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 Mutation Research 613 (2006) 76–102
www.elsevier.com/locate/reviewsmr
Community address: www.elsevier.com/locate/mutres
Review
The random amplified polymorphic DNA (RAPD) assay and

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related techniques applied to genotoxicity and
carcinogenesis studies: A critical review

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Franck A. Atienzar *, Awadhesh N. Jha
School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, Devon, UK
Received 13 February 2006; received in revised form 12 June 2006; accepted 12 June 2006
Available online 18 September 2006

al
Abstract on
More than 9000 papers using the random amplified polymorphic DNA (RAPD) or related techniques (e.g. the arbitrarily primed
polymerase chain reaction (AP-PCR)) have been published from 1990 to 2005. The RAPD method has been initially used to detect
polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. Despite
their extensive use, these techniques have also attracted some criticisms, mainly for lack of reproducibility. In the light of their
widespread applications, the objectives of this review are to (1) identify the potential factors affecting the optimisation of the RAPD
rs

and AP-PCR assays, (2) critically describe and analyse these techniques in genotoxicity and carcinogenesis studies, (3) compare the
RAPD assay with other well used methodologies, (4) further elucidate the impact of DNA damage and mutations on the RAPD
profiles, and finally (5) provide some recommendations/guidelines to further improve the applications of the assays and to help the
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identification of the factors responsible for the RAPD changes. It is suggested that after proper optimisation, the RAPD is a reliable,
sensitive and reproducible assay, has the potential to detect a wide range of DNA damage (e.g. DNA adducts, DNA breakage) as
well as mutations (point mutations and large rearrangements) and therefore can be applied to genotoxicity and carcinogenesis
studies. Nevertheless, the interpretation of the changes in RAPD profiles is difficult since many factors can affect the generation of
RAPD profiles. It is therefore important that these factors are identified and taken into account while using these assays. On the other
hand, further analyses of the relevant bands generated in RAPD profile allow not only to identify some of the molecular events
implicated in the genomic instability but also to discover genes playing key roles, particularly in the initiation and development of
r's

malignancy. Finally, to elucidate the potential genotoxic effects of environmental contaminants, a powerful strategy could be firstly
to use the RAPD assay as a screening method and secondly to apply more specific methods measuring for instance DNA adducts,
gene mutations or cytogenetic effects. It is also envisaged that these assays (i.e. RAPD and related techniques), which reflect effects
at whole genome level, would continue to complement the use of emerging technologies (e.g. microarrays which aim to quantify
o

expression of individual genes).

# 2006 Elsevier B.V. All rights reserved.
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Keywords: Randomly amplified polymorphic DNA (RAPD); Arbitrarily primed polymerase chain reaction (AP-PCR); DNA amplification
fingerprinting (DAF); DNA damage; Mutations; Genomic instability; Cancer; Eco-genotoxicology
Au

* Corresponding author at: UCB, Chemin du Forriest, B-1420 Braine-l’Alleud, Belgium. Tel.: +32 2 386 38 31; fax: +32 2 386 27 98.
E-mail addresses: franck.atienzar@ucb-group.com, fatienzar@infonie.fr (F.A. Atienzar).

1383-5742/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrrev.2006.06.001
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102 77

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2. Advantages, disadvantages and optimisation of the techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.1. Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.2. Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2.3. Optimisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.3.1. Reproducibility of profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.3.2. Effect of the annealing temperature on profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

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2.3.3. Influence of each PCR chemical on profiles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.3.4. Concluding remarks on the optimisation procedure of the assays . . . . . . . . . . . . . . . . . . . . . . . . . 82
3. The use of RAPD in genetic ecotoxicology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
3.1. AP-PCR and RAPD to detect DNA damage and mutations (genotoxicity investigations). . . . . . . . . . . . . . . 85

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3.2. RAPD to measure population effects (changes in genetic diversity or gene frequencies) . . . . . . . . . . . . . . . 86
3.3. Linking genotoxic and population effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
4. RAPD and AP-PCR to detect genomic instability in carcinogenesis studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.1. AP-PCR to detect genomic instability in malignant cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.2. RAPD to detect genomic instability in malignant cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.3. Further analysis of RAPD/AP-PCR changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
5. Comparison of RAPD assay with other methodologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

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5.1. Discrimination of closely related strains of bacteria with the RAPD assay . . . . . . . . . . . . . . . . . . . . . . . . 89
5.2. Microarrays and RAPD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5.3. Comparison with other genotoxicity and mutagenicity assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
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6. Recommendations in the context of genotoxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
7. Interpretation of changes in RAPD profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
7.1. Effects of DNA damage and mutations on profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
7.2. How to improve the interpretation of the RAPD assay and related techniques . . . . . . . . . . . . . . . . . . . . . . 96
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8. RAPD optimisation and interpretation of the data: a case study. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
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1. Introduction RAPD, AP-PCR and DAF are three semi-quantita-

tive methods which have been originally used in
The 1993 Nobel Prize for chemistry was awarded to genetic mapping, taxonomy and phylogeny [2,3,5,6].
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Dr. Kary Mullis for having invented the polymerase chain
reaction (PCR) [1]. This remarkable technology has
revolutionised the field of molecular biology and has
been used in diverse areas of research such as evolution,
o
For instance, the RAPD method has been used to detect
polymorphism in studies examining genetic diversity
[7–9], pedigrees [10], the construction of genetic maps
[11], the identification of cultivars [12], pest resistance

clinical medicine, forensic science, pathogen detection. genes [13], and sex markers [14]. In addition, the
th

Subsequently, new PCR based methods have been RAPD assay and related techniques have also been
developed. In particular, Williams et al. [2] and Welsh used in genotoxicity and carcinogenesis studies as
and McClelland [3] developed the random amplified mentioned later.
Au

polymorphic DNA (RAPD) and arbitrarily primed PCR
(AP-PCR), respectively. Similar techniques such as the
DNA amplification fingerprinting (DAF) has also been
developed [4]. As shown in Fig. 1, such methodologies
(principally RAPD) are quite popular. For instance, by
2005, 8493 RAPD papers were published as compared to
504 AP-PCR papers and 132 DAF papers. In addition,
Fig. 1 also shows that the number of RAPD papers
increased rapidly (from 1990 to 1996) but has since
settled at around 600–800 papers per year.
RAPD, AP-PCR and DAF are a modification of PCR
that provides an information-rich fingerprint of genomic
DNA. These techniques are based on the selective
amplification of genomic sequences that, by chance, are
flanked by adequate matches to an arbitrarily chosen
primer. If two template genomic DNA sequences are
different, the PCR products display different banding
patterns. Polymorphisms detected by these techniques
can be used as taxonomic markers in population studies
of a wide variety of organisms [15]. Although RAPD,
78 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102

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Fig. 1. Publications hit(s) obtained in OVID (http://www.ovid.com) database from 1990 to 2005. , and refers to RAPD, AP-PCR and DAF
papers, respectively. The search was performed with the following databases: BIOSIS Previews, Derwent Drug File, EMBASE and OVID Medline
by using the terminologies ‘‘random amplified polymorphic DNA’’, ‘‘arbitrarily-primed polymerase chain reaction’’ and ‘‘DNA amplification

al
fingerprinting’’ with the respective years.

AP-PCR and DAF are very similar techniques, they
on
often cause some ambiguity in terminology. There are
however some procedural differences which have been
clearly defined by Meunier and Grimont to distinguish
among theses techniques [16]. Table 1 provides an
rs
analysis, all clear/reproducible RAPD bands, irrespec-
tive of whether they are diagnostic or not, are
considered. Generally, the present/absent RAPD bands
are used to estimate similarity and/or diversity
measures. A RAPD band that is absent is just scored

overview of these procedural differences. From a ‘‘0’’ and will ultimately add value to the similarity or
practical point of view, it is clear that the RAPD assay, diversity indexes. In the genetic analysis (contrary to the
which does not require use of radionuclides or phenetic analysis), a band which is absent corresponds
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electrophoresis in acrylamide gels, is the easiest assay
to perform when compared to AP-PCR and DAF. In
addition, the fact that the RAPD assay allows the
visualisation of a wide range of PCR products may
explain why this assay is a preferred choice (Fig. 1).
In the field of ecotoxicology, most RAPD studies
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to a ‘‘not phenotypically expressed locus’’ and is scored
as the double recessive heterozygotic genotype. De
Wolf et al. published a review describing how the RAPD
technique can be applied in an ecotoxicological context,
providing information on all direct and indirect routes
through which toxicants may affect the genetic structure

describe the RAPD changes such as differences in band of populations [17]. In particular, they propose to
intensity as well as gain/loss of RAPD bands. This can introduce some statistical tools to infer RAPD based
be defined as diagnostic RAPD. The further analysis of diversity and similarity indices (i.e. phenetic numerical
o

RAPD patterns through phenetic and genetic analysis analysis) and to assess the genetic structure of exposed
has been rarely attempted. In the case of the phenetic populations (i.e. genetic numerical analysis). These
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Table 1
Procedural differences among AP-PCR, RAPD, and DAF
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RAPD AP-PCR DAF

Primer length (bp) 10–12 20 5–15
Primer concentration (mM) 0.3–3 3 3–30
Annealing temperature ( 8C) 30–36 (50a) 40 and 60 30
Number of cycles 40–45 40 35
Use of radioactivity No Yes (a32P-dATP-10 last cycles) No
Separation Agarose Polyacrylamide Polyacrylamide
Visualisation Ethidium bromide Radiolabelling Silver staining
Size range of PCR products (kb) 0.3–4 0.05–0.5 0.05–0.5
a
Typically, most RAPD papers use low stringency conditions but we use much higher annealing temperature (i.e. 50 8C for 10 mer primers).
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
information related to the phenetic and genetic analysis
have been reviewed comprehensively by De Wolf et al.
with appropriate examples [17] and therefore have been
excluded in this review.
There is increasing awareness of the need to evaluate
the effects of contaminants at the population level.
Genetic techniques offer a powerful approach to assess
contaminant-induced changes in populations. Belfiore
and Anderson [18] present a summary of genetic
assessment methods and a review of published studies
of genetic effects in field-exposed aquatic organisms
[18]. The authors caution that all these approaches are
greatly improved by careful experimental design that
includes adequate numbers of reference and contami-
nated sites and sample size. In addition, careful
exposure assessment is required, including site and
tissue chemistry, biomarker responses, and measures of
potentially deleterious effects, such as DNA damage,
reduced reproductive output or survival [18].
The RAPD assay and related methodologies have
also proved useful to detect genomic instability in
on
malignant cells (see Section 4). Genomic instability
encompasses molecular events such as point mutations,
genomic and chromosomal rearrangements, deletion,
and insertions. Of special interest regarding the
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application of these techniques to cancer is that the
amplified bands usually originate from unique
sequences rather than from repetitive elements. The
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RAPD and AP-PCR (as well as related techniques) are
very likely to detect genomic instability as the
malignant cell will produce a clone of dividing daughter
cells. Thus, the proportion of cells presenting the same
genomic instability is high and easy to detect. Finally,
further analysis of the relevant bands allows not only to
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identify some of the molecular events (e.g. allelic losses
and gains) implicated in the genomic instability, but
also to discover genes playing key roles in the initiation
o
and development of malignancy (e.g. oncogenes, DNA
repair genes, anti-oncogenes).
th
Providing a holistic picture and complementing the
earlier reviews [17,18], the objectives of this review are
to (1) identify the potential factors affecting the
Au
optimisation of the RAPD and AP-PCR assays, (2)
critically describe and analyse these techniques in
genotoxicity and carcinogenesis studies, (3) compare
the RAPD assay with other well used methodologies,
(4) further elucidate the impact of DNA damage and
mutations on the RAPD profiles, and finally (5) provide
some recommendations/guidelines to further improve
the applications of the assays and to help the
identification of the factors responsible for the RAPD
changes.
79
2. Advantages, disadvantages and optimisation
of the techniques
2.1. Advantages
The RAPD assay and related techniques present
numerous advantages over conventional methods such
as hybridisation-based protocols. In particular, no prior
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knowledge of the genome under investigation is
required. These assays require very little source
material and under certain circumstances, the analysis
can also be performed non-destructively which can be
co
useful for the screening of rare or valuable samples. In
addition, a single random oligonucleotide primer is used
which means that by employing different primers,
banding profiles can be rapidly generated that differ in
complexity. Another remarkable advantage is the high
level of the overall sensitivity of the technique. In
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addition, these technologies are relatively cheap and do
not require the use of specialised and expensive
equipment. Another advantage is that after optimisa-
tion, the RAPD assay is very reliable (see Section 2.3).
The assay also presents a number of advantages for the
detection of genotoxic effects (see Section 3). And last
but not the least, the RAPD method has the potential to
detect a wide range of DNA damage (e.g. DNA adducts,
DNA breakage) as well as mutations (point mutations
and large rearrangements). In the field of cancer
research, the RAPD assay and related techniques allow
the simultaneous detection and cloning of genomic
alterations, without previous knowledge of the altered
region (see Section 4).
2.2. Limitations
Despite having shown great utility for many applica-
tions, problems may occur in RAPD which diminish its
discriminative ability. The problems include the presence
of spurious amplification products in negative control
reactions not containing template DNA [19]. Some
studies have also criticised the technique on the basis that
it lacks reproducibility [20,21], and Mendelian inheri-
tance [22–24] because of the low stringency conditions
employed (i.e. low annealing temperature) which could
lead to spurious amplification. Most of the studies which
experienced some problems of reproducibility suggested
that the results were due to artefacts, contamination or
mutation in genomic DNA [22,23]. When RAPD profiles
are not reproducible, researchers generally claim that the
variation is due to artefact although no evidence of such
event is usually presented. Indeed, if profiles from
the same genomic template, or from different individuals
80 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
of the same species vary, the reproducibility of the assay
should be confirmed by repeating the PCR reaction by
using 2 different template concentrations, differing by at
least two-fold [25] (see also Section 2.3.3). This is not to
say that artefacts do not occur but that the use of 2 DNA
concentrations that differ by at least a factor of 2 can be
used to identify spurious or non-reproducible bands.
Concerns have also been raised over the suitability of
the RAPD assay for determining parentage because
non-parental bands may appear in offspring of known
pedigrees [23,24,26]. Ayliffe et al. [24] clearly
demonstrated that heteroduplex molecules formed
between allelic RAPD products are a potential source
of artifactual polymorphism that can arise during RAPD
analysis [24]. Riedy et al. [23] reported a high frequency
of non-parental bands from primates [23]. However,
other studies also reported non-parental bands but at a
very much lower frequency in beetles [26] and in flax
rust [24]. Nevertheless, the users of the technology
should keep this phenomenon in mind particularly for
pedigree analysis in highly polymorphic species. This is
on
particularly important given the fact that homozygotes
cannot be distinguished from heterozygotes [27].
The major challenge of identifying the precise
reasons for the changes which may be induced in the
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RAPD profiles may remain unresolved. Indeed,
different types of DNA lesions and mutations can
induce the same type of alterations in RAPD profiles
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(e.g. variation in band intensity, band appearance and
disappearance). Thus, other methods measuring geno-
toxic endpoints, such as detection of DNA adducts, gene
mutations or cytogenetic effects, are required for the
identification of changes at DNA level. In the field of
eco-genotoxicology, we also strongly encourage use of
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the RAPD in conjunction with parameters at higher
level of biological organization such as growth, fitness
and reproduction, as our studies have demonstrated a
o
link between these parameters and changes in RAPD
profiles in different aquatic organisms (see Section 3).
th
Another potential obstacle is that the use of RAPD for
comparative purposes relies on the assumption that
similarity of fragment size is a dependable indicator of
Au
homology [28]. Finally, it is important for all who use
the technique to appreciate that the RAPD method
generates qualitative or semi-quantitative data rather
than quantitative data.
2.3. Optimisation
2.3.1. Reproducibility of profiles
Lack of reproducibility of the RAPD assay in some
studies has led some researchers to suggest that RAPD
is not reliable. However, a variety of parameters should
be verified before drawing such a conclusion. For
instance, the quality of the extracted DNA plays a
central role, and genomic DNA of poor quality will
certainly lead to non-reproducible patterns. Similarly,
the estimation of DNA concentration is important,
because RAPD assays performed with very different
amounts of DNA will lead to different profiles (e.g. a
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majority of reproducible bands but some possible
differences in band intensity). In our hands, very low
DNA concentrations (OD < 0.1) can cause an over-
estimation of the DNA concentration and underestima-
co
tion of the 260/280 nm ratio. Obviously, other
parameters such as magnesium, dNTPs, Taq enzyme,
etc., also play important roles, and before beginning any
experiments, one should clearly understand the impacts
and effects such parameters have on RAPD profiles.
After rigorous optimisation of the RAPD reactions
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(and related assays), several studies have also confirmed
the reproducibility of these assays [29–34]. Those who
accept that RAPD is a reliable method claim that, with
practice and care, repeatability or reproducibility is
actually high [35]. However, it is also well established
that many conditions of the RAPD reaction procedure
may influence the results. For instance, the use of
different thermostable DNA polymerase, or thermal
cyclers can generate variable RAPD profiles [16,36,37].
In this context, a comparison of RAPD profiles among
laboratories should not be attempted unless RAPD
reactions are performed under strictly identical condi-
tions. In a recent study, the repeatability (intra-
laboratory) and reproducibility (inter-laboratory) of
the RAPD protocol were tested using 15 Yersinia strains
representing different RAPD patterns [38]. The authors
concluded that reproducible RAPD results could be
achieved by appropriate optimisation of the RAPD
protocol. In another study, the RAPD assay was
evaluated as a genotypic method for typing clinical
strains of Propionibacterium acnes [39]. After optimi-
sation, all isolates were typeable using the optimised
RAPD protocol which was found to be highly
discriminatory and reproducible. The authors con-
cluded that typing of P. acnes by optimised RAPD is a
valuable tool for the epidemiological investigation of
this species for which no other widely accepted method
currently exists. Finally, after RAPD optimisation,
Guclu et al. [40] also showed that various Sarcocystis
spp. were detected and differentiated in cattle [40].
Initially, when we performed the first RAPD reactions
(back in 1996) we encountered the problem of
reproducibility in positive controls as well as the
presence of bands (whose complexity depended on the
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
primer concentration) in negative controls. Ultraviolet
radiation treatment of each of the PCR constituents
(except the enzyme) did not improve the situation. DNA
exposed to a high level of UV cannot be amplified [41]
due to the presence of DNA photoproducts such as
thymine dimers which seriously hamper the processivity
of DNA polymerases [42]. In this way, contaminated
DNA is prevented from acting as template for
amplification. In addition, the major bands in negative
controls displayed poor reproducibility in comparison to
the profiles generated in positive controls. Thus, these
results suggested that in the absence of DNA template
spurious amplifications occurred as a consequence of
non-specific reactions such as primer-mediated poly-
merisation. Our first objective was thus to find the optimal
conditions which are required for the generation of
reproducible RAPD profiles from different individuals of
the same species. The parthenogenetically produced
offspring of the clonal organism, Daphnia magna, an
important tool for ecotoxicological studies, was chosen
in order to eliminate the potential confusion caused by
on
genomic changes due to sexual reproduction. However,
the optimisation work was also performed using various
members of the bacterial, plant and animal kingdom
prior to evaluation of the impact of environmental
rs
contaminants.
2.3.2. Effect of the annealing temperature on
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profiles
Despite having potential for many applications, the
arbitrary nature of the RAPD method has drawn criticism
owing to the low stringency conditions. Relatively low
annealing temperatures (34–36 8C) are used in RAPD to
ensure a maximal number of primer binding events and
r's
consequent generation of a large number of amplified
DNA fragments for analytical purposes. However, the
low stringency of the accompanying DNA hybridisation
o
can result in the formation of spurious amplifications
[20,21,24]. In this context, it was essential to significantly
th
increase the stringency of the RAPD reactions by
sequentially increasing the annealing temperature and/or
by using DMSO, a solvent which proved useful in this
Au
respect [43,44]. In the absence of DNA target, DMSO
reduced the spurious amplifications [41]. However,
DMSO could not be used in our PCR reaction because
it also decreased the performance of the PCR reactions in
the presence of genomic template. With selected primers,
reproducible DNA profiles, consisting of a large number
of amplified fragments with a heterogeneous size range,
could be produced at an annealing temperature of 50 8C
under optimised PCR conditions [33]. This contrasts with
the study of Williams et al. [2] who reported that
81
annealing temperatures above 40 8C prevented amplifi-
cation by most of the 10-mer primers tested. The purity
and yield of the reaction products depend on several
parameters, one of which is the annealing temperature. At
both sub- and super-optimal annealing temperature, non-
specific products may be formed, and the yield of
products is reduced [45]. Other studies have also reported
that RAPD patterns and related profiles can be generated
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at high annealing temperature (48–52 8C) [20,31,32] for
short primers. The main advantage of high stringency
conditions is that non-specific reactions are significantly
reduced. Thus, protocols that use a high annealing
co
temperature should always be preferred if the sensitivity
of polymorphism detection is not jeopardised. Studies
criticising the AP-PCR for its lack of reproducibility are
not reported in the literature probably because high
stringent conditions (annealing temperature of 60 8C) are
used. This keeps the production of non-specific reactions
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to a very low level. In this context, the users of the RAPD
assay are really advised to use high annealing
temperature such as 50 8C for 10-mer primers.
2.3.3. Influence of each PCR chemical on profiles
As clearly demonstrated by previous studies [30,46],
the influence of DNA purity on the success of PCR
reactions was also evident in our experiments. DNA of
good purity, free from other macromolecules and
possible inhibitory compounds produced clear and
diagnostic RAPD profiles. Indeed, the intensity of an
amplified band depends on the efficiency of the
interaction of the genomic sequence with the primer
during the initial steps [25]. In this context, it is likely
that when DNA is not properly purified, the proportion
of cell components increase and interfere with the PCR
events (e.g. annealing of the primers) and reagents (e.g.
primers and Taq DNA polymerase). For the bulk of our
experiments, genomic DNA prepared by a standard
phenol/chloroform extraction purification was suffi-
cient to produce RAPD profiles of good quality.
Identical patterns were obtained whether DNA was
highly purified (caesium chloride treated DNA) or not
(phenol/chloroform extracted DNA) [41]. When PCR is
performed to amplify a single copy of genomic DNA, it
is not always necessary to extract and purify the
genomic DNA [47,48]. However as RAPD amplifies
multiple loci, it is recommended to carefully extract
DNA although RAPD profiles can also be obtained
without purification of the genomic DNA [41,49]. For
instance, in our hands, the repeatability of the assay was
affected when D. magna was boiled and it was very poor
when the water flea was homogenised and diluted in
analytical grade water [41].
82 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
The concentration of DNA template is also crucial in
the production of reproducible genomic profiles, not only
to ensure the largest number of amplified bands and
therefore maximum discrimination, but also to confirm
the fidelity of the PCR reaction condition. If profiles from
the same genomic template, or from different individuals
of the same species vary, the reproducibility of the assay
should be confirmed by repeating the PCR reaction using
two different template concentrations, differing by at
least two-fold. If profiles still vary, then the results should
always be treated with scepticism. This safeguard against
concentration dependent spurious amplifications was
highlighted by Welsh et al. [25], but unfortunately has not
been adopted by many workers using the RAPD
technique. To check the reproducibility of a RAPD
profile, we generally use 5 and 20 ng of genomic DNA.
Our optimal range for genomic DNA (0.05–100 ng) is
generally in agreement with other studies [30–32].
However, it is important to recognise that variations in the
estimation of the DNA concentration of the same sample
by two experimenters may also be an important factor
on
depending on the method used. Furthermore, in our
hands, it was also clear that identical RAPD profiles were
obtained whenever the DNA extraction or PCR were
performed [41]. In another study, it was reported that
rs
DNA extractions at different times and amplification of
the same DNA on different days produced minor bands
that varied in intensity [30].
pe
The priming oligonucleotide is obviously crucial to
the success of any RAPD protocol, and our experiments
support the judicious choice of primer for each
application and DNA template. Nevertheless, in our
studies a set of 11 primers, have been successfully
applied to RAPD profiling of each genomic DNA
r's
template we have studied, covering the bacterial, plant
and animal kingdoms [33] (and Fig. 2). In this context,
this set of primers shows the potential to generate
o
RAPD profiles with any genomic DNA of sufficient
quality. Khandka et al. [21] reported that the optimal
th
concentration of every primer should be determined
empirically for each application. Our data do not
support this statement because RAPD profiles of high
Au
quality were obtained using different primers and
species, under the same optimised conditions [33].
With regard to the magnesium concentration, it has
been suggested that a relatively low magnesium ion
concentration prevents undesirable annealing events
within the PCR [50]. Our results however, showing
satisfactory reproducibility with Mg++ concentrations
between 3–6 mM, support other studies which recom-
mend the use of higher concentrations to improve the
stability of the RAPD patterns [30,31]. It was also
reported that 2.5 units of Taq DNA polymerase gave
more reproducible results compared to low concentra-
tions (e.g. 1 unit) [30,41]. Interestingly, Singh and Roy
[51] revealed that detection of mutation in the genome
of malignant tissue compared to normal tissue by
RAPD/AP-PCR depends upon the type of DNA
polymerases used (see Section 4).
In addition, one of our major goals was to minimise
py
the presence of spurious amplifications (primer poly-
merisation) generated in the absence of template DNA
[19,52]. By varying the dNTP concentrations between
otherwise identical reaction mixtures, it was shown that
co
‘dirty’ negative controls could be greatly reduced. A
dNTP concentration of 0.33 mM produced a large
number of bands in reactions containing template DNA
with a complete or partial reduction in spurious
amplifications in the reactions without genomic DNA
templates. When the RAPD methodology is used,
al
negative controls are not always presented in the
literature probably because of the presence of products
in reactions containing no target DNA. The thermo-
dynamic driving force for PCR is the molar excess of
reagents with respect to template. This situation leads to
the formation of primer dimers and other artefacts
which consume the stock of primers and enzyme with a
consequent reduction in the yield of the appropriate
products. Although methods such as the Booster PCR
[53] and the Hot-StartTM PCR [54] have been developed
to tackle these problems, these procedures are
performed within the heating block of the thermocycler,
it is not very practical and there is a considerable risk of
sample contamination.
2.3.4. Concluding remarks on the optimisation
procedure of the assays
In some studies, researchers reported that some PCR
parameters (e.g. magnesium concentration and anneal-
ing temperature) should be optimised for each species
and primer combination separately [55]. Our results
suggested that there was no need to have different PCR
conditions for each species and primer combination as
RAPD profiles of satisfactory quality were generated
using different species and 10-mer primers [33] (and
Fig. 2). Finally, for those interested in performing quick
optimisation, the Taguchi method can be used [56]. The
method is used for development trials during industrial
process design. The method has the advantage of
investigating the effects of a number of variables and the
interactions between them in a single experiment
containing a few reactions. For instance, Cobb and
Clarkson [57] used the Taguchi method to optimise the
RAPD reactions [57]. Although this method is useful, it
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102 83

seems however that more experiments are required to
fully optimise the RAPD parameters.
In conclusion, in our experience, the RAPD protocol
was found to generate high quality genomic DNA
profiles from phylogenetically different groups of
organisms (bacteria, plants and animals) using the
same set of primers [33]. Since RAPD reactions are
performed at a high annealing temperature, spurious
amplifications are kept to a minimum and consistent
profiles are generated when component concentrations
are within the predefined optimal range, contrary to
PCR reactions performed at low annealing temperature.
Thus, after rigorous optimisation, the RAPD method is
a reliable assay. Table 2 describes our optimal
conditions for each parameter. Finally, when optimisa-
tion is performed it is highly recommended to generate

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co
al
on
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o r's
th
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Fig. 2. RAPD profiles of (A) Daphnia magna, (B) Escherichia coli, (C) calf thymus, and (D) human placenta. Primers as indicated at the top of each
gel. Lanes 1 and 2: 20 and 5 ng DNA, respectively. M and M0 : 1 kb DNA ladder from BRL and Igi, respectively. The molecular sizes (kb) are
indicated on the left of each gel. Please see [33] for more details.
84 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102

py
co
al
on
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pe
o r's
th

Fig. 2. (Continued ).
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as many bands as possible. The more bands that are
visible in the fingerprint, the less likely it is that the
profile will be influenced by the presence or absence of a
particular product [25].
3. The use of RAPD in genetic ecotoxicology
There is a great concern about the effect of
environmental contaminants on the genetic make-up
of natural populations. One class of genetic effects
includes alterations to the structure and function of
DNA including DNA adducts, DNA breakage, and
mutations as a result of chemical exposure (genotoxic
effects). However, indirect genetic effects can also arise
as a consequence of the interactions of genotoxic agents
with DNA. The RAPD and similar technologies have
been used to detect not only DNA damage and
mutations but also changes in genetic diversity and
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
Table 2
Optimised conditions (range) to produce good quality RAPD profiles at high annealing temperature
PCR parameters
Annealing temperature
Mg++ concentration
dNTP concentration
Primer concentration (10-mer)
Units of native Taq DNA polymerase units
Amount of DNA
PCR buffer
Bovin serum albumin
Thermal cycling conditions
The conditions, we have currently used in our experiments are given in parenthesis.
gene frequencies. However, most of the studies indicate
that the observed changes in RAPD profiles which
occurred among the exposed population were the result
of either DNA damage and mutations or population
genetic effects (see below). For instance, Theodorakis
et al. [58], who has documented population genetic
effects, gave a number of explanations such as the low
frequency of genotoxin-induced mutations and the low
probability of the RAPD method to detect rare
mutations [58]. In addition, Theodorakis et al. [58]
stated that a band could be amplified even if many of the
primer binding sites were damaged [58]. However, we
have clearly demonstrated the impact of diverse DNA
damages on RAPD profiles under in vitro conditions
pe
[59] and the reversibility of RAPD profiles after
recovery experiments (considering that population
genetic effects are not reversible) [60]. Nevertheless,
it seems likely that, in these studies, the RAPD
technique detected genetic diversity as well as direct
DNA effects. It is however difficult to estimate or
r's
differentiate the real contribution of DNA damage,
mutations and population genetic effects.
o
3.1. AP-PCR and RAPD to detect DNA damage
and mutations (genotoxicity investigations)
th
The AP-PCR assay has been used for the detection of
mutations and DNA damage. For example, AP-PCR was
Au
used to detect g-ray-induced genetic damage in fish
embryos [61,62]. The methodology was also used to
investigate mutations in blood DNA sampled from fishes
exposed to N-methyl-N0 -nitro-N-nitrosoguanidine [63].
Lopez et al. [64] also used the AP-PCR technique to
analyse genomic damage in the mutagen-sensitive mus-
201 mutant of Drosophila melanogaster [64]. Another
study also using D. melanogaster revealed that the AP-
PCR assay was successfully used to detect genomic
instability that increased when there was partial or total
85
Optimised conditions
50–54 8C (50)
3–6 mM (3)
0.33–0.44 mM (0.33)
1.0–4.0 mM (2.0)
2.0–3.0 U (2.0)
0.05–100 ng (1–20)
py
1  (10 mM Tris–HCl pH 8.8 at 25 8C, 50 Mm KCl, 0.08% Nonidet P40)
2.5 mg
First cycle: 95 8C for 4 min, 39 cycles: 95 8C for 1 min, 50 8C for 1 min,
74 8C for 1 min, Last cycle: 95 8C for 1 min, 50 8C for 1 min, 74 8C for 10 min.
co
deficiency in DNA mismatch repair [65]. A more recent
study using the AP-PCR assay clearly indicates that
proliferating cell nuclear antigen (PCNA) is an important
factor to maintain genomic stability in germ line cells in
al
D. melanogaster [66]. For more details, please refer to the
review concerning the use of AP-PCR to detect exposure
to genotoxic chemicals [67] although in our view, the
on
review in fact deals with RAPD and not AP-PCR.
The first study measuring genotoxic effects using the
RAPD assay was performed by Savva et al. [68]. In this
study, the RAPD profiles generated from rats exposed to
rs
benzo(a)pyrene revealed the appearance and disappear-
ance of bands in comparison to control patterns [68].
These changes observed in the fingerprints of exposed
animals may be due to the presence of DNA adducts,
mutations or DNA strand breaks [68]. Since, the RAPD
method was also successfully used to detect ‘DNA-
effects’ induced by benzo(a)pyrene [69–71], metals such
as lead, manganese, cadmium and copper [72–74],
mitomycin C [75], 4-n-nonylphenol and 17-ß estradiol
[76], UV, X-ray, gamma radiations [77–81] and radio-
nuclides [82]. DNA effects include DNA damage (e.g.
DNA adduct, DNA breakage) as well as mutations (point
mutations and large rearrangements) and possibly other
effects (e.g. structural effects) at the DNA level that can
be induced by chemical or physical agents that directly
and/or indirectly interact with genomic DNA. Finally, it
is worth remembering that the RAPD assay is a
qualitative or at best a semi-quantitative method and
the nature and amount of DNA effects can only be
speculated unless changes occurring in RAPD profiles
are analysed (e.g. cloning, sequencing, probing).
In genotoxicity studies, the current approach
adopts comparison of RAPD profiles obtained from
control and treated population at a defined time (e.g.
after a few days of exposure). However, the RAPD
assay can be successfully used in kinetic studies.
Recently, we have further evaluated the potential of the
86 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
RAPD assay to measure benzo(a)pyrene [B(a)P]-
induced DNA changes, and repair (in kinetic experi-
ments) as well as transgenerational effects in the water
fleas, D. magna [60]. Qualitative and quantitative
changes were observed in RAPD profiles generated
not only from the B(a)P exposed Daphnia but also from
previously treated organisms during recovery experi-
ments (suggesting that the DNA effects were fully
repaired or reversed). In addition, some of the B(a)P-
induced RAPD alterations detected in parental D. magna
were also observed in the offspring patterns. This
suggested that DNA alterations that occurred in germ
cells were transmitted to the next generations. In
conclusion, this study shows that the RAPD assay can
be useful to qualitatively assess the kinetics of DNA
changes, repair and transgenerational effects. Other
studies have further analysed the changes occurring in
RAPD profiles obtained from the treated population.
For instance, the RAPD assay was used to identify
several markers which revealed genetic differences
between contaminated and reference western mos-
on
quitofish populations [82]. Further analysis of these
markers by Southern blot showed that they were highly
conserved DNA sequences suggesting that these loci
may play an important role in molecular process [83].
rs
3.2. RAPD to measure population effects (changes
in genetic diversity or gene frequencies)
pe
As already mentioned, indirect genetic effects can
arise as a consequence of exposure to physical or
chemical stressors on the genetic composition of the
population, in terms of genetic variability or the
distribution of allele frequencies [84]. These effects
r's
can lead to an increase in mortality, an impairment in
reproduction and, possibly, to genetic bottlenecks or the
introduction of novel selective pressure in a hetero-
o
geneous population [85,86]. Consequently, this could
result in an alteration in genetic variability or allele
th
frequencies [87,88]. The RAPD method was used to
detect genetic diversity among populations which had
been exposed to environmental contaminants, including
Au
well-known genotoxins [58,84,89–91]. Nadig et al. [89]
reported that fish populations in the contaminated sites
were consistently less genetically distant from each other
than they were from each of the reference sites [89]. The
results of another study suggested that RAPD based
measures of genetic diversity may be suitable for
development as a sensitive means of directly assessing
the impact of environmental contaminants upon ecosys-
tems [90]. Finally, Theodorakis et al. [58] indicated that
the probability of survival and degree of DNA strand
breakage in radionuclide-exposed mosquitofish were
dependent on RAPD genotype, and were consistent with
the hypothesis that the contaminant-indicative RAPD
bands were markers of loci which imparted a selective
advantage in radionuclide-contaminated environments
[58].
The RAPD assay has been used to determine the
population genetic structure of Littornia littorea along a
py
pollution gradient [91]. The results suggested that
selection, rather than bottleneck effects, induced by
the polluted area are responsible for the genetic
patterning. Bagley et al. [92] also used the RAPD and
co
AFLP (amplified fragment length polymorphism) to
measure genetic diversity in populations exposed to
pollutants. However, due to lack of reproducibility of the
RAPD assay, they have chosen the AFLP methodology
for future work. Another study revealed as well that
agrochemicals (mainly triadimefon and ammonium
al
bicarbonate and their metabolites) affected soil microbial
community diversity at DNA level detected by the RAPD
assay [93].
3.3. Linking genotoxic and population effects
Substantial progress has been made in the last two
decades to evaluate the impact of physical and chemical
genotoxins in aquatic organisms at different level of
biological organisations, from the molecular to the
population level. Recent advances in our attempt to
evaluate these effects in aquatic organisms have been
reviewed by Jha [94]. While the majority of the studies
utilising molecular techniques have attempted to cover
the areas of gene expression following exposure to
specific environmental contaminants (e.g. endocrine
disrupting agents), adaptation, phenotypic plasticity,
multixenobiotic resistance and effects of so-called
chemosensitizers in the aquatic organisms, not much
efforts have been directed towards using these techniques
to evaluate direct effects of environmental contaminants
on the DNA.
In some of our studies, we have compared the
sensitivity of the RAPD assay with some fitness
parameters [69,72,78]. It was thus necessary to quantify
the changes in RAPD profiles to make a comparison with
the fitness parameters. In order to do these comparisons,
each separate DNA effect observed in RAPD profiles
(disappearance of bands, appearance of new bands and
variation in band intensities) was scored +1. The genomic
template stability (%) was calculated as ‘100  (100(a/
n))’ where ‘n’ is the number of bands detected in control
DNA profiles and ‘a’ the average number of changes in
DNA profiles [69,72,78]. To compare the sensitivity of
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
each parameter (genomic template stability with other
fitness parameters), changes in these values were
calculated as a percentage of their control value (set to
100%). It is proposed that alterations to RAPD profiles
due to genotoxic exposure can be regarded as alterations
in genomic DNA template stability, and that this measure
of genotoxic effect can be directly compared with
changes in key Darwinian fitness parameters. Our data
clearly suggest that genomic template stability can be
more sensitive than growth parameters and of at least
equal or even greater sensitivity than other fitness
measures obtained in B(a)P or copper exposed D. magna
[69,72]. In another study, the sublittoral macroalgae
Palmaria palmata was exposed to both ambient and
elevated irradiances of UV-B [78]. The results showed
that the genomic template stability was as sensitive as
other parameters including changes in chlorophyll
fluorescence, in vivo pigment absorbance, and thallus
growth. Some other rare studies (but not using RAPD)
have also measured effects induced by genotoxins at
different level of biological organisations. For instance,
on
genotoxic, cytotoxic and developmental effects of tri-n-
butylin (TBT) were measured in Platynereis dumerillii
and M. edulis [95,96]. Following these studies, the
toxicity of TBT to adult organisms was further deter-
rs
mined using a suite of biomarkers designed to detect
cytotoxic, immunotoxic and genotoxic effects [97].
Traditionally, genetic ecotoxicology studies have
pe
either focused on the direct effects of genotoxins on
DNA or on genetic diversity due to indirect effects.
However, it is clear that using both approaches would be
advantageous for three reasons according to Theodorakis
[98]. Firstly, at the population level, concurrent responses
between changes in population genetic structure and
r's
elevated levels of DNA damage may provide evidence
that the population genetic changes are influenced by
exposure to genotoxic chemicals. Secondly, if the
o
frequencies of alleles or other genetic markers differ
between genotoxicant-contaminated and reference popu-
th
lations, associations between relative amount of DNA
damage and genotype may provide evidence that these
changes are due to genotoxicant-induced selection.
Au
Thirdly, genetic analysis of gene flow may provide
insight into patterns of dispersal that could obscure
differences between contaminated and reference popula-
tions.
4. RAPD and AP-PCR to detect genomic
instability in carcinogenesis studies
Numerous studies have already shown that the
RAPD or AP-PCR could be used as powerful tools to
87
detect genomic instability in cancerous cells. It is worth
mentioning as well that the amplified bands usually
originate from unique sequences rather than from
repetitive elements. Furthermore, the amplification is
semi-quantitative, in that the intensity of an amplified
band is proportional to the concentration of its
corresponding template sequence [25]. The degree of
aneuploidy of a tumour cell genome is reflected in the
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intensities of bands, compared to those from normal
diploid genome from the same individual. In this
context, by carefully adjusting the concentration of the
template DNA, it is possible to detect losses or gains in
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the number of copies of a sequence by changes in the
intensity of a band [25]. This property is very useful in
many studies, such as the investigations of the genetic
events that occur in the process of transformation from
normal to cancer cell. The strategy consists in
comparing the RAPD profiles obtained from normal
al
and cancerous cells obtained from the same individual.
In this context, the potential genetic diversity that can be
observed among individuals is bypassed. The RAPD
and AP-PCR (as well as related techniques) are very
likely to detect genomic instability, because the
cancerous cell will produce a clone of dividing daughter
cells. This won’t be the case e.g. when an organism is
exposed to a genotoxin or when the % of altered cells is
low and may not be detectable if below about 2% [79].
Recently, Risques et al. [99] discussed the different
approaches (including AP-PCR) that have been used to
assess cumulated genetic alterations of colorectal
cancer [99]. The authors suggest that future studies
should address the analysis of a large series of samples
with a wide array of techniques in order to provide
enough information to establish associations between
the different types of genomic damage, the clinico-
pathological parameters of the tumours and the survival
of the patients [99].
4.1. AP-PCR to detect genomic instability in
malignant cells
To our knowledge, Peinado et al. [100] were the first to
use the AP-PCR methodology to detect somatic genetic
alterations in tumours of the colon and rectum [100]. AP-
PCR bands showing decreased and increased intensities
in tumour tissue DNA, relative to normal tissues, have
been cloned after re-amplification with the same arbitrary
primer. RFLP and Southern blot analyses showed that
these DNA sequences have undergone allelic losses and
gains, respectively, in the tumour cell genome. The
authors also conclude that the AP-PCR provides the basis
for an alternative molecular approach to cancer
88 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
cytogenetics. In 1993, Ionov et al. [101] successfully
used the AP-PCR assay to show that 12% of colorectal
carcinomas carry somatic deletions in poly(dA.dT)
sequences and other simple repeats [101]. They
concluded that these mutations reflect a previously
undescribed form of carcinogenesis in the colon
mediated by a mutation in a DNA replication factor
resulting in reduced fidelity for replication or repair. In
another study, Kohno et al. [102] have searched for novel
genetic alterations in small-cell lung carcinoma cell lines
by using the AP-PCR assay [102]. Similarly, high
incidence of allelic loss on chromosome 2q was detected
in colorectal carcinoma and neuroblastoma. This
suggested the presence of a novel tumour suppressor
gene at 2q33, which is involved in the development of
several human cancers. In summary, these first three
studies illustrated the usefulness of the AP-PCR assay to
detect allelic losses or gains [100], deletion of one or a
few nucleotides [101] and large deletions [102]. For more
details concerning the use of AP-PCR in cancer research,
please refer to the review of Navarro and Jorcano [103].
on
In the review, the authors highlight the advantages of the
AP-PCR assay such as the detection (i) of mutations, (ii)
moderate gains in DNA that cannot be detected by other
methodologies (iii) and cloning of alterations in a single
rs
experiment [103]. They also discuss some characteristics
of the AP-PCR assay and review some of its achieve-
ments in cancer research [103].
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More recently, Mukherjee et al. [104] used the AP-
PCR assay to determine genomic instability and
apoptosis (detection of DNA fragmentation) in brain
tumour cells [104]. In another study, three different
techniques, namely microsatellite markers, compara-
tive genomic hybridization (CGH) and AP-PCR, were
r's
applied to screen renal cancers for genetic alterations
[105]. The AP-PCR was also used to detect genomic
instability in 23 patients with B-cell chronic lympho-
o
cytic leukaemia [106]. Liang et al. [107] used the AP-
PCR assay to screen for altered methylation patterns in
th
genomic DNA and to isolate specific sequences
associated with these changes [107]. The use of
CGH combined with AP-PCR can be a successful
Au
approach to detect genetic abnormalities [108–110].
The AP-PCR has also been used to detect genomic
instability in a glioblastoma cell line [111], hepato-
cellular carcinoma and fibrolamellar carcinoma (a
variant of hepatocellular carcinoma) [112,113], med-
iastinum fibrosarcoma [114], primary mediastinal B-
cell lymphoma [108,109], MALT lymphoma in
stomach [115], breast [116,117], lung [118–120] and
gastric carcinomas [121]. Interestingly, Salem et al.
[122] used a methylation-sensitive (Ms) AP-PCR
technique to scan genomic DNA in colon and bladder
cancer cells for differential methylation patterns and
identified a hypermethylated band corresponding to a
highly conserved transcription regulatory factor
involved in embryogenesis ([122] but see also
[107]). Such approaches adopting AP-PCR technique
are important as the mechanisms underlying de novo
methylation of CpG islands in human cancer remain
py
almost completely unknown.
4.2. RAPD to detect genomic instability in
malignant cells
co
To our knowledge, the first studies using the RAPD
assay for the detection of genomic instability in brain
and lung cancer were performed by Dil-Afroze et al.
[123] and Ong et al. [124], respectively. The detected
alterations in brain tumor DNA included the loss of a
al
normal band, appearance of a new band and amplifica-
tion of a pre-existing band [123]. The authors concluded
that the RAPD assay which covers the range (0.4–2 kb)
is very complementary to existing DNA fingerprinting
methods, such as microsatellite mapping (less than
0.4 kb) and Southern blotting (over 2 kb) for detecting
genetic alterations in tumors. More recently, the RAPD
assay was used to assess the overall genomic instability
in liver cancers in transgenic mice [125], hepatocellular
carcinoma [126–128] and oral squamous cell carcino-
mas [129,130]. In other studies, Singh and collaborators
[131,132] showed the ability of RAPD to detect DNA
sequences representing genetic alterations in stilbene
estrogen-induced cancer cells, including losses of
heterozygosity and homozygous deletion and insertion
mutations [131,132]. Singh and Roy [133] who also
studied genomic instability in breast cancer cells by
RAPD analysis showed that an insertion of a 1270 bp
amplified fragment was observed in 81% of the tumour
samples [133]. Finally, other studies have also
successfully used the RAPD assay to detect genomic
instability in primary human astrocytic tumors (glio-
mas) [134,135], pituitary adenomas [136], skin cancers
[137], colorectal tumors [138–140], head and neck
squamous cell carcinoma [141], mouse hepatoma cells
MH-22a [142], breast and Wilm’s human cancer tissues
[51,143], cervical intraepithelial neoplasia [144] and
silica- and cadmium chloride-transformed BALB/c-
3T3 cell lines [145].
4.3. Further analysis of RAPD/AP-PCR changes
Classically, the users of the RAPD or AP-PCR assay
compare profiles obtained from normal and malignant
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
cells. Any differences between the profiles are then
attributed to genomic instability. To determine the
molecular mechanisms underlying the basis of such
changes, it is clear that further investigations are required.
For instance, the relevant bands can be cut, re-amplified
and used as probe to screen the genome extracted from
control and cancerous cells. Such an approach has
allowed different studies to determine events, occurring
in the genome of the malignant cells, such as allelic losses
and gains [100], somatic deletions in poly(dA.dT)
sequences [101], allelic loss on chromosome 2q [102].
The analysis of such allelic losses in greater detail may
reveal the presence of a tumour suppressor gene or anti-
oncogene [116]. Thus, this could lead to the description
of new genes or possibly to genes with not well-
characterised functions. In the same way, the further
analysis of gains of chromosomes may reveal the
presence of oncogene [119] or more generally genes
implicated in the initiation and progression of cancers
[130]. Kuchiki et al. [114] demonstrated that detection of
DNA alterations by AP-PCR fingerprinting without any
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previous knowledge of the genes and subsequent analysis
of radiation hybrid panels could lead to easy identifica-
tion of candidates for genes involved in carcinogenesis
[114]. In another study, the cloning and sequencing of
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RAPD amplified loci revealed that one mutated locus had
significant sequence similarity with the hamster Cyp1A1
gene [131]. Similarly, Singh and Roy [133] showed that
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an extra band which appeared in RAPD profiles of
malignant cells showed a significant DNA base sequence
similarity (93%) with one of the breast tumour-specific
human expressed sequence tag [133]. Misra et al. [134]
reported the identification, cloning and characterization
of a RAPD amplified fragment which was lost in a glioma
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[134]. The band was found to be homologous with a
group of sequences, which are probably derived from the
human endogenous retrovirus-K (HERV-K). Finally,
o
more recently, the RAPD assay revealed a gain of
approximately 400 bp in oral squamous cell carcinomas
th
[130]. The analysis of the fragment revealed the presence
of three genes and one of the genes, namely the
AKAP220 which plays a role in regulating the Rb
Au
pathway, was overexpressed in the malignant cells [130].
It is hoped that such studies will further stimulate
research in this area involving changes in AP-PCR/
RAPD fingerprints to determine the genes implicated in
carcinogenesis processes.
Chromosome assignment of fingerprint bands is
essential for molecular karyotyping of cancer by AP-
PCR DNA fingerprinting. This can be done by cloning
and in situ hybridization. However, Yasuda et al. [146]
have developed a technique for the simultaneous
89
chromosomal assignment of multiple human DNA
sequences from DNA fingerprints obtained by AP-PCR
[146]. Radioactively labelled human AP-PCR products
are hybridized to DNA fingerprints generated with the
same arbitrary primer from human/rodent monochro-
mosome cell hybrids after electroblotting to a nylon
membrane. This method is called simultaneous hybri-
dization of AP-PCR DNA fingerprinting products
py
(SHARP). The subchromosomal localisation of AP-
PCR bands can be mapped using radiation hybrids
panels [147]. For instance, using this approach which
represents a molecular tool of high resolution for cancer
co
cytogenetics, Malkhosyan et al. [147] have generated in
this manner a molecular karyotype of metastatic colon
cancer [147]. This amplotype shows that sequences
from several chromosomes undergo both losses (1, 4, 9,
14, and 18) and gains (6, 7, 12, and 20) in over half of
the tumours. These reports suggest that molecular
al
cytogenetic studies complement AP-PCR/RAPD work.
In conclusion, non repairing or misrepairing of DNA
lesions such as double strand breaks, inter-or intra-
strand cross links cause genomic instability that may
lead to development of malignancy [148]. Tumour
development and progression involve multiple genomic
alterations including deletions, rearrangements, ampli-
fications and point mutations of relevant genes.
Genomic instability is a key characteristic of malignant
cells. The RAPD assay and related techniques such as
the AP-PCR represent excellent tools to screen the
genome of malignant cells for many reasons. Firstly,
these methodologies can detect most types of mutations
that occur in tumours as well as gain in DNA. Secondly,
the detection and cloning of DNA alterations is possible
in a single experiment. Thirdly, no information on the
genomic DNA under study is necessary. Fourthly, it is
relatively inexpensive and no special equipment is
required. Finally, further analysis of the relevant bands
allows not only to identify some of the molecular events
(e.g. allelic losses and gains) implicated in the genomic
instability but also to discover genes playing key roles
in the initiation and development of malignancy.
5. Comparison of RAPD assay with other
methodologies
5.1. Discrimination of closely related strains of
bacteria with the RAPD assay
Renibacterium salmoninarum is an obligate patho-
gen of salmonid fish. There is no effective vaccine or
chemotherapy against this organism which causes a
bacterial kidney disease (BKD), which can be fatal
90 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
under the appropriate conditions [149]. This bacterium
appears to possess remarkable biochemical uniformity
and no reliable serological means of distinguishing
between isolates has been found [150]. A study of 40
isolates of R. salmoninarum from North America in
which multilocus enzyme electrophoresis was used
indicated that the level of genetic diversity was low
[151]. There are a variety of DNA-based methods
available (such as the 16S–23S rRNA intergenic spacer
(ITS)) [152] for differentiating between isolates, strains,
and species of bacteria. An alternative method to
distinguish among the bacteria is to use the RAPD
assay. Our data revealed that RAPD analysis is a better
method for discriminating between isolates of R.
salmoninarum [8,9]. In our study, R. salmoninarum
isolates from a variety of sources, some with identical
16S–23S spacer region DNA sequences, could be
distinguished on the basis of RAPD patterns. Pre-
viously, R. salmoninarum had defied attempts to find a
reproducible way to differentiate among isolates and
our study was the first one which revealed the genetic
on
diversity among isolates. In another study, RAPD
analysis of strains of Bacillus cereus revealed also a
remarkable diversity which was not revealed by rRNA
or tRNA ITS-targeted PCR [153].
rs
5.2. Microarrays and RAPD
pe
In the field of gene expression analysis, DNA
microarray technology is having a major impact on
many different areas including toxicology [154] and
ecotoxicology [155,156]. In the future, DNA chips with
probes corresponding to the chromosomes will prob-
ably be available facilitating the detection of genomic
r's
alterations. Will the researchers still use intensively the
RAPD assay in the context of these high-tech
methodologies? The answer is probably yes for at least
o
two reasons, apart from being relatively cheaper. First
of all, the RAPD assay screens the whole genome and
th
principally non codant DNA regions, contrary to
microarrays and SNP which study changes at the gene
level. In addition, the simultaneous detection and
Au
cloning of the alterations would be difficult to achieve
with DNA arrays. Thus, it seems that the RAPD assay
would remain a popular choice, complementing newly
emerging technologies.
5.3. Comparison with other genotoxicity and
mutagenicity assays
It has been suggested that the RAPD assay can detect
mutations if they occur in at least 2% of the tested DNA
[79]. Indeed, Jones and Kortenkamp [79] demonstrated
that a polymorphic band corresponding to mutant DNA
sequences began to become apparent at a concentration
of 1/50 of non-mutant DNA [79]. This is in agreement
with some of our preliminary data [41]. Nevertheless,
mutations and DNA damage can only be detected by
RAPD if they occur at specific loci. Interestingly,
genotoxins are also known to interact with genomic
py
DNA at specific sites [157,158] which lead to hot spot
DNA damage and potentially to hot spot mutations
(point mutations and genomic rearrangements). Indeed,
point mutations can be produced after replication of a
co
damaged base [159]; moreover, DNA damaging agents
contribute to the instability of the genome by
introducing recombination-prone sites at DNA leading
to extensive chromosomal lesions and rearrangements
of genes and their regulatory sequences [160,161].
Compared to other mutagenicity tests such as the Ames
al
test or the hprt (hypoxanthine phosphoribosyl transfer-
ase) test in mammalian cell lines, it is clear that the
RAPD assay is less sensitive [79]. However, in these
well-validated mutagenicity assays, a relatively small
target is investigated whereas RAPD examines muta-
tions in the whole genome. Until now, there has been
very little data evaluating the potential of RAPD assay
to detect genotoxic effects with some well-known
genotoxicity tests. Data from Castano and Becerril [71]
support the sensitivity of the RAPD technique for the
detection of benzo[a]pyrene-induced DNA damage.
Indeed, a concentration of 0.05 mg/L of B(a)P was
sufficient to induce changes in RAPD profiles in fish-
derived cells whereas 0.1 mg/L of B(a)P was required to
detect micronuclei [162], chromosome aberrations
[163] or alterations in the variation coefficient DNA
content in the G1 phase [164]. In a recent study, the
genotoxic, cytotoxic and developmental impact of
tritiated water (0.37–370 kBq/ml) were investigated in
the embryo-larvae of marine mollusc Mytilus edulis
[81]. Genotoxic effects were assessed using cytogenetic
and molecular approaches which included the induction
of sister chromatid exchanges (SCEs), chromosomal
aberrations (Cabs) and RAPD. The data revealed that
Cabs and SCEs were significantly different from the
control at all doses and at the three highest doses,
respectively. Some RAPD changes were also detected at
all doses when compared to control profiles. Thus, in
our study, the RAPD assay is more sensitive than the
detection of SCEs and as sensitive as the detection of
Cabs [81]. Finally, Becerrill et al. [75] have suggested
that the RAPD technique is very useful for studying
genetic alterations in fish cells because most accepted
genotoxic assays cannot be performed due to the large
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
number and small size of fish chromosomes, as well as
the long cell cycle associated with most fish species
[75].
The assessment of exposure levels of genotoxic
agents and the genetic consequences requires the
application of a set of inter-related techniques capable
of providing both qualitative and quantitative informa-
tion [165]. However, when little or no reliable
information is available on the mode of action of a
potential genotoxin, the RAPD can be particularly
advantageous in comparison to quantitative methods.
For instance, earlier studies have failed to prove the
presence of oestrogen-DNA adducts [166] and oestro-
gens were found to be negative in short-term assays for
the induction of gene mutations, irrespective of whether
this was measured in prokaryotic [167] or eukaryotic
cells [168,169]. Some of our data clearly revealed that
DNA effects were generated by oestrogens [76]. It is
now well established that a wide range of DNA damage
and mutations are induced by oestrogens [170]. In
addition, RAPD may be especially useful for gathering
on
evidence of genotoxicity in the field particularly when
no chemical studies have been performed on a site and
to assist in the recognition of clean and polluted areas.
In the field of ecotoxicology, methods such as 32P-
rs
postlabelling [171,172] and the Comet assay [173,174]
are widely used. In eco-genotoxicity studies, to
elucidate the potential genotoxic effects of environ-
pe
mental contaminants, a powerful strategy could be
firstly to use the RAPD assay as a screening method and
secondly to use more specific methods measuring for
instance DNA adducts, gene mutations or cytogenetic
effects. A question may be raised whether RAPD
method compare favourably with these well established
r's
methods? We have shown that one B(a)P adduct in
1.5  105 normal bases (as detected by the 32P-
postlabelling) had significant effect on RAPD profiles
[59]. However, since the 32P-postlabelling method can
o
detect DNA adducts at concentrations as low as one
adduct in 109–1010 normal nucleotides [175,176], it is
th
clear that other comparative data are necessary
(especially with low amount of DNA adducts) to
Au
compare the sensitivity of both RAPD and 32P-
postlabelling methods. It is however anticipated that
the 32P-postlabelling assay is probably more sensitive
than the RAPD methodology.
Overall, our data suggest that RAPD is a sensitive
assay for the detection of genotoxin-induced changes at
the DNA level. Indeed, alterations in RAPD profiles
were obtained after exposure of diverse aquatic systems
to low concentrations of physical and chemical
genotoxic agents under in vivo conditions (e.g. mussel
91
embryo larvae exposed to 0.37–370 Bq [81], algae
irradiated to 1.3 W/m2 UV-A [78], larvae barnacles
exposed to 0.1–10 mg/L 4-n-nonyl phenol or 10 mg/L
17b-estradiol [76], Daphnia exposed to 12.5 mg/L
B(a)P [69] or 15 mg/L copper [72]. Nevertheless,
studies comparing the RAPD method with routinely
used genotoxicity/mutagenicity assays are necessary to
further evaluate the potential of the RAPD assay for the
py
detection of DNA damage and mutations.
6. Recommendations in the context of
genotoxicity studies
co
If the RAPD assay is used in genotoxicity studies, the
choice of the species and level of genomic diversity
among individuals of the same species are of
fundamental importance. Indeed, it is necessary to
know the level of genomic diversity among individuals
al
of the same species before launching any genotoxicity
studies. Based on visual inspection of the RAPD
patterns presented in Fig. 3, the data indicate that the
reproducibility of RAPD profiles (among individuals)
was high when the algae, P. palmata and the worm, P.
dumerilii were used. However, this was not the case
when the mussel, M. edulis was employed. The data
revealed also that the specificity of the mussel RAPD
profiles was confirmed by using two different con-
centrations of DNA which gave similar patterns with
different primers. Originally, we have mainly used
clonal organisms (Daphnia magna) to tackle the
problem of reproducibility. Depending on genetic
diversity in individuals within a species, different
strategies can be applied to measure the effects of
induced DNA alterations and mutations. If the RAPD
patterns generated by individuals of a species are
reproducible, then the profiles obtained from an
exposed population of the same species can be directly
compared to control patterns. On the other hand, if
individuals produce truly polymorphic profiles, then a
non destructive method must be used [177]. For
instance, it may be possible to obtain haemolymph or
blood samples from an organism before and after
exposure to environmental contaminants. In this case,
RAPD profiles generated can be compared and the
differences can be attributed to the direct or indirect
effects of the contaminants.
A practical problem associated with the use of RAPD
is its application under field studies in particular after
pollution accidents. A convenient strategy could be to
look at DNA effects in diverse aquatic invertebrates
(e.g. mussels, crabs) and vertebrates (e.g. fish) collected
from uncontaminated sites. Non-invasive biological
92 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102

material (e.g. haemolymph, blood) could be individu-
ally collected from each of the species before
introducing them in polluted and reference sites. Acute
and long term DNA effects could then be evaluated by
sampling biological material from these species at
intervals or as per the requirement to generate RAPD
profiles along with other quantitative genotoxicity
assays (e.g. Comet assay, micronucleus test) for
7. Interpretation of changes in RAPD profiles
7.1. Effects of DNA damage and mutations on
profiles
This section provides an overview of the possible
effects of DNA damage and mutations on RAPD
profiles and attempts to identify some of the factors

py
comparison. which may induce changes in RAPD profiles. A

co
al
on
rs
pe
o r's
th
Au

Fig. 3. Reproducibility of RAPD profiles among three marine species: (A) Palmaria palmata, (B) Platynereis dumerilii, and (C) Mytilus edulis.
Primers as indicated at the top of each gel. M: 1 kb DNA ladder (BRL). The molecular sizes (kb) are indicated on the left of each gel. Each number
represents a single animal. (D) RAPD profiles reproducibility generated by four M. edulis individuals (used in Fig. 3C) using 20 and 5 ng DNA.
Lanes 1–4 and 10 –40 : 20 and 5 ng genomic DNA, respectively. PCR reactions were performed under the optimised conditions [33]. Please see [41] for
more details.
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
on
rs
pe
r's
Fig. 3. (Continued ).
o
summary of the possible effects of DNA adducts, DNA
th
breakage, and mutations (point mutation and large
rearrangements) on RAPD patterns is displayed in
Table 3 and Fig. 4. Table 4 also gives a summary of the
Au
changes on RAPD profiles when different effects were
generated under in vitro conditions [59]. The presence
of DNA damage and mutations within a primer binding
site will alter the DNA fingerprint that is obtained from
undamaged but otherwise identical DNA. However,
such a situation is expected to arise less frequently than
changes in RAPD patterns which occur as a conse-
quence of the direct effect of DNA damage within the
DNA contained between the primer binding sites
(Table 3). The main reason for this is that primer
93
py
co
al
binding sites are small (10 bp) compared to the
intervening DNA of the amplicon (maximum 3–
4 kb). The direct effect of DNA lesions (e.g. when
the Taq DNA polymerase encounters a type of DNA
damage) is expected to have a greater influence on the
RAPD profiles than point mutations, because such
mutations need to arise in the primer binding site to
directly affect the patterns. This statement does not take
into consideration the fact that the structural effects of
point mutations may induce some changes in profiles
(please see below). It has to be stressed that the
stringency of the conditions may greatly influence the
results; the higher the stringency, the more likely it will
be to detect a single base change within the priming site.
94 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102

Table 3
Direct effect of DNA damage and mutation on RAPD profiles
DNA alterations Consequences Effects on RAPD profiles Probability to
detect the
lesion by RAPD
!By pass ! processivity affected !Decrease in band intensity/band loss Medium
!By pass ! processivity not affected !No effect
DNA adduct !Block ! dissociation enzyme adduct !Band loss Low
!Block ! dissociation ! more free Taq !Increase in band intensity Medium

py
!Block ! no dissociation !Band loss Low
!No primer/DNA association !Band loss Low
(adduct within the priming site)
DNA breakage

co
!Block ! dissociation enzyme DNA !Band loss Low

!No primer/DNA association !Band loss Low

!Creation of a new annealing site !Appearance of band Low

al
!Loss of pre-existing annealing site !Band loss Low
Rearrangement
!New priming site !Appearance of band High
on
For more details please refer to the text. *: DNA adduct; : DNA breakage; : Taq DNA polymerase; : DNA synthesis; : point
mutation; : 10-mer primer.
rs
pe
o r's
th
Au

Fig. 4. Structural effect of DNA damage and mutation on RAPD profiles. For more details please refer to the text. *: DNA adduct, *: mutation,
: DNA breakage, : 10-mer primer, : exponential amplification, : linear amplification, - - -: DNA synthesis, &: arrest of the
DNA synthesis, : hairpin loop, +: appearance of a band, and -: disappearance of a band.
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
Table 4
Summary of the consequences of diverse types of DNA effects on RAPD profiles
DNA effects Changes in RAPD profile
Disappearance of bands
Non random DNA Major event
breakage (restriction)
Random DNA Major event (high molecular
breakages (sonication) weight bands)
Benzo(a)pyrene Major event (mainly high
induced adducts molecular weight bands)
Pyrimidine dimers Major event (mainly high
molecular weight bands)
Random mutations No
Change in primer sequence
All DNA effects were generated under in vitro conditions except for the creation of random mutations (see [59] for more details).
When the Taq DNA polymerase encounters a DNA
adduct, there are a number of possible outcomes
including blockage, by-pass and the possible dissociation
of the enzyme/adduct complex which will cause changes
on
in RAPD profiles (Table 3). The other direct effects on
RAPD profiles caused by DNA breakage, mutation, and
rearrangement are also illustrated in Table 3. A breakage
which occurs in the DNA template between two opposite
rs
primers may result in a loss of an amplicon whereas
genetic rearrangements and point mutations may be
responsible for either a loss or creation of new annealing
pe
sites which could result in the disappearance or
appearance of new amplicons, respectively.
According to Jones and Kortenkamp [79], a poly-
morphic band corresponding to mutant DNA sequences
began to become apparent at a concentration of 1/50 of
non-mutant DNA [79]. As a result, a new band can appear
r's
if at least 2% of the DNA is affected, as mentioned earlier.
In terms of probability of occurrence, rearrangements are
the main factors influencing RAPD profiles and
o
particularly those leading to band appearance
(Table 3). Point mutations can also influence RAPD
th
patterns but the probability of having point mutations in
the priming sites is low in comparison to the occurrence
of rearrangements. DNA damaging agents contribute to
Au
the instability of the genome in terms of chromosomal
lesions and rearrangements [160,161] which would
eventually lead to the appearance or disappearance of
bands in the RAPD profiles. Variation in band intensities
due to DNA adducts are also likely to be detected by the
RAPD assay (Tables 3 and 4). In addition, events such as
gene amplification and DNA methylation are also likely
to affect RAPD profiling. Finally, it is worth mentioning
that the combination of all events at the DNA level
reported in Table 3 (e.g. DNA adducts, breaks, point
95
Appearance Decrease in Increase in
of bands band intensity band intensity
No No No
Minor event Major event Minor event
py
Minor event Major event Minor event
No Major event Minor event
co
No No No
New RAPD profile
mutations and rearrangements) will lead overall to even
al
greater changes in RAPD patterns than those due to
individual lesions.
DNA damage and mutation could also induce
structural changes in the DNA template which could
lead to changes in RAPD patterns. During PCR some
regions of the genomic DNA, such as GC rich sequences
with strong secondary structures, may not be suffi-
ciently denatured to allow amplification to proceed. An
example of the structural effects induced by DNA
damage and/or mutation is illustrated in Fig. 4. After
RAPD amplification the control genomic DNA pro-
duces two amplicons, bands A and B. It is also shown
that not all the 10-mer-primers will lead to exponential
amplification (i.e. primer 2 and 5) due to the presence of
strong secondary structure in the form of a hairpin loop
in the control genomic DNA (Fig. 4). For example, as a
result of the structural rearrangements a new product is
synthesised (band C) whereas amplicon B is lost
(Fig. 4). DNA damage such as bulky adducts [178,179]
and mutations [180] are known to induce structural
changes. For instance, Bowditch et al. [180] reported
that mutations which stabilise or destabilise the
secondary structure may be detectable as RAPD
polymorphisms even though they do not reside within
the primer binding site [180]. In these cases, the RAPD
assay potentially can detect DNA damage and muta-
tions which affect secondary structure over a much
greater area than that covered by the primer binding
sites. This also suggests that the sequence of the primer
binding site, which is small in comparison to the
intervening DNA, is frequently unchanged in RAPD
polymorphisms [180]. Finally, the results from one of
our studies led us to suggest that, in addition to
chemical-induced DNA damage and mutations, factors
96 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
such as variation in gene expression, steady levels of
genetic alterations and changes in metabolic processes
could induce some changes in RAPD patterns [70].
Nevertheless, DNA damage and mutations appear to be
the main factors influencing RAPD patterns [70]. The
successful application of the RAPD method to detect
DNA alterations and mutations is related to the fact that
the genotoxin-induced DNA damage does not occur
randomly [158]. Hot spot interactions between geno-
toxins and genomic DNA have often been reported
[181,182] and in these cases the same structural changes
will be induced in most of the damaged cells. This
situation is very likely to allow the detection of DNA
damage and mutations by the RAPD method.
7.2. How to improve the interpretation of the RAPD
assay and related techniques
As mentioned in Section 7.1, the interpretation of the
changes in RAPD profiles in genotoxicity studies is not
always easy since DNA damage and mutations can both
on
affect RAPD patterns. Thus, it would be helpful to
develop strategies to determine the cause of the RAPD
changes. After comparison of RAPD profiles generated
from the control and the treated population, a further
rs
step could consist in comparing the RAPD assay
generated with the control and DNA subjected to
different physical or chemical treatments. Such treat-
pe
ments could be for instance the repair of DNA lesions.
Thus, the persistent bands (different from control
profiles) could be attributed to mutations whereas the
bands that disappear (due to the in vitro treatment) could
be attributed to DNA damage. By comparing RAPD
profiles before and after treatment, a direct relation
r's
could be possible between bands and DNA effects.
Salem et al. [122] used a methylation-sensitive(Ms)
AP-PCR technique to scan genomic DNA for
o
differential methylation patterns and identified a
550 bp band that was hypermethylated in a majority
th
of colon and bladder cancer cells [122]. This
technique relies on digesting genomic DNA with
methylation-sensitive and insensitive restriction
Au
enzymes prior to AP-PCR amplification. Matched
normal and tumour DNAs were compared to identify
differential methylation. Using this technique, novel
CpG islands were found to be frequently hypermethy-
lated in bladder and colon tumours [107]. The users of
the Ms AP-PCR have demonstrated that this technique
is a rapid and efficient method that can be used to
screen for altered methylation patterns in genomic
DNA and to isolate specific sequences associated with
these changes.
8. RAPD optimisation and interpretation of the
data: a case study
Weinberg et al. [183] reported a major increase in
mutation rate in the children of Chernobyl liquidators as
a result of their fathers’ exposure to ionizing radiation
[183]. The authors used the RAPD and the interSSR-
PCR methodologies to draw such conclusions. However,
py
Jeffreys and Dubrova [184] claimed that the data was
invalid because the RAPD is an unreliable technology
[184]. According to them, these mutants were not
validated and had no obvious molecular basis. They may,
co
instead, have arisen as PCR artefacts or through non-
paternity or sample mix-up. They recommend that,
unless these mutants can be validated, Weinberg et al.
[183] withdraw their remarkable claims. As pointed out
in Section 2.3, it is clear that after rigorous optimisation,
the RAPD assay is a reliable technology. Unfortunately,
al
in Weinberg’s paper, no information was provided
concerning RAPD optimisation. In addition, before
using a source of DNA, it is highly recommended to
understand the variation of RAPD patterns among
control samples (see Section 6 for more details). In this
case, this means that the systematic analysis with the
RAPD assay of control human samples from different
families (pedigree study with mother, father and
children) would have certainly helped. In addition,
Weinberg et al. [183] did not use two DNA concentra-
tions that differ by at least a factor of 2 to check the
reproducibility of the patterns, as recommended by
Welsh et al. [25]. However, Weinberg et al. [183] claim
that each of the RAPD changes was checked by running
three independent PCR runs [183]. If it is the case, it is
likely that the extra bands are real but we believe that the
problems might come from the interpretation related to
these bands. As already mentioned in this review, in
pedigree studies, non-paternal bands could be due to
heteroduplex molecule formation between allelic RAPD
products [24]. The problem is particularly evident when
the RAPD was used to assess paternity in primates
according to Riedy et al. [23] who reported a high
frequency of non-parental bands from primates [23].
Although Riedy et al. [23] did not mention anything
about optimisation and did not use two DNA concentra-
tions, it is possible that some of the bands were due to
heteroduplex formation between allelic RAPD products
[24]. Other explanations concerning the appearance of
the non parental bands are also described by Jeffreys and
Dubrova [184].
It is possible therefore that some of the non parental
bands reflect DNA mutations in germ cells. Never-
theless, this means that the frequency of mutations is
 F.A. Atienzar, A.N. Jha / Mutation Research 613 (2006) 76–102
probably lower than originally calculated by Weinberg
et al. [183] due to some confounding factors as
mentioned above. This case clearly indicates that when
RAPD is used, it is necessary to be as careful as possible
by (i) optimising RAPD reactions, (ii) knowing the
potential variation among control samples, (iii) using 2
DNA concentrations that differ at least by a factor 2
(when variation is observed or not) [25], and (iv)
interpreting the changes in RAPD profiles (bearing in
mind that most of the time diverse factors can have an
impact on the RAPD assay).
9. Conclusion
In conclusion, whilst RAPD and related techniques
(e.g. AP-PCR) have been used extensively for diverse
studies, application of these techniques has also attracted
criticisms with respect to its reproducibility, despite
several advantages. However, it emerges that most of the
criticism relates to lack of proper optimisation and
validation of the techniques in different cell types and
on
species, prior to their applications under in vivo or in vitro
conditions. Nevertheless, the RAPD assay and related
assays offer great promise especially for the determina-
rs
tion of genetic damage under in vivo conditions in wild
species and for the evaluation of genomic instability in
the process of carcinogenesis. Whilst a large number of
pe
new technologies and assays are developing to profile the
gene expression pattern either following exposure to
environmental contaminants or during the process of
malignant development, the RAPD based techniques
offer great promise for future and would continue to
complement other new and well-established techniques
in population genetics, genotoxicity and carcinogenesis
r's
studies.
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