs should be riods of time or frozen for longer times. Alternately,
avoided as this promotes the formation of primer UNG can be removed by extraction with organic
oligomers (primer dimers). Such artifactual products solvents or digested with proteases.
are themselves templates for PCR amplification and The 30 end of a PCR primer should be perfectly
compete with the desired products for deoxynucleo- complementary to the template DNA. Failure of the
tide triphosphates (dNTPs), polymerase, and prim- 30 end to hybridize results in inefficient amplification.
ers. This leads to a decreased yield of the desired Internal sequences are less critical. Provided a suit-
product and the presence of nonspecific products able annealing temperature is used, degenerate primers
that may complicate analysis. (primers that consist of a pool of different but closely
A primer oligomer forms when two or more prim- related oligonucleotides) may be used to ensure that
ers anneal to each other and are extended during primers will anneal to highly variable sequences or
PCR. The double-stranded product acts as a template sequences that are only partially known. These
during subsequent rounds of amplification and com- primers are designed from amino acid sequences or
petes with the desired product. In general, low an- from a comparison of similar genes from other
nealing temperatures, high enzyme concentrations, organisms. Sequences at the 50 end of the primer are
and high primer concentrations increase the proba- least critical. Providing the hybridizing portion of
bility of primer oligomerization. However, it is the primer is long enough to ensure annealing to
stringency during the initial cycle of PCR that is the template DNA, nonhomologous 50 extensions
most critical for primer oligomer formation, as well (regions that do not correspond to the sequence of the
as for nonspecific PCR products in general. Various template) can be used to introduce restriction enzyme
techniques for maintaining high stringency up until sites into PCR products to be cloned or to add other
the point when primers and enzyme are mixed sequences such as phage RNA polymerase promoters.
together have been described (generally called Hot Multiplex PCR enables the detection of multiple
Start). Hot Start methods involve mixing of enzyme gene sequences within the same reaction. Because
and primers only after reactions have reached a several sets of PCR primers must function in the
246 POLYMERASE CHAIN REACTION

same reaction without interference, primer design Proteins (gelatin or bovine serum albumin) are some-
and optimization of reaction conditions are critical. times included at similar concentrations as nonionic
Primers are often labeled with detectable groups to detergents for the same reason. Other components
facilitate post-PCR analysis. Radioactive isotopes, that have been reported to increase PCR product
haptens such as biotin, or fluorescent dyes are the yields or specificity on specific templates include
most widely used. Labels attached to the 50 end of a betaine, dimethylsulfoxide, formamide, and glycerol.
primer have little or no effect on amplification.
Thermal Cycling Equipment
Many heat-stable polymerases used for PCR exhibit
a template-independent activity that adds deoxynuc- The PCR process involves thermal cycling between
leosides (predominantly deoxyadenosine) to the 30 denaturation, annealing, and extension temperatures
ends of all double-stranded molecules in the reaction. (Figure 1). Each incubation step is a segment, and a
These A-overhangs must be filled in prior to blunt-end complete round of denaturation, annealing, and ex-
cloning of PCR products. Alternatively, special vectors tension is a cycle. First, samples are heated to a tem-
are available that have single 30 T-overhangs at the perature adequate to melt the double-stranded DNA
cloning site ready for insertion of the PCR product. template and any secondary structure in the primers.
Choosing a polymerase without this activity or ad- Denaturation is most commonly performed at 941C,
justing reaction conditions to either minimize or maxi- although temperatures of 97–991C may be required
mize the extent of A-overhangs may produce sharper for templates with high GC content. After the first
peaks in high-resolution electrophoretic analysis. several cycles, denaturation temperature can be low-
ered to 90–921C to reduce loss of polymerase activity
Deoxynucleoside triphosphates dNTPs are typically through heat denaturation during the course of PCR.
used at a concentration of B200 mmol l  1 each dNTP Next, samples are cooled to an annealing temperature
in a PCR reaction. Excessively high concentrations at which the primers will hybridize to their target
promote nonspecific product formation. Modified sequences. The choice of annealing temperature de-
dNTPs are sometimes used to label PCR products pends on primer Tm, but is usually in the range of
with radioactive or fluorescent markers or with 50–601C. Finally, the samples are heated to the ex-
haptens such as biotin, fluorescein, or digoxygenin. tension temperature (68–721C). If primers are
The modified dNTP is typically used at a designed to permit efficient annealing at 60–681C,
concentration (0.1–1 mmol l  1) much lower than the annealing and extension steps can be combined
the unmodified dNTP (B200 mmol l  1). Probes can into one step. Typical PCR amplification is per-
be easily generated using PCR amplification with a formed over 20–30 cycles, although 40 or more
labeled nucleotide, followed by removal of the cycles may be used with template DNA at very low
unincorporated label. initial concentration or with otherwise marginal
amplifications involving, for example, degenerate
Buffer components Several reagents containing buff- primers or poor-quality template DNA.
er ions, monovalent salts, and divalent cations re- Although it is possible to perform 20 or even 40
quired for polymerase activity, have been described cycles of PCR manually using sandbaths, waterbaths,
for use in PCR amplification. The most widely used or heat blocks and physically moving the samples
buffer consists of 10 mmol l  1 Tris–HCl (pH 8.3 at from one to the other at timed intervals, the tedium
room temperature) and 50 mmol l  1 KCl. At the involved makes this approach impractical. Many dif-
extension temperature (721C), the pH of this Tris ferent types of machines are commercially available to
buffer falls to 7.2, near the optimum for Taq DNA perform thermal cycling. Some cyclers use robotic
polymerase. Sulfate-containing buffers such as arms to transfer a rack of PCR samples from one heat
20 mmol l  1 Tris–SO4 (pH 8.5–9.0 at room tempera- block to another. Other machines use resistive heating
ture) and 20 mmol l  1 (NH4)2SO4 are also widely elements, compressors, water circulators, fan-forced
used. Monovalent cations (K þ or NH4þ ) are included air, high-intensity lamps, Peltier elements, or combi-
to adjust ionic strength. DNA polymerases require nations of these heating and cooling devices. Micro-
divalent cations for activity, and PCR reactions processors enable the user to program the instrument
contain MgCl2 (1–5 mmol l  1). In general, higher for time and temperature of each segment, and the
MgCl2 concentrations promote nonspecific primer number of cycles to be performed.
annealing and nonspecific product amplification. Thermal cyclers that can monitor progress of the
Reaction buffers usually include a low percentage amplification reaction while it is taking place are
(0.1–0.5%) of nonionic detergent such as Igepal CA- called real-time thermal cyclers. Real-time instru-
630, Tween 20, or Triton X-100 to minimize adsorp- ments are designed to monitor fluorescence from
tion of polymerase to the surfaces of the PCR tube. labels whose emission intensity is proportional to the
POLYMERASE CHAIN REACTION 247

amount of amplified DNA. Flexibility varies greatly of the PCR product reflects the sequence of the
among the available machines, but all instruments original DNA template. A range of DNA polymerases
can measure fluorescence at least once during the is commercially available, and certain enzymes have
annealing segment of every cycle. Intercalating dyes demonstrably higher fidelity. In addition to intrinsic
such as ethidium bromide or SYBR Green that are differences among the various heat-stable polymerases,
selective for double-stranded DNA provide the sim- fidelity is influenced by a number of factors. It is
plest method. Probes that hybridize adjacent to one therefore possible to increase or decrease the number
another on one strand of the amplicon can be of mismatches between the product and initial tem-
designed to have a fluorescence energy transfer do- plate. Conditions of low fidelity may be chosen to
nor and acceptor that will be close enough for energy introduce random point mutations into a PCR prod-
transfer if and only if hybridization occurs. Emission uct. More general applications benefit from accurate
from the hybridized donor- and acceptor-labeled amplification. Fidelity is improved by keeping poly-
probes is monitored during each cycle. A third ap- merase, dNTP, and MgCl2 concentrations as low as
proach is to use the 50 nuclease activity of Taq DNA practical, using the same concentrations of each of
polymerase to cleave a probe labeled with a fluoro- the four dNTPs, maintaining an annealing tempera-
phore and a quencher. When Taq DNA polymerase ture near the Tm of the primers, and programming a
(and some, but not all, other heat-stable polymerases) short extension segment and as few cycles as practical.
encounters the probe, it cuts it, dissociating the fluoro-
Cross-Contamination
phore and quencher. As with energy transfer methods,
amplification is detectable as an increase in fluores- Because PCR primers are incorporated into each
cence. In addition to monitoring fluorescence, real-time molecule of PCR product, amplicons are themselves
thermal cyclers provide data analysis for quantitation suitable templates for amplification. Re-amplifica-
of initial template concentration. Real-time PCR has tion of previously amplified DNA is a major problem
become the standard for quantitative PCR. for PCR. To reduce the likelihood of contamination
Post-PCR Analysis
of reagents, separate laboratory work areas should
be designated for reagent preparation, sample prepa-
Unless real-time thermal cyclers are used, some form ration, reaction assembly and thermal cycling, and
of manipulation or analysis of PCR products must be post-PCR analysis. Depending on the analytical rigor
performed. In preparative applications, amplicons are required and the resources available, well-isolated
inserted into a suitable vector and propagated by rooms for each step of the process will be beneficial.
standard molecular biological methods. In analytical In the research laboratory, however, it is more com-
applications, some form of DNA size or sequence mon to rely on separate work areas and equipment
analysis is performed. In many cases, high-perform- for pre- and postamplification work and gowning of
ance liquid chromatography or electrophoresis on laboratory personnel in cleanroom garments. Labo-
agarose or polyacrylamide gels to confirm that the ratory equipment, especially pipettors and shared
amplicon is the expected size is sufficient. In clinical equipment, and laboratory personnel are the most
testing, hybridization of an oligonucleotide probe common sources of PCR product contamination.
complementary to a sequence between the PCR prim- Clinical laboratories or other analytical laboratories
ers is more commonly used to confirm specificity of the now employ physical separation of work areas and
amplified DNA. Probe hybridization can be combined equipment in addition to a biochemical method using
with DNA size analysis by electrophoresis or can be the enzyme uracil DNA glycosylase (UNG) to
performed using methods analogous to immunoassays. prevent amplicon contamination. If PCR reactions
are performed with dUTP rather than dTTP, ampli-
Considerations cons will be susceptible to digestion by UNG. This
method has been called PCR sterilization. The need
Fidelity
to avoid contamination with amplified DNA consti-
For preparative applications and the analysis of tutes an incentive to use real-time PCR for qualitative
single-base changes, fidelity of the DNA polymerase as well as quantitative assays. With real-time PCR,
is important. The fidelity of the DNA polymerase in a tubes containing amplified DNA need never be
PCR reaction determines the similarity between the se- opened. No matter what precautions are taken,
quence of the PCR product and the original template. negative controls containing no DNA template must
Taq DNA polymerase sometimes incorporates an in- always be included with each batch of PCR reac-
correct base in the growing DNA strand, and this error tions. If low-copy detection is the assay goal, then
is propagated during subsequent rounds of cycling. multiple negative controls are required to adequately
The higher the fidelity, the more closely the sequence test for amplicon contamination.
248 POLYMERASE CHAIN REACTION

Reverse Transcription PCR Amplification of viral genes forms the basis of


PCR-based detection systems for viral pathogens.
PCR can be used to detect and quantify RNA if RNA
The human immunodeficiency virus (HIV), which is
is first reverse-transcribed to complementary DNA
responsible for AIDS, is currently detected primarily
(cDNA). Reverse transcriptases from Maloney murine
by immunological means. However, as with most
leukemia virus (MMLV-RT) or avian myeloblastoma
viral infections, it takes some time following infec-
virus (AMV-RT) are commonly used. Either the buffer
optimal for the reverse transcription (RT) or the PCR tion for antibodies to develop, and there is therefore
a period when a patient infected with the virus would
buffer is suitable in most cases, and reverse transcrip-
appear negative by conventional tests. A PCR assay
tion is typically performed at 37–421C for 15–60 min.
can be used to detect the presence of the virus in
Because these enzymes are heat-labile, they are in-
this case. Proviral DNA that has integrated into the
activated during the first denaturation segment of
genomes of leucocytes can be detected, or the viral
PCR. Heat-stable reverse transcriptases enable reverse
RNA genome can be detected in plasma by RT-PCR.
transcription at higher temperatures to denature RNA
Identification of HIV by PCR methods has played an
secondary structure. In the presence of Mn2 þ , some
DNA-dependent DNA polymerases such as the en- important role in AIDS diagnostics and research, as
well as improved safety testing in blood banks. PCR
zyme from Thermus thermophilus (Tth DNA poly-
assays have also been used in epidemiological studies
merase) have activity on RNA templates. Using MnCl2
and in the identification of other retroviruses such as
for reverse transcription can enable cDNA synthesis
human T-lymphotropic virus types 1 and 2 (HTLV-1
with the same enzyme used for PCR. Unless a balance
and HTLV-2). PCR procedures have also been
between MnCl2 and MgCl2 is carefully identified,
developed to detect human papillomaviruses. This
Mn2 þ must be removed or chelated, and Mg2 þ must
diverse group of viruses can cause a number of dis-
be added prior to PCR. RT-PCR can be performed
within intact cells (in situ PCR) to identify cells ex- eases. A PCR assay can distinguish the relatively
harmless strains from those that cause serious dis-
pressing particular genes or to assess the presence of
eases such as cervical cancer. Current laboratory
disease-related genes. Thermal cyclers are available to
methods for the diagnosis of human enteroviruses,
automate the process on microscope slides.
which cause infection in children, are slow and lack
sensitivity. Detection is hindered by the low level of
Applications viruses present in clinical specimens. PCR tech-
nology, however, provides a rapid and accurate
Clinical Diagnostics
diagnostic test. PCR assays have also been used
The main clinical application of PCR technology is in to detect coronaviruses, cytomegalovirus, human
the diagnosis of disease, and in many cases the speed herpesvirus type 6, adenovirus, Epstein–Barr virus,
and simplicity of PCRs have revolutionized clinical rotaviruses, human parvovirus, and herpes simplex
diagnosis. Bacteria that are difficult or impossible to viruses, among many others. In addition to detection,
culture on artificial media can now be detected by PCR assays can be designed to differentiate between
PCR. The causative organism of syphilis, Treponema different strains or serotypes of a particular virus.
pallidum, can be detected by a PCR assay in which a Parasitic infections cause important human diseas-
gene encoding a specific membrane protein is ampli- es such as leishmaniasis and malaria. About 40% of
fied. PCR is being employed to investigate the the world’s population is at risk from malaria, and
pathogenicity of Mycoplasma genitalium. As few as drug-resistant strains have emerged and are spread-
four organisms can be detected by this method. PCR- ing rapidly. As well as being helpful in diagnosis of
based detection assays have also been developed for disease, a PCR assay can distinguish between drug-
Mycobacterium tuberculosis, an organism that takes resistant and drug-sensitive strains, thus enabling
up to several weeks to identify by conventional effective treatment to be prescribed.
means owing to its slow growth on artificial media. Many PCR diagnostic kits are now commercially
M. tuberculosis, the bacterium that causes tubercu- available. Hoffman-La Roche has introduced kits for
losis, infects about one-third of the world’s popula- Chlamydia, M. tuberculosis, HIV, hepatitis C virus,
tion and is most prevalent in developing countries. and others. It is likely that an even wider range of
Recently, this organism has re-emerged as a signifi- PCR diagnostic kits will become available, and PCR
cant pathogen in the developed world, especially testing will continue to have a huge impact in the
among the poor and homeless, and antibiotic-resist- clinical diagnostic laboratory. Despite the simplicity
ant strains are developing. PCR tests have become and rapidity of PCR from the perspective of the re-
essential for rapid diagnosis and epidemiology of search laboratory, it is viewed as a complex test in
diseases such as tuberculosis. the clinical laboratory. The expenses of highly
POLYMERASE CHAIN REACTION 249

trained personnel and commercial PCR kits coupled be of enormous benefit. The availability of PCR
with concerns about false positive test results due to technology creates an incentive for the development
contamination with previously amplified DNA have of noninvasive prenatal sampling techniques. Be-
so far confined this promising technology to clinical cause amniocentesis and CVS carry a certain amount
research and specialty testing labs. Eventually, im- of risk, less invasive sampling procedures are desir-
proved technology and assay automation will enable able. A small number of cells of fetal origin circulate
PCR testing to be as common in diagnostic labora- in the maternal blood. Methods to purify these cells
tories as immunoassays. have been described. Mutations and polymorphisms
on the Y chromosome already can be tested using
maternal blood samples without the need to purify
Molecular Genetics
fetal cells. It seems likely that in the future PCR tests
In addition to being an invaluable tool for the de- will be used for prenatal diagnosis of a whole range
tection and diagnosis of infectious diseases, PCR of genetic diseases from a sample of maternal blood.
tests have also proved useful in the diagnosis and PCR testing is currently available for preimplanta-
analysis of genetic diseases. Many genetic disorders tion genetic diagnosis for in vitro fertilization. One cell
are caused by specific mutations, and the fragment of is removed from an eight-cell zygote for PCR testing.
DNA involved can be amplified by PCR. DNA se-
quencing, high-resolution size analysis on sequencing
Basic Research
gels, single-strand conformation polymorphism anal-
ysis, Southern blotting and probe hybridization, PCR has become an indispensable tool in the molec-
allele-specific oligonucleotide probes, and other tech- ular biology laboratory. Many traditional methods
niques are used to test for the presence of mutations. and protocols have now been replaced by PCR-based
This approach has been used to detect mutations techniques. One of the most common techniques
causing diseases such as sickle cell anemia, beta- employed by molecular biologists involves the
thalassemia, cystic fibrosis, and many others. Genetic cloning of DNA fragments into plasmid vectors.
diseases caused by trinucleotide repeat expansion The plasmids are maintained in bacterial strains.
can be tested using PCR followed by high-resolution To determine which bacterial colony contains the
electrophoretic analysis on sequencing gels. A com- plasmid of interest, transformant colonies can be
mercial test for fragile X syndrome employs novel analyzed by PCR using primers designed from the
PCR reagents and thermal cycling conditions to vector (plasmid) DNA, bracketing the insert. In this
enable amplification of (CGG) repeats up to 800 way, colonies can be screened to find one containing
repeat units in length in the same reaction containing a plasmid with an insert of a particular size, obvia-
a normal allele (usually 29–31 repeat units). In gen- ting the need to screen transformants by plasmid
eral, PCR should be considered a versatile template DNA preparation followed by restriction enzyme
onto which can be superimposed known DNA digestion. PCR can also be used to screen libraries for
sequences for any disorder, whether genetic or infec- clones containing a particular DNA fragment or
tious in nature, to rapidly configure a high-per- gene. This strategy dispenses with the filter hybrid-
formance test. ization step and can be both quicker and more
PCR is routinely used in the analysis of human sensitive than traditional methods. PCR is also
leucocyte antigen (HLA) polymorphisms (HLA typ- invaluable in mutagenesis, radioactive, and nonra-
ing) to assess donor compatibility prior to bone mar- dioactive labeling of DNA fragments, and many
row or organ transplantation. The use of PCR in other applications. In addition, the technology
transfusion medicine is also being investigated, and developed for PCR amplification has been applied
the basis of a system for ABO blood typing using to the extremely important techniques of DNA
PCR has been described. sequencing and gene expression analysis.
PCR is being used in phylogenetic and evolutio-
nary studies. PCR amplification and screening of
Prenatal Diagnostics
ribosomal RNA (rRNA) genes and mitochondrial
Prenatal diagnosis of genetic disease is normally car- DNA have been used to estimate relationships be-
ried out on a sample of amniotic fluid obtained by tween species and subspecies. Besides studies on the
amniocentesis or by chorionic villus sampling (CVS). relationships of contemporary species, PCR allows
Conventional diagnostic tests for diseases such as studies on extinct species because it can be performed
cystic fibrosis can take as long as 2 weeks. Results of on material in museum collections. Amplification
a PCR test can be available in 1 day. If the option to and sequencing of mitochondrial DNA are also used
terminate a pregnancy is taken, this time savings can in evolutionary studies.
250 POLYMERS / Natural Rubber

Forensics See also: Amplification Reactions. DNA Sequencing.


Electrophoresis: Nucleic Acids. Forensic Sciences:
PCR has become an essential tool in forensic science. DNA Profiling. Nucleic Acids: Chromatographic and
Many methods that exploit differences in DNA to Electrophoretic Methods; Immunoassays.
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San Diego: Academic Press.
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POLYMERS

Contents
Natural Rubber
Synthetic
Polyurethanes

used in tires and related products. Natural rubber,


Natural Rubber which contains polyisoprene as the main constituent,
is present in over 1000 plant species. However, the
A Krishen and M A Schafer, The Goodyear Tire & vast majority of the world’s natural rubber produc-
Rubber Company, Akron, OH, USA tion is derived from the tree Hevea brasiliensis. Writ-
ten references to natural rubber date back to the late
& 2005, Elsevier Ltd. All Rights Reserved.
fifteenth century following the voyages of Columbus,
although certain articles made of the rubber were
used earlier in South America.
Introduction Serious investigations of rubber were first made in
During 2001, the world’s natural rubber consump- the mid-1700s by Charles de la Condamine and
tion was 7.07 million metric tons and 75% of it was Francois Fresneau, who refer to it as caoutchouc,

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