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PCR-Based Methods for Mutation Detection

Chapter · January 2005

DOI: 10.1385/1-59259-928-1:065

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 PCR-based Methods for Mutation Detection (including Real-Time PCR)

Running head: PCR-based mutation detection methods

Ian M. Frayling, MA, MB, BChir, PhD, MRCPath

Consultant in Clinical Genetics and Director of Clinical Genetics Laboratory

Institute of Medical Genetics,

University Hospital of Wales,

Heath Park,

Cardiff, CF14 4XW, UK

Phone: (44)29 2074 4203 Fax: (44)29 2074 7603

E-mail: [email protected]

Emma Monk, BSc

Clinical Molecular Geneticist

East Anglian Regional Genetics Service,

Molecular Genetics Laboratory,

Addenbrooke’s Hospital, Cambridge, UK

Rachel Butler, BSc(Hons), MRCPath

Consultant Clinical Scientist

Head of Molecular Genetics Laboratory

Institute of Medical Genetics, Cardiff, UK

1
1. Introduction

1.1. Overview

Although many, even most, methods of mutation detection depend upon the PCR, in

the majority of techniques the PCR itself does not detect the actual mutation. Rather,

the PCR generates an amplicon which is then analysed by some other method to find

possible mutations within the amplicon, e.g. conformation-based techniques such as

SSCP or DGGE, or sequencing. However, there are some methods in which a

modified PCR, or similar, does act as the primary mutation detection system, albeit

that for example, some type of electrophoresis may be needed to separate the

subsequent amplicons. These include real-time PCR, the Amplification Refractory

Mutation System (ARMS), quantitative fluorescent PCR (QF-PCR), and a derivative

of the oligoligation assay, Multiplex Ligation-dependent Probe Amplification (MLPA).

Also discussed is the single nucleotide primer extension assay, and a proprietary

derivative of it called ProntoTM. Because it uses a DNA polymerase in a post-PCR

extension step it can be deemed to fall into the group of PCR-based methods of

mutation detection. A primary limitation of these methods is that, with a few

exceptions, they are only suitable for testing for mutations that have been previously

detected and characterised by other techniques.

2. Real-time PCR

2.1. Introduction

Real-time PCR is the only purely PCR-based method of mutation detection, in that it

does not require any adjunctive technique, such as electrophoresis. As is described

below, the PCR is directly monitored within the reaction tube and the PCR primers

themselves are designed to be specific for a particular sequence or sequences.

Thus, real-time PCR is only of use in detecting previously described mutations found

by some other technique.

2
In real-time PCR the exponential phase of PCR is monitored as it occurs, using

fluorescently labelled molecules.(1) During the exponential phase the amount of

PCR amplicon DNA present in the reaction tube is directly proportional to the amount

of starting material specific to the PCR primer pair (or, target sequence). Thus, the

amount of emitted fluorescence is directly proportional to the amount of amplicon,

which in turn is proportional to the starting amount of target sequence.(2, 3) If

required, this can then be used to measure target copy number.

Real-time PCR needs to be distinguished from reverse transcription PCR (RT-PCR),

although, for example, RT-PCR can be used in conjunction with real-time PCR to

measure gene expression as well as test for mutations.(4, 5)

There are two types of real-time PCR, that which uses non-specific DNA binding

dyes such as ethidium bromide and SYBR green I, and that which uses labelled

probes: these are discussed below.

2.2. Real–time PCR: non-specific DNA binding dyes

Non-specific DNA binding dyes simply allow determination of the presence or

absence of an amplicon, without giving any information regarding the precise nature

of the product. SYBR Green I is a dye that emits fluorescence when it is bound to

double stranded DNA. As the PCR proceeds and the copy number of the product

increases, the amount of intercalated SYBR Green I will also increase, raising the

level of emitted fluorescence in direct proportion to the copy number.(6) Most

applications of real-time PCR have been to measure gene expression, or detect

pathogens, although there are applications specific to medical genetics.(7, 8, 9, 10)

2.3. Real–time PCR: labelled probes

There are three main kinds of labelled probes for use in real-time PCR: Cleavage (5’

Exonuclease) based, Molecular Beacons, and FRET probes (Fig. 1).

2.3.1. Cleavage based probes

3
Cleavage based probes are the most widely used real-time PCR probes and depend

upon the 5’ to 3’ exonuclease activity of Taq DNA polymerase (Fig. 1a). This is also

known as the Taqman® assay. Forward and reverse primers are bound to the target

DNA sequence, and a fluorescently labelled probe specific for the wild-type or mutant

sequence is bound downstream of the forward primer. The probe is an

oligonucleotide which has a fluorescent reporter dye at the 5’ end and a quencher,

typically TAMRA, attached to the 3’ end. It is modified to prevent extension occurring

from the 3’ end. When the probe is intact, the proximity of the quencher reduces the

fluorescence emitted by the reporter dye. During PCR, the forward primer is

extended by the Taq DNA polymerase until it reaches the probe. At this point the

exonuclease activity of the Taq DNA polymerase displaces and cuts up the probe,

releasing the reporter dye and the quencher. Once they are no longer in close

proximity the reporter dye emits fluorescence of a particular wavelength that can be

detected.(11)

2.3.2. Molecular Beacons

Molecular Beacons are self-complementary single stranded oligonucleotides that

take up a hairpin loop structure (Fig. 1b).(12, 13) They consist of a probe

homologous to the target sequence, flanked by sequences that are homologous to

each other. Attached to one end is a reporter dye (FAM, TAMRA, TET, or ROX) and

to the other, a quencher, usually DABCYL. When the beacon binds to the target

sequence the quencher and reporter are moved apart, and fluorescence can be

emitted. Unlike in the cleavage based assays, where fluorescence is detected during

the elongation phase of PCR, with molecular beacons, fluorescence is detected

during the annealing phase.(14, 15, 16)

2.3.3. FRET probes

FRET (Förster or Fluorescence Resonance Energy Transfer) probes are two

separate fluorescently labelled oligonucleotides, one with a 5’ donor molecule and

4
the other with a 3’ acceptor molecule attached: one is specific for target (i.e. wild-type

or mutant), the other is common (Fig. 1c). Only when these are placed within 1-5 bp

of each other can energy be transferred from the donor to the acceptor, which then

emits fluorescence.(17, 18)

2.3.4. Scorpion primers

Scorpion primers are a form of molecular beacon, but unlike them they act by a

unimolecular, rather than a bimolecular mechanism (Fig. 1d).(19, 20, 21) Such a

unimolecular mechanism is both faster and more efficient than a bimolecular

mechanism, and direct comparison with equivalent cleavage (TaqMan) and

Molecular Beacon based tests would suggest that Scorpion primers perform best. By

combining two Scorpion probes in multiplex PCR it is possible to probe for two

mutations simultaneously.

In its original form (Fig. 1d), a Scorpion primer has, from 5’ to 3’: a fluorophore at its

5’ end; a stem and loop oligonucleotide sequence, the loop part of which is

complementary to a sequence within the PCR amplicon; a quencher dye (e.g. methyl

red); a PCR stopper/blocker section (e.g. hexethylene glycol; to prevent extension of

the opposite strand beyond this point); a conventional target-specific oligonucleotide

PCR primer section.(22) After the initial PCR cycle, the loop section of the Scorpion

probe is able to intramolecularly base-pair with its complementary sequence within

the amplicon. Theoretical advantages of Scorpions probes is that they are more

specific, give a stronger signal, i.e. improved signal:noise ratio, and act faster than

comparable Molecular Beacons or TaqMan probes, which appears to be borne out in

practice.

Another version of the Scorpion probe system is the duplex format, whereby two

primers complementary to each other, one with a fluorophore and the other with a

quencher, are used instead of a single stem-loop Scorpion probe (Fig. 1e).(23) Like

stem-loop Scorpions, this format requires the complex synthesis of non-standard

5
oligonucleotides.(22) However, the advantage of duplex Scorpions is that, like

TaqMan probes, the quencher and fluorophore become completely separated during

the process, unlike stem-loop Scorpions and Molecular Beacons where some degree

of Förster resonance energy transfer tends to quench the signal. The background is

also theoretically lower because of higher quenching due to proximity of the

fluorophore and quencher, compared with TaqMan probes. This has led to the

development of FRET duplex Scorpions.(23) Duplex Scorpion probes are easier to

synthesize compared with stem-loop Scorpions, but nonetheless their synthesis is

more involved than that for standard fluorescently-labelled oligonucleotides.

So far there have not been many medical applications of Scorpions published, but

those that have include known mutation detection in cystic fibrosis (CFTR/ABCC7),

quantitation of HIV-1, splice variant analysis of calpain 3 (21, 23, 24, 25, 26). Some

interesting applications to plant pathogen and food science have been presented.(27,

28)

2.4. Information

There is plenty of information on the internet related to real-time PCR, in particular

we would recommend:

http://www.pcrlinks.com/variants/real-time_pcr.htm

http://www.protocol-online.org/prot/Molecular_Biology/PCR/Real-Time_PCR/

http://home.att.net/~dorak/genetics/realtime.html

http://molecular-beacons.org/

2.5. Equipment for real-time PCR

Real-time-PCR is performed on thermal cyclers that have an integral optical system

to detect fluorescence of different wavelengths. The optical system is connected to a

6
computer with software that can analyse the fluorescence data to produce the

exponential curves or allelic discrimination data required.

2.6. Uses of real-time PCR

The main uses for real-time PCR in a molecular diagnostic laboratory are for SNP

genotype analysis and sequence copy number determination.

For SNP genotyping and small mutation testing, two differently labelled probes are

designed, one for the wild type allele and one for the mutant allele. The mismatch

between the wild type allele and the mutant probe facilitate competitive hybridisation.

Therefore, fluorescence will only be detected when the correct probe binds the target

sequence. This sort of assay can be utilised where specific mutations are being

analysed, such as in sickle cell disease, or as part of a cystic fibrosis screen.(11, 14,

17, 18, 20, 23, 29, 30)

Real-time PCR can also be used to determine the copy number of specific target

sequences, such as the Peripheral Myelin Protein 22 gene (PMP22). By multiplexing

the primers and probes for PMP22 with the primers and probes for a control

sequence, known to be present in 2 copies, accurate measurement of the PMP22

copy number can be made. This determines whether a patient has one, two or three

copies of PMP22. Patients with only one copy of PMP22 have hereditary neuropathy

with liability to pressure palsies (HNPP), while patients with three copies have

Charcot-Marie-Tooth disease (CMT).(3, 24, 25, 26, 31, 32, 33)

An interesting application of real-time PCR has been the detection of DNA

methylation. Expression of the DNA mismatch repair enzyme MLH1 is lost in

approximately 20% of colon cancers, and is associated with methylation of the

gene’s promoter (see Chapter 32, section 1.2.2.4. for more details). Other tests for

methylation, dependent on cleavage at or binding of primers to, single specific CpG

sites do not necessarily give results representative of the global methylation status of

7
single DNA molecules. However, a real-time PCR assay (MethyLight), in which all

the primers involved, forward, reverse and reporter, bind to sites containing at least

three CpGs each has been designed (34, 35, 36). Thus, the MethyLight assay only

reports methylation when all of the CpG sites on a particular DNA molecule are

methylated, which is likely to be more biologically relevant.

Real-time PCR also has many other applications in the fields of clinical microbiology,

food microbiology, tumour biology, gene therapy and gene expression.(4, 27, 28, 37,

38)

2.7. Advantages of real-time PCR

The main advantage of real-time PCR is the speed with which samples can be

analysed, as there are no post-PCR processing steps required. PCR can be

performed in a 96 or 384 well format, and reactions can be multiplexed, leading to a

high throughput of samples. The analysis of results is very simple and this

contributes to it being a much faster and simpler method for analysing gene copy

number compared with many other current methods, such as Multiplex Amplifiable

Probe Hybridisation (MAPH), Southern blotting and semi-quantitative PCR.(39)

Another major advantage of having no post-PCR steps is that real-time PCR is a

closed tube method of analysis, which greatly reduces the chance of sample

contamination, errors from mistakes in tube tranfers, or amplicons escaping into the

laboratory environment: all important factors in the molecular diagnostics laboratory.

2.8. Limitations of real-time PCR

When using non-specific real-time PCR methods, such as with SYBR green I, side-

reaction products other than the desired amplicon, such as primer dimers, are also

detected. Careful optimisation using melting curve analysis is required to give a clear

distinction between the correct amplicon and any unwanted PCR products. The

8
inability to differentiate between specific and non-specific products limits non-specific

real-time PCR to single amplicons, i.e. the PCR cannot be multiplexed.

As there is a limited availability of fluorescent dye combinations, a maximum of 4

dyes can be currently used per reaction tube. This limits the extent to which

multiplexing can be performed.

It has to be borne in mind that the capital investment in real-time PCR is expensive,

that the probes, too, are expensive compared with conventional oligonucleotides, and

that an investment must also be made in staff training and expertise, especially probe

design.

A final limitation to be considered is that the quality and accuracy of the data

produced is very dependent on the sample preparation and the quality of the DNA.

Very careful probe design and optimisation are required to get good results,

particularly for SNP analysis. This means that careful planning and work up is

required in order to set up a reliable and accurate assay. The inclusion of

appropriate controls is mandatory.

3. The Amplification Refractory Mutation System (ARMS)

3.1. Introduction

All PCRs depend on the annealing of oligonucleotide primers to specific sites in

target DNA, thus allowing amplification of a unique sequence. For the polymerase to

extend the primers they must be perfectly annealed to the target sequence at their 3’

ends. The Amplification Refractory Mutation System (ARMS) exploits this by utilising

a primer which is designed such that its 3’ end matches, for example, one of two

alternatives at a mutant nucleotide.(40) ARMS is ideally suited to the detection of

point mutations and small insertions/deletions, and at its simplest involves two PCRs,

one for each allele (Fig. 2). Thus, an ARMS PCR is allele specific, and earlier

9
alternative names for the process have reflected this, e.g. Allele Specific PCR

(ASPCR), and PCR Amplification of Specific Alleles (PASA).(41, 42) A number of

ARMS variants have been produced and these are discussed below.

Additional mismatches near the 3’ end of ARMS primers helps to increase the

specificity of reactions, but too many mismatches can cause excessive

destabilisation. Differentiation of alleles is said to be problematic when the 3’

mismatch is C:A or involves a T, but this is not our experience.(43) The most

discriminatory mismatches appear to be A:G/G:A, closely followed by Py:Py. It is

wise to test the performance of a new ARMS PCR by varying, for example, primer

concentration, buffer, magnesium concentration, annealing temperature and PCR

additives such as DMSO. Hot-start Taq is worth the extra cost. As always, novel

applications require rigorous working up to test inter alia their specificity and

sensitivity. It is essential to include a control PCR, that is not allele specific, in the

same tube as the ARMS reaction to avoid a null result. If a number of ARMS

reactions are multiplexed then this requirement is obviated.

3.2. Double ARMS

Double ARMS utilises a pair of allele-specific primers, instead of one specific and

one common primer as per the standard ARMS test. The two polymorphic or mutant

sites must be close enough to each other to allow a PCR to work, although given the

distance that can be spanned by a long range PCR, several kb, this is not the

constraint it perhaps once was. Double ARMS enables the haplotyping of an

individual in the absence of DNA from relatives. To do this four ARMS PCRs must

be set up: if, say, locus 1 has two alleles, A and B, and locus 2 has two alleles, C and

D, then these PCRs will be A × C, A × D, B × C, and B × D. Double ARMS can be

useful for haplotyping doubly heterozygous individuals, to check, for example, if two

mutations are in cis or trans, to distinguish a carrier of a recessive disorder from an

10
individual at risk of being affected. It has been used to haplotype alleles at closely

linked loci, e.g. HLA and rhesus blood groups.(44)

3.3. MS-PCR

In some diagnostic applications it is possible to include both allele-specific primers,

one for each alternative at the mutant or polymorphic site, which then together with

the common primer allows each amplicon to act as a control for the other: in this

instance, however, there must be a way of distinguishing the two amplicons, either by

differential size or labelling.(45, 46, 47, 48, 49)

Advantages of MS-PCR include the fact that only a single PCR needs to be carried

out and there is no need for a separate internal control. Sensitivity is improved

because of the competitive nature of the PCR. Design of MS-PCR primers is

somewhat more involved than simple ARMS assays, but this is not a significant

negative factor.

3.4. Multiplex ARMS

In conditions where there are a restricted number of common mutations, then it may

be efficacious to perform a test that simultaneously tests for as many as possible of

them, i.e. multiplex ARMS. It is exemplified by commercially available kits to detect

particular CFTR mutations that are prevalent in particular populations, e.g. the

Elucigene CF20 kit which detects 20 different mutations.(44, 49, 50) It can be a

difficult technical challenge to design primers that do not interfere with one another,

but still work in a multiplex ARMS. Amplicons are best distinguished by being either

of differing sizes, or differentially labelled, or both.

3.5. Advantages and limitations of ARMS

ARMS assays are generally quick, cheap and simple to devise and work up.

Inherently, they are not suited to screening for unknown mutations, but only

mutations detected initially by some other technique. They do not require any

11
unconventional PCR equipment, although the method used to detect amplicons can

vary acording to that locally available or desired, e.g. from simple agarose gel

electrophoresis, to detection on an automated capillary fluorescent DNA analyser.

ARMS reactions are reasonably easily multiplexed, and can also be used to

determine haplotypes.(51) In terms of sensitivity, they are generally able to detect

one mutant allele in 40 normal alleles. As with all oligonucleotide-based tests, care

must be taken at the design stage to avoid known polymorphisms that may affect

primer binding, e.g. CFTR ΔF508, and similarly caution must be exercised in

interpretation bearing in mind that a hitherto unknown sequence variant may exist in

a patient, and it interferes with, or appears to be a known mutation.(52)

4. Quantitative fluorescent PCR (QF-PCR)

4.1. Introduction

Quantitative fluorescent PCR (QF-PCR) is based on the principle that the amount of

amplicon produced by a PCR in its exponential phase, i.e. before the yield has

reached its plateau, is proportional to the starting amount of target.(53) Thus, such

PCRs are carried out with generally no more than 24 cycles. Fluorescent detection

of such amplicons, via labelling of one of the primers, facilitates detection and

quantitation of the PCR product using a fluorescent DNA analyser, either gel, or

better, capillary-based. No processing of the PCRs is necessary, other than the

usual preparation of samples for running on such analysers. Validation of such

assays, using samples tested by an independent method, is, of course, necessary.

4.2. Uses of QF-PCR

QF-PCR is well suited to the rapid detection of aneuploidies in antenatal

amniocentesis or chorion villus samples and is in wide use.(54, 55, 56, 57) FISH is

perhaps slightly less reliable than QF-PCR for the detection of aneuploidy in such

12
situations, and QF-PCR also has the benefit of easy automation. However, while

FISH reagents are considerably more expensive than those needed for QF-PCR, the

capital cost of a fluorescent microscope is less than a fluorescent DNA analyser,

though neither can be described as cheap.(58) QF-PCR has also been applied to

RhD testing, Duchenne muscular dystrophy testing, and has also revealed that TP53

deletions can cause Li-Fraumeni syndrome.(59, 60, 61)

5. The Oligonucleotide Ligation Assay (OLA)

5.1. Introduction

Oligonucleotide primers which have annealed to adjacent sites on single-stranded

DNA, such that there is no gap between them, can be covalently joined together by

DNA ligase: the oligoligation assay (OLA). Successive cycles of denaturation,

annealing and ligation result in a linear amplification of the target sequence, in

contrast to the exponential increase in PCR. The adjacent ends of the primers must

be perfectly matched with the target sequence, otherwise ligation will not occur.

Thus, a single nucleotide or a few adjacent nucleotides can be interrogated.(62, 63)

Multiplexing allows a number of nucleotides and/or mutations to be detected in a

single reaction tube.(64) Nowadays, one of the primers usually has a fluorescent

label so that the product can conveniently be detected on a fluorescent DNA

analyser. Although OLA is not strictly a PCR-based method, a new method has been

developed in which an initial OLA is followed by a PCR, called Multiplex Ligation-

dependent Probe Amplification (MLPA). As described below, MLPA is ideally suited

to dosage testing of multi-exon genes. A similar technique, but in which the initial

OLA is replaced by a PCR (Universal primer quantitative fluorescent multiplex:

UPQFM) has also been described.(65)

5.2. Multiplex Ligation-dependent Probe Amplification (MLPA)

13
Multiplex Ligation-dependent Probe Amplification is a hybrid technique that combines

an OLA with a quantitative fluorescent PCR (QF-PCR). It is ideally suited to the

measurement of exon copy number in multi-exon genes, but could be used for the

quantitative assessment of any locus.(66)

The basis of MLPA is covered in Chapter 32, section 1.3.2., but in brief, genomic

DNA is denatured and a mixture of oligonucleotide probes is hybridised to the DNA

(Fig. 3). Each MLPA probe consists of two oligonucleotides which take part in a one

cycle OLA reaction. The two oligonucleotides have generic sequences attached, to

the 5’ end of the upstream primer, and to the 3’ end of the downstream primer. Thus,

after the initial OLA step, a PCR is then carried out using primers complimentary to

the generic sequences on the ends of the OLA primers. In this way multiple different

target sequences can be interrogated, but which are all amplified using the same,

generic, PCR primers, with equal efficiency. As one of the generic primers is

fluorescently labelled, then the fluorescence due to a particular amplicon, i.e. MLPA

probe, is proportional to the amount of starting target DNA, and thus copy number

can be determined. The lengths of the various probes are engineered so that

individual probes differ by a few bp and can be easily separated on a fluorescent

DNA analyser.(66)

5.3. Advantages and uses of MLPA

MLPA is quick, cheap and relatively simple. Probes can be easily designed for novel

applications, although a wide range of clinically useful kits are manufactured (see

http://www.mrc-holland.com/). Its use in detecting exon copy number changes in

DNA mismatch repair genes, in Hereditary Non-Polyposis Colorectal Cancer

(HNPCC), is covered in chapter 32 (sections 1.3.2., 4.3.1. and 5.3.1.).(67, 68, 69)

5.4. Limitations of MLPA

14
Kits are currently manufactured by only a single commercial company, though they

are open to suggestions for new applications. Like any dosage technique, MLPA is

sensitive to DNA quality and quantity. It is best to perform tests in duplicate and be

suspicious of discordant results. Control probes are included in MLPA kits from

MRC-Holland and notice should be taken of them and the manufacturer’s

instructions.

If a point mutation or polymorphism occurs within an MLPA probe binding site it may

result in failure of ligation, and hence appear as deletion of a whole exon. Deletion of

two or more contiguous exons is unlikely to be due to this effect, but apparent

deletion of a single exon might be. For this reason another complementary

technique should be used to confirm the nature of the mutation if it appears to be

deletion of a single exon. This could be by using an alternative probe (pair of MLPA

primers), real-time or long range PCR, Southern blot or mRNA analysis. It might also

be useful to sequence the exon in question.

6. Primer Extension

6.1. Introduction

The single nucleotide primer extension assay is in widespread use. It is the basis of

a proprietary technique: ProntoTM.(70) An amplicon is produced in a conventional

PCR and residual dNTPs are eliminated using alkaline phosphatase. The amplicon

is denatured and a specific primer binds to one of the two DNA strands. At its 5’ the

primer has a covalently linked label. Depending on the target sequence a

biotinylated dNTP is added to the 3’ end of the primer by DNA polymerase. The now

biotinylated primer can bind to streptavidin-coated wells in a 96-well plate and after

washing a horseradish peroxidase conjugated antibody to the primer label is added.

Binding of the HRP conjugated antibody is manifest by colour development in that

15
well and the mutation causing addition of the specific dNTP is detected. Different

fluorescent labels can be linked to different dNTPs, such that with an appropriate

detector different mutations at the same nucleotide can be distinguished, e.g. K-ras.

6.2. Equipment for primer extension

Aside from a conventional PCR thermocycler, results can be scored either visually or

with an ELISA plate reader, if a numerical output is desired.

6.3. Uses of primer extension

In common with other PCR-based methods of mutation detection, primer extension is

suited to the detection of defined mutations. The commercially available tests using

ProntoTM concentrate on common point mutations in both dominant and recessive

disorders, in particular those prevalent in the Ashkenazi Jewish population.(70, 71,

72)

6.4. Advantages and limitations of primer extension

Primer extension is a gel-free mutation detection system of low to high-throughput. It

only requires commonly available equipment. Although visual scoring is probably

adequate for low throughput applications, medium to high throughput benefits from

using an ELISA reader.

Limitations include that ProntoTM kits are currently only manufactured by a single

commercial company (http://www.savyondiagnostics.com/), a restricted range of

applications is available, mostly based on conditions due to founder mutations in the

Ashkenazi Jewish and Mediterranean populations. (70, 71, 72) However, a large

number of in-house primer extension assays have been developed.(e.g. 73, 74) Like

most PCR-based methods of mutation detection, primer extension is only able to

detect defined mutations found by another technique, i.e. it is not a method able to

screen amplicons for any mutation.

16
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26
Figure 1

Sequence specific Real-time PCR methods

a) Cleavage (5’ Exonuclease) based assay

Probe
Forward primer
R Q

Reverse primer

R
Q

Fluorescence
R
Q

Light emission from the reporter fluorophore (R) is quenched because of its proximity

to the quencher (Q). Cleavage by Taq polymerase separates the reporter and

quencher allowing fluorescence.

27
Figure 1

b) Molecular beacons

Molecular beacon

R Q

Fluorescence
R Q

Forward primer

Reverse primer

Light emission from the reporter fluorophore (R) is quenched because of its proximity

to the quencher (Q), brought about by the self-complementary 5’ and 3’ ends of the

molecular beacon causing it to take up a hairpin loop structure. Thermal

denaturation and annealing allows the central, loop section of the molecular beacon

to bind to its target sequence in the PCR amplicon. Thus, the reporter and quencher

become sufficiently separated to allow fluorescence. In this system the probe is not

subject to cleavage by Taq polymerase.

28
Figure 1

c) Fluorescence Resonance Energy Transfer (FRET) probes

D A

A Fluorescence
Forward primer D

Reverse primer

The acceptor fluorophore (A) is unable to fluoresce until it is within 1-5 bp of the

donor fluorophore (D), which occurs when the two FRET probes anneal to the

specific PCR amplicon.

29
Figure 1

d) Scorpion primers

Reverse primer
Scorpion primer

R Q

Fluorescence Heat denature

R Q

Anneal*

R Q

Extend*

R Q

R Q Heat denature

Fluorescence
R Anneal*
Q

Stabilisation

30
*Reverse primer and target DNA strand omitted for clarity.

Light emission from the reporter fluorophore (R) of the Scorpion primer is quenched

because of its proximity to the quencher (Q), brought about by the self-

complementary of the stem sections, causing it to take up a hairpin loop structure.

Thermal denaturation, annealing, and polymerase extension allows the central, loop

section of the Scorpion primer to bind to its target sequence in the PCR amplicon,

thus stabilising separation of the quencher and fluorophore. Thus, the reporter and

quencher become sufficiently separated to allow fluorescence. In this system the

probe is not subject to cleavage by Taq polymerase.

31
Figure 1

e) Duplex Scorpion primers

R
Reverse primer
Q
Duplex

Scorpion

Fluorescence Heat denature

Q R

Q R
Anneal*

Q R
Extend*

R
Heat denature

Fluorescence
R Anneal*

Stabilisation

32
Light emission from the reporter fluorophore (R) is quenched because of its proximity

to the quencher (Q), brought about by the complementarity of the two Scorpion

primers: the probe primer has the fluorophore attached (R). Thermal denaturation

separates the two primers allowing fluorescence, but annealing brings them back

together again causing quenching. However, in subsequent cycles annealing of the

probe sequence to its intramolecular complement in the amplicon is favoured over

binding to the quencher primer. Thus, the reporter and quencher become sufficiently

separated to allow fluorescence. In this system the probe is not subject to cleavage

by Taq polymerase.

33
Figure 2

The Amplification Refractory Mutation System (ARMS)

Forward primer ‘A’

Allele

Reverse (common) primer

Forward primer ‘B’

Allele

Reverse (common) primer

The forward primer is designed at its 3’ end to match allele B, but not A. Thus, if

allele B is present in the target DNA an amplicon will be produced in the PCR. A

complimentary ARMS PCR in which the forward primer is designed to match allele A,

but not B, is also performed.

34
Figure 3

Multiplex Ligation-dependent Probe Amplification (MLPA)

Annealing

Ligation

PCR

Fluorescence
A Detection

35
Figure 4

Pronto assay

PCR product

Biotin-labelled

dNTP
B
B
5’-labelled primer
L C
C

B
L
C
HRP

A conventional PCR is carried out, followed by enzymatic degradation of

unincorporated dNTPs. A 5’-labelled primer is then annealed to the PCR and DNA

polymerase and a biotinylated dNTP added. The biotinylated dNTP is only added to

the primer if it matches the target amplicon. The reaction mix is then added to a

streptavidin coated well and biotinylated primers are thus bound; unbound primers

are washed away. A horseradish peroxidase-linked antibody to the 5’ primer label is

then added, and detected by colour development measured visually or by an ELISA

plate reader.

36
 37

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