Download Polymerase chain reaction and its applications

Current Diagnostic Pathology (2003) 9, 159--164
c 2003 Published by Elsevier Science Ltd.
doi:10.1016/S0968 - 6053(02)00102-3
MINI-SYMPOSIUM: ADVANCES IN LABORATORY PRACTICE
Polymerase chain reaction and its applications
N. Bermingham and K. Luettichw
w
Department of Pathology, Education and Research Building, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland; and
Department of Dermatology, Weill Medical College of Cornell University,1300 York Avenue, New York, NY10021, USA
KEYWORDS
PCR;
RT-PCR;
primer design;
gene expression;
applications
Summary The polymerase chain reaction and its expanding numbers of modif|cations have become a mainstayin diagnostic and research medicine.The technique allows
amplif|cation of nucleic acid sequences both for the purposes of disease and pathogen
detection and also for the preparation of hybridization probes and sequencing templates. PCR mimics the in vivo process of DNA replication with a sensitivity which enables detection of a single target sequence in 106 genomes. The principles of standard
PCR, together with its more widely-used variations, are reviewed and their main applications outlined.
c 2003 Published by Elsevier Science Ltd.
Since its development in the 1960s and 1970s,1 amplif|cation of nucleic acid sequences has revolutionized much
of diagnostic and research molecular science. The polymerase chain reaction (PCR) is now a basic part of a
large proportion of assay protocols. In this article, the
principles of PCR are reviewed and the main PCR variations and applications are outlined.
PCR has become a mainstay of the use of molecular
science in diagnosis and research. Central to molecular
diagnostics and assays are the nucleic acids -- the building
blocks of nature. Nucleic acids [deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA)] form the basis for
storage and processing of the genetic information; abnormalities of their structure and function are responsible for the majority of disease, both inherited and
acquired, and the detection of such alterations is the
aim of a large proportion of laboratory molecular
medicine.
STRUCTURE OF DNA AND RNA
DNA is a stable, double-stranded polymeric molecule
that exists mainly as a right-handed double helix.The basic component is the nucleotide, which comprises one of
four paired deoxyribonucleotides, each consisting of a
sugar molecule, a base and a triphosphate group. The
backbone of DNA is the deoxyribose sugar connected
Correspondence to: NB.Tel.: +3531 809 3701; Fax: +3531 837 0687;
E-mail: [email protected]
by phosphate groups. Phosphodiester bonds between
the 30 -carbon atom of one sugar and the 50 -carbon atom
of the next give the backbone its structure and its 50 to 30
direction. By convention, the sequence of nucleotide
bases is written in this direction. Linked to the 10-carbon
atom of each sugar is one of four possible bases. The
bases of DNA and RNA are heterocyclic aromatic rings
with a variety of substituents. Adenine and guanine are
purines (bicyclic) while cytosine, thymine and uracil are
monocyclic pyrimidines.
Structurally, DNA is described best using the Watson--Crick model of base pairing; two opposing DNA
strands (image/mirror image) wind around a common
axis forming a double helix, thus positioning bases to
the inside with phosphate groups and sugar moeities
turned outwards. Both strands are connected to each
other via hydrogen bonds formed between base pairs
where adenine always interacts with thymine (or uracil)
and cytosine pairs with guanine. The sequence of one
chain determines the other, making the two chains complementary. RNA differs from DNA in that the sugar
molecule is ribose rather than deoxyribose, and the base
uracil is substituted for thymine.
DNA, which contains the genetic information, is transcribed to information-transmitting molecules of RNA
[messenger RNA (mRNA)].These mRNA molecules subsequently serve as a template for gene products where
protein is synthesized (translation). A sequence of three
nucleotide base pairs represents a code for an amino acid
(codon), and this codon sequence determines a sequence
of amino acids (a protein).
160
PCR
PCR is the in vitro enzymatic synthesis and amplif|cation
of specif|c DNA sequences.2 PCR technology began with
the discovery of the f|rst DNA polymerase around 1955.
The enzyme was purif|ed in 1958, but automation and
modern PCR technology was not developed until 1983.
The discovery of thermostable polymerase enzymes revolutionized PCR making automation and rapid reactions possible.
PCR mimics the in vivo process of DNA replication. It
begins with one molecule of the double-stranded DNA
to be amplif|ed, called the target or template DNA. Following separation (denaturation) of the target sequence,
a pair of short synthetic DNA sequences called oligonucleotides or primers is bound to the template (annealing/
hybridization).These serve as a starting point for the addition of nucleotides by DNA polymerases. Here, the
original template determines the sequence of nucleotides added. In vitro, this process can be achieved by cyclical alterations of temperature facilitating the DNAstrand separation, hybridization of primers and polymerization as follows.
First, target DNA is separated into two strands by
heating to 92--981C. The temperature is then reduced
to between 37 and 551C to allow the primers to anneal
(the actual temperature depends on the primer lengths
and sequences). Following annealing, the temperature is
then increased to 60 --721C for optimal polymerization.
In the f|rst polymerization step, the target is copied from
the primer sites for various distances on each target molecule until the beginning of Cycle 2, when the reaction is
heated to 951C again which denatures the newly synthesized molecules. In the second annealing step, the primer
can bind to the newly synthesized strand, and during
polymerization can only copy until it reaches the end of
the f|rst primer. Thus at the end of Cycle 2, some newly
synthesized molecules of the correct length exist. In subsequent cycles, these soon outnumber the target molecules and increase two-fold with each cycle. If PCR was
100% eff|cient, one target molecule would become 2n
after n cycles.3 In practice, 20 -- 40 cycles are commonly
used.
If a pair of oligonucleotide primers can be designed to
be complementary to the target molecule such that they
can be extended by a DNA polymerase towards each
other, then that target region can be greatly amplif|ed.
Due to the extreme amplif|cation achievable, it has been
demonstrated that PCR can sometimes amplify as little
as one molecule of the starting template;4 PCR can also
detect one copy of DNA in 106 genomes. Thus, any
source of DNA that provides one or more target molecules can, in principle, be used as a template for PCR.
This includes DNA prepared from blood, tissue, forensic
specimens, palaeontological samples or in the laboratory
from bacterial colonies or phage plaques. Whatever the
CURRENT DIAGNOSTIC PATHOLOGY
source, PCR can only be applied if some sequence information is known so that primers can be designed.5
Primers
In most applications, it is the sequence and combination
of the primers that determine the overall assay success.
Therefore, primer design should follow certain rules:6
primers should lie within highly conserved regions of
the genome of interest; they should be 14 -- 40 nucleotides long with a balanced G+C content of around 50%;
they must not be complementary to each other (to prevent primer dimer formation) and should lack secondary
structure.
Primers are designed to anneal to opposite strands of
the target sequence so that they will be extended towards each other by addition of nucleotides to their
30 ends. Short target sequences amplify more easily so
often this distance is o500 base pairs, but with optimization, PCR can amplify fragments of 410kbp in length. If
the sequence to be amplif|ed is known, the primer design
is relatively easy owing to the multitude of computational tools for primer design available today. If it is not
known, then primer design is more diff|cult but may still
be possible by using degenerate primers.
DNA polymerases
The f|rst enzyme to be used was the large fragment of
DNA polymerase I from Escherichiacoli (the Klenow fragment) for primer extension at 371C. This enzyme had a
number of drawbacks as it is not stable at high temperatures and therefore required repeated addition after the
denaturation step of each cycle. In addition, target DNAs
of greater than a few hundred base pairs could not be
amplif|ed. A major advance came with the discovery of
the thermostable Taq DNA polymerase, f|rst found in
Thermus aquaticus.7 Optimal PCR extension occurs at
721C withTaq polymerase in the presence of magnesium
ions and dNTPs, and the enzyme remains active at this
temperature even after repeated exposures to 941C during the denaturation step. This polymerase is more specif|c, produces less background in PCR than the Klenow
fragment, and can amplify targets up to 10 kb.
A variety of DNA polymerases have also been characterized from other Thermus species such asTth (Thermus
thermophilus) and Tfl (Thermus flavus).
Cyclers
As mentioned before, microprocessor-controlled machines called thermal cyclers/thermocyclers allowed for
the automation of PCR and are now the most convenient
method for performing PCR in the laboratory. Modern
cyclers feature user-def|ned, temperature-controlled
PCR AND ITS APPLICATIONS
sample blocks or wells providing both uniform temperature adjustment during cycling and high cycling time
reproducibility.
PCR reactions are not usually 100% eff|cient and
the reaction conditions need to be varied to improve
eff|ciency. This is very important when trying to amplify
a particular target from a population of other sequences,
e.g. one gene from genomic DNA. The usual parameters
to vary include the annealing temperature and the
magnesium concentration.
TYPES OF PCR
Standard PCR
This entails the amplif|cation of a specif|c sequence, as
outlined above, and is used routinely to prepare more
DNA targets for subsequent analyses.
Multiplex PCR
161
ing fragments are ligated under conditions that favour
intramolecular circulation of individual fragments. If a linearized vector DNA of known sequence is present at
this stage, the DNA fragments will be incorporated into
the vector and can then be analysed by targeted amplif|cation with primers complementary to fragment-flanking vector sequences.
Differential PCR
This entails the simultaneous amplif|cation of a gene of
interest and a control gene. Both genes are amplif|ed in
the sample reaction tube and, following electrophoresis,
the products are then analysed using a densitometer
where the intensity of the gene of interest is compared
with the intensity of the control gene. Originally, this
technique was used to detect gene copy number
changes, for example c-erbB2 in cancer samples, but has
found a more widespread application for semiquantitative genetic assays.10
In situ PCR
This involves the addition of a number of different primer
pairs to the same reaction mixture for the detection of
multiple abnormalities or infectious agents in the same
assay. Multiplex PCR is particularly helpful to detect mutations, deletions, insertions and re-arrangements in
clinical specimens.
This is a method which combines PCR amplif|cation of
target DNA/RNA with localization of these sequences
in intact tissue or cytological specimens. This technique
entails amplif|cation taking place followed by direct visualization of positive areas/cells using in situ hybridization.
Reverse transcriptase-PCR
TaqMan assays
This begins with the conversion of RNA to complementary DNA (cDNA) by a reverse transcriptase enzyme.
This is an extremely sensitive means of analysing gene
expression.8
TaqMan assays exploit the fact that Taq polymerase possesses a 50 --30 exonuclease activity in addition to its 50 -30 -polymerase activity.TaqMan PCR differs from conventional PCR by the addition of a third oligonucleotide,
called the TaqMan probe, to the PCR reaction. The TaqMan probe is a dually labelled oligonucleotide complementary to the PCR product to be amplif|ed. A
fluorescent dye, called reporter, is covalently linked to
the 50 end of the probe whereas another dye, the
quencher, is linked to its 30 end. As long as the probe is
intact, fluorescence emission is negligible due to Forstertype energy transfer. During PCR cycling, both primers
and probe anneal to their target sequences. While elongating the primers, Taq polymerase will reach the 50 end
of the probe and hydrolyse it thus releasing the reporter
from the influence of the quencher. An increase in fluorescence can be measured, and fluorescence data can then
be analysed qualitatively and quantitatively.
Nested PCR
This is a form of re-amplif|cation of a PCR product. In
other words, two separate amplif|cations are used to improve the outcome of a PCR. The f|rst PCR uses a set of
primers that yields a large product which is then used as
a template for the second amplif|cation. The second set
of primers anneals to sequences within the initial product producing a second smaller product. Nested PCR
increases the specif|city of the reaction since formation
of the f|nal product depends upon the bonding of two separate sets of primers and because two sets of amplif|cation (each of the order of 25 cycles) are used.9
Inverse PCR
This allows the amplif|cation of DNA sequences that lie
outside of known sequence boundaries.Genomic DNA is
f|rst digested into small fragments with a restriction enzyme that cleaves genomic DNA frequently. The result-
APPLICATIONS OF PCR
There are two main types of PCR application:
1. Analytical PCR -- often from unique samples, e.g.
detection of infectious agents in patient samples,
162
genetic analysis, tumour diagnostics, research and
forensics.
2. Preparative PCR, e.g. synthesis of hybridization
probes and sequencing templates (Tables1 and 2).
PCR analysis of gene mutations allows both nature and
location of the mutation to be determined. It is ideally
suited to providing numerous DNA templates for mutation screening. Partial DNA sequences at the genomic or
cDNA level from a gene associated with disease enable
gene-specif|c PCR reactions to be designed. Amplif|cation of the appropriate gene segment then allows rapid
testing for the presence of associated mutations in large
numbers of individuals.11
Restriction fragment length polymorphisms
Restriction fragment length polymorphisms (RFLPs) designate genetic variations in alleles characterized by the
presence or absence of a specif|c restriction site. Such
polymorphisms can be typed using Southern-blot hybridization. A DNA probe representing the locus is hybridized against genomic DNA samples that have been
digested with the relevant restriction enzyme and size
fractionated using agarose gel electrophoresis. The resulting restriction fragments represent the two alleles
corresponding to the absence or presence of the restriction site. PCR can provide an alternative to this method;
primers can be designed to flank the polymorphic restriction site and used for amplif|cation of genomic
DNA. The resulting PCR products can then be digested
with the appropriate restriction enzyme and analysed by
gel electrophoresis. RFLP is a technique useful in the determination of clonality, loss of heterozygosity (LOH)
and, more recently, methylation.
CURRENT DIAGNOSTIC PATHOLOGY
A wide variety of PCR-based methods can be used to
test for known mutations. Small insertions or deletions
can be detected by designing primers complementary
to regions flanking the mutation site, and distinguishing
the normal and mutant alleles by gel electrophoresis. If
the mutation changes a restriction site, then mutant
and normal alleles can be distinguished by amplifying
across the mutant site and digesting the PCR product
with the relevant restriction endonuclease.
Even if the mutation does not result in a restriction
site difference, it may be possible to exploit the difference between wild-type and mutant by amplif|cationcreated restriction site PCR. Oligonucleotide primers
can be designed to discriminate between target DNA sequences that differ by a single nucleotide in the region of
interest. Genomic DNA can be amplif|ed using a primer
that creates a specif|c restriction site within either one of
the alleles that is not present in the other allele. Sequence variation can then be analysed using restriction
digestion.This method can be used to type specif|c alleles
at a polymorphic locus and is called allele-specif|c PCR.
In another form of allele-specif|c PCR, a primer is designed to have a mismatched nucleotide at the extreme
30 end. Since specif|c and eff|cient priming of DNA synthesis is crucially dependent on correct base pairing at the
30 end, mispaired sequences are less readily amplif|ed. As
a result, one of the alleles can be selectively amplif|ed for
further analysis. This method has found particular use in
detecting a specif|c pathogenic mutation and is also
called amplif|cation refractory mutation system (ARMS)
test.12
Short tandem repeats
Short tandem repeats (STRs, microsatellites) comprise a
def|ned region of DNA containing multiple copies of
Table 1 Applications of PCR
1. Identif|cation and analysis of mutations in eukaryotic DNA
-- DNA deletions and insertions can be detected by a change in the size of the PCR product.
-- Failure to produce any PCR fragment indicates thatthe primer is found in an area of deletion.
-- Mutations can be identif|ed by hybridizing PCR-generated DNA fragments to radioactively labelled RNA probes and digesting
the DNA--RNA complexes with RNase A.Digestion will occur if the complex contains any mismatches.
-- Localization of mutations has also been achieved by examining PCR products using denaturing gradient gel electrophoresis
(DGGE).This separates about 50% of DNA strands o1000 bp in length that have single base changes.
2. Detection of amplif|ed oncogenes -- differential PCR
--The oncogene of interest and a single-copy reference gene are co-amplif|ed in the same PCR tube.Gene amplif|cation is
determined by comparing the intensity of the oncogene PCR product with that of the amplif|ed reference single-copy gene.
3. Detection of pathogenic organisms in clinical samples
4. Identif|cation of biological and forensic samples
5. Gene polymorphisms
6. Gene expression
-- Growth factors during wound healing.
-- Gene expression during embryogenesis.
-- Quantitation of gene expression in various organs, e.g. dystrophin.
PCR AND ITS APPLICATIONS
Table 2 Summary of general applications of PCR
Typing genetic markers -- RFLPs, STRPs
DNAtemplates for mutation screening
Detection of point mutations
cDNA cloning
Genomic DNA cloning
Genome walking
DNA sequencing
Gene expression studies
In vitro mutagenesis
163
naturation, annealing and DNA synthesis, as in PCR.The
difference from conventional PCR is that only one primer
is used and ddNTPs are present in the reaction mixture.
The product will accumulate linearly rather than exponentially due to the presence of only one primer. Fluorescent labelling systems with the use of different
fluorophores in the four base-specif|c reactions, and
automated detection technology allows for accurate
automated DNA sequencing.13
Mutagenesis
short sequences of bases which are repeated a number
of times. The number of repeats, typically between one
and f|ve nucleotides long, varies among individuals in the
population. STRs can be typed conveniently by PCR. Primers are designed from sequences known to flank a specif|c STR locus, permitting amplif|cation of alleles whose
sizes differ by integral repeat units. The PCR products
can then be size fractionated by gel electrophoresis.
Cloning
The major advantages of PCR as a cloning method are its
rapidity, sensitivity and robustness. DNA cloning by PCR
can be performed in a matter of hours, compared with
cell-based cloning which may take weeks. Cell-based
cloning of PCR amplif|cation products, however, is often
required to permit subsequent structural and functional
studies.Various plasmid-cloning systems are used to propagate PCR-cloned DNA in bacterial cells; once cloned,
the insert can be cut out and analysed further.
DNA sequencing
DNA sequencing usually involves enzymatic DNA synthesis in the presence of base-specif|c dideoxynucleotide
(ddNTPs) chain terminators. The DNA to be sequenced
is used in a single-standed form, and DNA polymerase
synthesizes new complementary DNA strands.The reactions are carried out using one or more labelled nucleotides and a sequencing primer. In addition, the mixture
contains the base-specif|c ddNTPs, which differ from
normal dNTPs in that they lack a hydroxyl group at the
30 -carbon position as well as at the 20 -carbon position.
When incorporated into a growing DNA chain, the
ddNTP cannot participate further in phosphodiester
bonding at its 30 carbon and therefore causes termination of chain synthesis beyond that point in the chain.
Double-stranded DNA templates can be used in standard sequencing by denaturing the DNA prior to oligonucleotide binding. However, the quality of sequences
produced may be poor. In cycle sequencing (linear amplif|cation sequencing), a thermostable DNA polymerase is
used together with a temperature cycling format of de-
Mutagenesis is a technique of changing the base sequence
of DNA in order to test its effect on gene or DNA function. Mutagenesis can be carried out in vitro or in vivo. In
vitro, a gene may be cloned and single-stranded recombinant DNA recovered. A mutagenic oligonucleotide primer is then designed which has, at the intended
mutation site, a single base difference, coding for the intended mutation.This is then allowed to prime new DNA
synthesis to create a complementary full-length sequence containing the desired mutation. The new DNA
is then used to transform cells and to study the effects of
the mutation.
Genetic testing
In terms of genetic testing, information can be acquired
in two ways: direct testing, where a sample is tested to
see whether a certain genotype is present in that individual; and gene tracking, where linked markers are used
in family studies.
Direct testing is the optimal means of laboratory diagnosis, and its role expands as more and more specif|c genetic abnormalities are identif|ed. The gene and
abnormality of interest must be known, as well as the relevant normal (wild-type) sequence. Direct testing is almost always done by PCR, and can be applied to a wide
range of tissue samples including blood, buccal scrapes,
chorionic villous samples, hair or semen etc. in forensic
samples, archived pathological specimens and Guthrie
cards.
Regardless of the application, of paramount importance in all types of PCR is rigorous laboratory technique
to prevent contamination and false-positive results. As
PCR is so sensitive, contamination with even small
amounts of exogenous material can result in its amplif|cation. A number of precautions can be taken to minimize this risk including separate rooms for preparation
and post-PCR analysis, the wearing of protective clothing and gloves, and ultraviolet-mediated DNA crosslinking. From nucleic acid extraction to performance of
the amplif|cation steps, care must be taken to preserve
sample integrity, and the inclusion of positive and negative controls in each assay is crucial to result validation.
164
CURRENT DIAGNOSTIC PATHOLOGY
DNA-based diagnosis, therefore, relies heavily on
PCR, both for geneticists, research scientists and microbiologists/virologists. Detection of infectious agents can
be performed using PCR techniques for an increasingly
large number of pathogens, both for diagnostic and prognostic purposes (e.g. human papillomavirus testing in
cervical intra-epithelial neoplasia). PCR-based techniques are also central to the screening and diagnosis of
genetic disorders such as cystic f|brosis, and for the diagnosis of cancer and determination of recurrences and
prognostic factors. PCR generation of sequences also
forms the basis for microarray technology (gene chips),
a dynamic and rapidly expanding area in research. The
role of PCR remains central in the setting of increasing
importance of molecular techniques in diagnostic
pathology.
PRACTICE POINTS
*
*
*
*
*
PCR forms the basis of much of diagnostic and research medicine
Amplif|cation of both DNA and RNA (RT-PCR) sequences can be performed allowing examination of
gene expression
A large range of sample types may be examined,including forensic or paleontological specimens
Various modif|cations of PCR have allowed for vast
expansion of the diagnostic and research uses
The development of fluorogenic probes and realtime quantitative PCR allows greater automation
of the technique without the requirement of subsequent detection steps
REFERENCES
1. Templeton N S. The polymerase chain reaction--history, methods
and applications. Diagn Mol Pathol 1992; 1: 58--72.
2. Erlich H A. PCR Technology: Principles and Applications for DNA
Amplification. New York: Stockton, 1989.
3. Mullis K B. The unusual origin of the polymerase chain reaction. Sci
Am 1990; 4: 56--65.
4. Li H, Gyllenstein U B, Cui X, Saiki R K, Ehrlich H, Arnheim N.
Amplification and analysis of DNA sequences in single human
sperm and diploid cells. Nature 1988; 335: 414--417.
5. Turner P C, McLennan A G, Bates A D, White M R H. Instant
Notes in Molecular Biology. BIOS Scientific Publications, 1997.
6. Taylor G R. Polymerase chain reaction: basic principles and
automation. In: McPherson M J, Quirke P, Taylor G R (eds). PCR:
a Practical Approach. Oxford: IRL Press, 1992.
7. Chien A, Edgar D B, Trela J M. Deoxyribonucleic acid polymerase
from the extreme thermophile Thermusaquaticus. J Bacteriol 1976;
127: 1550--1557.
8. Baumforth K R N, Nelson P N, Digby J E, O’Neill J P, Murray P G.
The polymerase chain reaction. J Clin Pathol Mol Pathol 1999; 52:
1--10.
9. Kawasaki E S. Amplification of RNA. In: Innis M A, Gelfand D H,
Stunski J J (eds). PCR Protocols, a Guide to Methods and
Applications. New York: Academic Press, 1990; 21--27.
10. Reimer T, Luettich K, Gerber B. Epidermal growth factor receptor
gene and c-erbB-2 gene amplification in ovarian cyst fluid. Obstet
Gynaecol 1996; 88: 967--972.
11. Stachan T, Read A P. Human Molecular Genetics. Bios Scientific
Publishers Ltd, 1999.
12. Newton C R, Graham A, Heptinstall L E et al. Analysis of any point
mutation in DNA. The amplification refractory mutation system
(ARMS). Nucleic Acids Res 1989; 17: 2503--2516.
13. Wilson R K, Chen C, Ardalovic N, Burns J, Hood L. Development
of an automated proceedure for fluorescent DNA sequencing.
Genomics 1990; 6: 626--634.