JOURNAL OF PATHOLOGY, VOL.

178: 239-248 (1996)

REVIEW ARTICLE

MICROSATELLITES AND PCR GENOMIC ANALYSIS

JOHN KORETH, JOHN J. O'LEARY AND JAMES O'D. MCGEE

University of Oxford, Nujield Department of Pathology and Bacteriology, The John RadchfSr Hospital,
Oxford OX3 9D U, U.K.

SUMMARY
Microsatellites form a significant proportion of the growing family of repetitive DNA sequences, widely dispersed in the human
genome. Due to their ubiquity, PCR (polymerase chain reaction) typability, Mendelian co-dominant inheritance, and extreme
polymorphism, microsatellites have assumed an increasingly important role as markers in the genome. Apart from their obvious
applications in genome mapping and positional cloning, these markers have been applied in fields as disparate as tumour biology,
personal identification, population genetic analysis, and the construction of human evolutionary trees. Microsatellites are associated with
human disease, not only as markers of risk but also directly in disease aetiopathogenesis, providing new insights into nowMendelian
inheritance; the replication, repair, and mutation of eukaryotic DNA; the regulation of gene transcription; and protein-protein
interactions. These insights have resulted in novel paradigms for oncogenesis and neurological disease.
KEY worms-microsatellite; STR; PCR; genome; loss of heterozygosity; RER; evolution

INTRODUCTION (kb). Minisatellites are found in euchromatic regions of

Microsatellites' (also called simple sequence length
polymorphisms: SSLPS;~ and short tandem repeat poly-
morphisms: STRs3) can be defined as tandem arrays of
short stretches of nucleotide sequences, usually repeated
between 15 and 30 times.4 Some workers have made a
distinction between microsatellites [2 base pair (bp)
repeats] and short tandem repeats (STRs) (3-5 bp
repeat^),^ but for the purposes of this review, they will be
considered together. This review will comprise three
sections: an overview of the current 'state of the art'
regarding microsatellite origin, evolution, distribution,
and function; a discussion of methodological consider-
ations for microsatellite PCR analysis; and applications
of microsatellite analysis, with emphasis on conceptual
advances and clinical applications.
MICROSATELLITE ORGANIZATION
Microsatellites belong to the family of repetitive non-
coding DNA sequences, which can be classified as
follows. (i) Satellite sequences: arrays with repeat sizes
ranging from 5 to 100 bp, characteristically organized in
clusters up to 100 megabases (Mb). These are located in
the heterochromatin near chromosomal centromeres
and telomeres and are not as variable in size within
populations as the other members of this family.c8 (ii)
Minisatellite sequences: arrays with repeat sizes of
15-70 bp which range in size from 0.5 to 30 kilobases
Addressee for correspondence: James O'D. McGee, University of
Oxford, Nuffield Department of Pathology and Bacteriology, The
John Radcliffe Hospital, Oxford OX3 9DU, U.K.
CCC 0022-341 7/96/030239-10
0 1996 by John Wiley & Sons, Ltd.
the genome and are highly variable in repeat size within
population^.^,^ (iii) Microsatellite sequences: arrays with
a repeat size of 2-6 bp, highly variable in size but
ranging around a mean of 100 bp. Microsatellites are
found in the euchromatin and allele sizes in populations
characteristically exhibit multiple size classes distributed
about the population mean.1°
Microsatellites were initially identified in eukaryotic
genomes as stretches of dT-dG alternating sequence
with varying length.11J2 Subsequent reports demon-
strated the PCR typability of dT-dG.dC-dA dinucleo-
tide repeat microsatellites and showed the Mendelian
co-dominant inheritance of the size polymorphisms. l , I 3
In order of decreasing abundance, dA, dA-dC, dA-dA-
dA-dN, dA-dA-dN, and dA-dG repeats were identified
as the most frequent sequence motifs in human micro-
~ate1lites.I~ Disregarding (dA), multimers, whose size
polymorphisms are difficult to type by PCR, (dC-dA),
microsatellites are estimated to number between 35 000
and 100000 copies in the human genome-a marker
density of approximately one microsatellite every
100 000 bp, even by the most conservative estimate^.'^.'^
It must be noted that although widely distributed,
microsatellites are not uniformly spaced along chromo-
somes, being underrepresented in subtelomeric
regions.16 The informativeness (polymorphic infor-
mation content or PIC) of dinucleotide microsatellites
increases with increasing average number of repeats,
PIC values ranging from 0 for 10 or fewer repeats to
0.8 for 24 or more repeats.I5 The human genome is
estimated to contain approximately 12 000 (dC-dA),
microsatellites with P I 0 0 . 5 (7000 of which have
PIC 2 0.7). I5 Tri- and tetranucleotide microsatellites
have been identified at a frequency of 1 every 300-
Accepted 3 October I995
240
500 kb on chromosome X (theoretically, they should
number 400 000 with repeat number >7 in the genome;
a frequency of 1 every 10 kb).' About half of the
microsatellites in the study were polymorphic and their
informativeness correlated with repeat number.
The origin and function of repetitive sequences is not
clear at present. The initial occurrence of short repeat
sequences could be due to chance (in a random sequence
the probability of a [dC-dA], repeat is 1/256), or they
could have arisen as mutations from the poly (dA),
sequences at the 3' end of adjacent Alu sequence^.'^^'^
The selective prevalence of (dC-dA), repeats can be
explained by the methylation of dC residues at 5'dG-
dC3, sequences normally present in the genome.
Methylated dC residues can be deaminated, producing a
transition of dC to dT. This process can lead to an
increased abundance of 5'dG-dT3' motifs and their
complementary 5' dC-dA3' motifs.17 Subsequently, the
expansion of the repeat can occur due to strand slippage
during DNA replication, creating length polymorphisms
differing by a few repeats at a time. Additional sequence
motifs may then arise by mutation of the expanded
d C 4 A repeat^.'^,^^ The frequencies at which changes in
repeat number occur at microsatellite loci are much
higher than normal mutation rates, ranging from l o - *
to l o - ' per g e n e r a t i o ~ i . ' ~Di
. ~ Rienzo
~ et aI.l9 report
that a step-wise mutation model, involving a frequent
change in repeat number by one or two repeats at a time,
and rare large changes in repeat number, represents two
distinct classes of mutation at microsatellite loci. This
slippage synthesis model can explain the relation
between defective DNA repair and microsatellite
instability seen in some disease states (discussed
below).
It was initially thought that repeat sequences pos-
sessed a functional role in the genome, either directly via
a role in gene regulation2() or indirectly as hot spots for
recombination,21 their mutagenic potential enhancing
the long-term evolutionary potential of the species.
While, in general, no definite function can be ascribed to
microsatellite sequences, in specific instances CAG tri-
nucleotide repeats are transcribed (to polyglutamine
tracts; discussed below); DNA binding proteins specific
to di- and trinucleotide repeats have been identified**
and one of the repeats may act as a site of nucleosome
assembly in i~itro.*~ Recent thinking emphasizes the
'selfish' role of repetitive sequences (reviewed by
Charlesworth rf ~ 1 . * ~in) , light of the fitness loss that
they cause the organism. It may be that in the majority,
these sequences are maintained solely by their ability to
replicate and expand in the genome, within the limits
established by the negative selection pressure of the
fitness loss that they may cause.
MICROSATELLITE PCR
The PCR amplification of microsatellite repeats con-
forms broadly to the general principles of genomic DNA
amplification described by Saiki.25Like standard PCR,
the reaction mixture comprises sample DNA (template),
two oligonucleotide primers, the four deoxynucleotides,
J. KORETH ET AL.
a buffer containing magnesium chloride, and the DNA
polymerase, all combined in a single tube. Methodologi-
cal considerations peculiar to microsatellites (especially
dinucleotide repeats) include the following:
Template DNA
DNA can be extracted from a variety of pathological
materials including formalin-fixed tissues, paraffin-
embedded archival blocks, histopathological and cyto-
logical smears, and aspirate^.^^^^^ Extraction protocols
range from proteinase K digestion followed by repeated
phenol chloroform extraction and ethanol precipitation
to simply boiling tissue sections.28.29 Usually the
extraction method is determined by the type of clinical
material available. This often results in template DNA
of variable quality and low target copy number. Such
DNA degradation results in a limitation on the fragment
size consistently amplifiable (<400 bp for paraffin-
processed material).30 Microsatellite PCR under these
circumstances is often demanding and reactions need to
be empirically optimized with each new primer pair,
even when using published parameters as a starting
point.
Primers
Litt3' recommends the use of 20mers with a base
composition in the range of 35-55% GC, trying to match
the %GC of the two primers to within 5 per cent.
Additional precautions need to be taken against primer
complementarity, secondary structure, and homology to
Alu consensus sequences, which are often near micro-
satellite^.^^,^^ Siting primers as close to the microsatellite
repeat as possible minimizes the size of the allelic
fragments, promoting efficient amplification and rapid
fragment separation and scoring. In practice, primer
sequences are often accessed from published reports, or
electronically from public databases like the Genome
Database (GDB). These sequences often differ in length
and %GC composition from the above criteria, though
differences in primer length usually lead to correspond-
ing T, values for the two primers. Optimal annealing
temperatures range from 3°C to 12°C above theoretical
T,, values and require empirical optimization. Primer
concentrations of between 0.1 and 0 . 3 , are ~ ~generally
suitable for microsatellite PCR, the individual value
being optimized for each primer pair."
dNTPs
Deoxynucleotide concentrations between 20 and
100 p~ each (individually optimized for each primer pair
for the balance between yield, fidelity, and specificity)
are suitable. These concentrations are lower than the
standard concentrations (200 , u M ) , ~ due
~ to the greater
specificity and fidelity required for microsatellite PCR .
PCR buffers
We find that easiest approach to individual optimiz-
ation is to prepare six 1OX buffers ( 1 0 0 m ~Tris-HC1,
 MICROSATELLITES AND PCR CENOMIC ANALYSIS
500mM KC1, 1 per cent Triton X-100) at two magne-
sium concentrations (15 and 30 mM) and three pH values
(pH 8.0, 8.5 and 9.0). Simultaneous amplification under
the same cycling conditions with these six buffers is a
useful first step in optimization. The addition of
adjuncts like bovine serum albumin, glycerol, forma-
mide, and ammonium sulphate have been reported to
enhance the yield and specificity in some cases and may
be attempted if the first optimization step is not wholly
sati~factory.~~
Instrumentation
For successful, reproducible microsatellite PCR, ther-
mal cyclers need to provide high ramp rates and uniform
temperatures across the block, with well-to-well tem-
perature variation of less than 0.5”C. The use of 0.2 ml
thin-walled tubes and total reaction volumes as low as
1Opl enhances heat transfer and reduces thermal lag.
Thermal cycling parameters
The scoring of allelic fragments can be severely
affected by the presence of spurious bands due to
mispriming (i.e., the cross-annealing of primers to non-
target sequences). It is therefore important to use
annealing temperatures that are as high as possible and
to reduce extension times to the minimum, given the
known nucleotide incorporation rate of the DNA
polymerase (35-100 nucleotides per second at 70-80°C
for Tuq p ~ l y m e r a s e )and
~ ~ the size range of the allelic
fragments. ‘Hot start’ PCR,33-35 where the template,
dNTPs, and primers mix is heated to 80-85°C before
adding an essential component of the reaction such as
Tuq polymerase, and ‘heat soaked’ PCR,36 involving
incubation of the DNA template at 94°C for 30 min
before adding Tuq polymerase, dNTPs, and primers,
are recommended as means of increasing specificity,
especially with low copy number or poor quality
template. Similarly, ‘touchdown’ PCR,37 with high
initial annealing temperatures, reduced by 1-2°C in
each successive cycle, has been suggested as a means
of bypassing the need to optimize thermal cycling
parameters individually for each primer set3*
Detection and scoring of microsatellites
Highly sieving gel electrophoresis (usually through
polyacrylamide gels) is the standard means of resolving
PCR products. It has been suggested that the most
reliable and unambiguous approach to typing micro-
satellites is to probe the PCR products with a specific
repeat oligonucleotide, so that only the specific
sequence-containing products are d e t e ~ t a b l e but
, ~ ~ this
is required mainly for reaction optimization. A problem
common to all detection methods, especially with
dinucleotide repeats, is the presence of additional
(‘stutter’) bands beside the microsatellite band, differing
by 1 or 2 bp. The major mechanism postulated for this is
‘slipped strand mispairing’ (akin to slippage synthesis
mechanisms for microsatellite size expansion and
sequence evolution), the multiple repeats permitting
24 1
slippage of the copied strand on the template, producing
fragments with two-nucleotide spacing. Other postu-
lated mechanisms are: failure of the polymerase to
read through the repeats, the 3’ terminal addition of
nucleotides by the polymerase, and the different rates of
migration of the (dC-dA), and (dGAT), strands (when
both strands are labelled).30~40
Radioactive and non-radioactive methods are used
for detection.
(a) Radioactive methods-Radiolabelling has tra-
ditionally been the ‘gold standard’ for the detection and
quantitation of PCR-amplified microsatellites, either by
the direct incorporation of a single labelled deoxy-
nucleoside triphosphate like [a-32P]dCTPduring thermal
cycle (internal labelling) or a single 5’ [ Y - ~ ~ P I ~ A T P
end-labelled primer in the PCR mix (end labelling).
While the internal labelling method is easier to perform
and more sensitive, the end-labelled approach minimizes
additional bands and produces cleaner results. The
products are resolved on sequencing gels, fixed, dried,
and autoradiographed, with or without intensifying
screens. To ensure response linearity, it is important that
X-ray film is preflashed to O.D. 0.10-0.20 at 540 nm and
autoradiographed at - 7 0 T , whenever intensifying
screens are used.41 With weak /I-emitters (e.g., 35S),
fluorography using an organic scintillant offers increased
sensitivity, but similar considerations of pre-exposure
and low temperatures apply.41 Optical densitometry is
now standard for quantitative analysis of PCR products.
( b ) Non-radioactive methods
(i) Ethidium bromide
Resolution of products on acrylamide gels, either
non-denaturing4* or denaturing ‘ s e q ~ e n c i n gtype
’ ~ ~ gels,
followed by ethidium bromide staining is the simplest
means of visualization. The disadvantages include low
sensitivity (only > 10 ng of double-stranded DNA can
be detected)44 and subjective quantitation, unless
additional image processing is ~ n d e r t a k e n . ~ ~
(iij Silver staining
The use of silver stains to visualize DNA offers
advantages of sensitivity over ethidium bromide (detect-
ing pg quantities of DNA in a sequencing format) and
has been used for qualitative assessment of microsatellite
allelic bands.45 However, there are problems with
variable background and non-linear deposition of silver.
While microsatellite allelic quantitation by silver stain-
ing is reported in the l i t e r a t ~ r ein
, ~our
~ experience, in a
non-denaturing polyacrylamide format, band intensity
on silver staining does not correlate well with radioactive
RFLP quantitation, unlike ethidium bromide staining
(unpublished data).
(iiij Fluorescence
The advantage of analysing multiple polymorphic loci
by using an automated DNA sequencer was described
by Skolnick and Wallace in 1988.47In 1992, Ziegle et ul.
reported the use of automated DNA sizing technology
for genotyping microsatellite loci, using a four-colour
242 J. KORETH ET A t .
fluorescence technique.48 In this method, fluorescent
phosphoramidites are linked to the 5’ end of one of the
primers in the PCR assay. The labels used include FAM
(blue), JOE (green), TAMRA (yellow), and ROX (red).
In addition, newly introduced fluorescently labelled
dNTPs (R110, blue; R6G, green; TAMRA, yellow) can
be used for internal labelling in the PCR. The PCR
products, fluorescently labelled, are separated on a poly-
acrylamide gel and detected when excited to fluoresce by
a laser. The fluorescence, appropriately filtered, is
detected by a photomultiplier or charged couple device
(CCD) camera and analysed by computer software. This
system, though expensive, offers considerable advan-
tages in terms of sizing accuracy (a size standard can be
run in the same lane), quantitation of amplified product
and response linearity (with a larger dynamic range
than autoradiography film), and enhanced throughput
(PCR multiplexing both during amplification and
electrophoresis). A recent report has examined the
reproducibility and sensitivity of fluorescent versus auto-
radiographic methods, has concluded that fluorescence
based technology is at least as accurate as standard
radiolabelling techniques, and recommends its use in
high-resolution genomic analy~is.~’
MICROSATELLITE APPLICATIONS
Genome analysis
An important goal of the Human Genome project is
to construct a physical map of the human genome,
consisting of unique genomic landmarks at an average
spacing of 100 kb. When completed (projected for 1998),
the map will consist of 30 000 marker loci, distributed
evenly throughout the genome.50 The National Insti-
tutes of Health (NIH) had an interim goal of a 2-5
centimorgan (cM) map by 1995, achieved with the 1994
Comprehensive Human Linkage Consortium (CHLC)
map.S1 Such dense maps provide an invaluable resource
of the localization and isolation of any human DNA
sequence of interest.
When genome maps were first mooted, restriction
fragment length polymorphisms (RFLPs) were proposed
as the DNA markers of choice by Botstein et u L . , ~ who
~ ies’, which test whether a disease and an allele show
suggested that 150 RFLP markers (a marker density of
20 cM, implying a recombination fraction 0=0.2) would
permit reliable detection of genes for dominant diseases.
Apart from their low heterozygosity, RFLPs have
the disadvantage of requiring the slow and laborious
techniques of Southern blotting. Also, since they do not
commonly associate with coding sequences in the
genome, RFLP maps only incorporate a small number
of cloned genes. Furthermore, the most informative
members of this class, VNTRs (variable number of
tandem repeats) or minisatellites tend to cluster near the
ends of chrom~somes.~’The obvious advantages of
microsatellites-high heterozygosity, ubiquity through
the genome (often adjacent to coding sequences, though
rarely transcribed), and PCR typability, combined with
ease of multiplexing-make them obvious candidates for
markers of choice in genome maps. However, since they
are underrepresented in chromosome centromeres and
Table I-Progress in human genome maps I‘’
Resolution
Year Group Marker Loci No. (CM)
1981 Keatslz7 Classical 53 16
1987 CRI”* RFLP 403 10
1992 Genethons4 CA 814 4.4
1992 NIH/CEPHs3 Mixed 1416 3.0
1994 Geneth~n’*~ CA 2066 2.9
1994 CHLC5’ Mixed 5840 0.7
telomeres, minisatellite data are complementary for uni-
form genome coverage by markers.s4 It is not surprising,
therefore, that there has been a sudden increase in the
number of microsatellite markers described in public
databases of the human genome. For instance, the
number of Genome Database (GDB) marker loci scora-
ble by PCR with heterozygosities exceeding 69 per cent
has increased from 989 in 199355to 3025 in 1995 (maps
listed in Table I). Concurrently, the 1994 CHLC human
linkage map lists 3617 microsatellite polymorphisms of a
total 5840 mapped loci, a marker density of 0.7 c M . ~ ’
An obvious application of such a map is the genetic
dissection of disease traits, using the tool of linkage
analysis. Owing to the enhanced density of microsatellite
markers, linkage analysis is now not limited to simple
(monogenic) Mendelian traits, more than 400 of which
have been mapped to date (reviewed by Lander and
S ~ h o r k ~but
~ ) has
, been applied to a number of geneti-
cally heterogeneous traits with some success, either
demonstrating linkage in a single pedigree, though not
in others (psoriasis and early-onset Alzheimer’s disease),
or applying a priori considerations in case selection
(mapping BRCAl to chromosome 17 q).S7-59
In addition to traditional linkage analysis, which tests
whether a chromosomal region shows correlated trans-
mission within a pedigree, mapping disease traits can
utilize alternative approaches such as ‘allele sharing’
methods, where one tries to prove that the inheritance
pattern of a chromosomal region is not consistent with
random Mendelian segregation and ‘association stud-
correlated occurrences in a population. Allele sharing
has been effectively used by Todd and colleagues in their
genome-wide study of type I diabetes, identifying 18
chromosomal regions with evidence for linkage, includ-
ing at least two and possibly three new genes (ZDDM3,
ZDDMS, and possibly chromosome 18 loci).6o Associ-
ation studies played a key role in implicating the apoli-
poprotein E gene in both late-onset Alzheimer’s disease
and heart disease and the angiotensin converting enzyme
gene in myocardial i n f a r ~ t i o n . ~ l - ~ ~
Allelic imbalance analysis
Tumour allelotyping, the genotypic analysis of all 23
pairs of human chromosomes for regions of interstitial
deletion, was introduced by Vogelstein et d h 5
Concep-
tually, it is based on Knudson’s paradigm of deleted
anti-oncogenes, validated by the cloning of tumour
 MICROSATELLITllS AND PCR GENOMIC ANALYSIS
Table II-Chromosome region losses associated with sporadic tumours
Lung' 3 3 * 135
Urinary tract
Renal134
Gastrointestinal system
Colorecta1133
Gastric' 33
Pancreas13z
Hepatocellular carcinoma'33
Salivary carcinomas'36
Male and female genital tract
C e r v i ~ a l37~ ~ . ~
Ovarian'38
Testicular 133
Prostate132
Skin
Melanoma'39
Nervous system
Gli~rna'~~
AstrocytomaI3'
Neur~blastoma'~'
suppressor genes like Rb at these deleted r e g i ~ n s . ~ " J j ~
Loss of heterozygosity (LOH), the loss of an allelic band
in tumour vis-a-vis constitutional DNA, is recognized as
indicative of putative tumour suppressor genes. Initially
RFLP-based, microsatellite PCR is now the method of
choice for most allelotyping studies. A further innova-
tion is the concept of 'allelic imbalance', developed by
Cawkell et U I . ,for
~ ~the analysis of tissue microdissected
from histopathological sections, where the quantitation
of the minute amounts of DNA extracted is not possible.
Since, in such circumstances, the differentiation of allelic
amplification from allelic loss is not feasible, the neutral
term 'allelic imbalance' (AI) is preferred. It is calculated
as a ratio of ratios, the numerator and denominator
being ratios of the intensities of the two allelic peaks in
the tumour and constitutional DNA. A pviovi cut-off
values, based on estimates of tumour cell load in the
sections, are used to define AI. At least 30 per cent
tumour cell load is required to identify AI/LOH in a
heterogeneous tumour specimen, though recent reports
using flow cytometry claim that LOH can be identified
in specimens with as few a s 10 per cent tumour cell^.^^,^^
Using microsatellite LOH/AI analysis in the quest for
new anti-oncogenes is a major focus of cancer research
(Table 11); for instance, the recent identification of a
novel locus on chromosome 1 lq,71deleted in two-thirds
of sporadic breast cancers (Koreth et al., in press).
Micvosatellites in human disease
( a ) Micvosatellite expansion in disease-Repeat
sequence mutation may be a common cause of human
disease, particularly those that follow dominant inherit-
ance. Initially, such associations were restricted to
243
IP, Iq, 3p, 6q, llp, llq, 13q, 16q, 17p, 17q
3p, 5q, 8p, Ilp, 13q, 17p
trinucleotide repeats,72but subsequently, associations of
multiple forms of cancer with either various unstable
simple tandem repeats, or rare alleles of the H-Ras
minisatellite repeat73 have been described. In myotonic
dystrophy (MD) and the fragile X syndrome, expansion
of repeats in sufferers has been d e ~ c r i b e d . ~This ~,~~
expansion is linked to parental copy number, which
tends to be at the high end of the normal range. It is now
believed that the repeat itself predisposes to mutation-a
cis process called 'dynamic m ~ t a t i o n ' ~ ~ - bvirtue
y of a
relationship between repeat copy number and instabil-
ity. In fact, with parental copy number >80, massive
expansion of repeats is seen in affected children. The
mechanism of mutation, as postulated by Richards and
S ~ t h e r l a n dis, ~distinct
~ for low and high copy number
repeats. Small repeat number alleles are less likely to
undergo major slippage during replication, as only
one single-stranded break is likely per allele (based on
Okazaki fragment lengths). Such a fragment, anchored
by unique sequences at the 5' end, undergoes minor
slippage to produce fragments altered by a few repeats.
Larger fragments (>80 repeats) can have two single-
stranded breaks. Such fragments, free at both ends, can
slip significantly to cause massive expansion. Alterna-
tively, the extruded CAG or CCG repeat strand could
undergo conformational changes, creating 'hairpin'
structures which permit massive slippage.77In the fragile
X syndrome, expansion of a CCG trinucleotide repeat
(>230 copies)78 in the 5' untranslated region of the
FMRl gene on chromosome X causes methylation at the
CpG residues both in the repeat and in the adjacent
FMRl promoter, stopping transcription of the gene-a
loss of function m ~ t a t i o n .A~similar
~ . ~ ~aetiology is seen
in myotonic dystrophy, where the degree of expansion of
244 J. KORETH ET AL.

Table 111-Microsatellites associated with human disea~e'~,*~

Repeat sequence Association Reference

Mononucleotide HPNCC 91, 142-144

Dinucleotide
Trinucleo tide
Dinucleotide Various human cancers 102
Trinucleotide
Tetranucleotide
CCG Fragile X syndrome
FRAXA 80
FRAXE 145
FRAXF 146

CAG Spinal and bulbar muscular atrophy 82

Myotonic dystrophy 14
Huntington's disease 147
Spinocerebellar ataxia (type 1) 148
Dentatorubral pallidoluysian atrophy 83, 149
RED-1 150
Machado Joseph Disease 151
Haw River Syndrome 152

non-coding 3' trinucleotide repeats is associated with
disease severity and age of ~nset~~-possiblydue to
preferential nucleosome assembly on the expanded
repeat sequence2' or the sequence-dependent shift to
'triad' DNA conformation,*' with subsequent impli-
cations for gene transcription and RNA secondary
structure. Conversely, in neurodegenerative disease (e.g.,
spinocerebellar atrophy, Kennedy disease), expanded
coding CAG repeats are translated as excessively poly-
glutaminated proteins-a gain of f ~ n c t i o n . * ~Poly-
.*~
glutamine-rich proteins may cause disease by gradual
cross-linkingx4 or aggregation by a polar zipper-like
mechanism.85 Similar mechanisms of gain of protein
function may be operative in most of the neuro-
degenerative disorders shown in Table I11 (reviewed by
Sutherland and Richards86). Interestingly, fundamental
developmental processes may be regulated by micro-
satellite size polymorphisms. Coward et aLx7 have
reported that CAG repeat number at a polymorphic site
in the open reading frame (ORF) of the sex-determining
gene Sry on the Y chromosome is correlated with the
degree of sex reversal in inbred mouse strains (reviewed
by Either**).
( h ) Microsatellite instability (MSI) in disease-
Truns-acting factors (proteins) also affect microsatellite
stability. Unlike the cis effects mentioned previously,
irans factors affect microsatellites across the genome and
tandem repeat instability is not causal in disease. These
factors were elucidated during the search for causal
mutations in hereditary non-polyposis colorectal cancer
(HNPCC), an autosomal dominant syndrome with a
predisposition to colorectal and endometrial cancers and
other epithelial t u m ~ u r s . In
* ~HNPCC kindreds, linkage
to the marker D2S123 on chromosome 2p was reported
and it was simultaneously noted that instability of
microsatellite repeat number was present in micro-
satellites scattered throughout the It is
estimated that the total number of mutations at micro-
satellite loci in replication error positive (RER+)
tumour cells could be up to 100-fold that in RER -
cells, suggesting a mutation affecting DNA replication
or repair predisposing to replication errors.92 Studies in
mutator mutants of Succhuromyres cerevisiae and
Escherichia coli showed that mutations in the mismatch
repair genes P M S l , M L H l , or MSH2 (S. cerevisiae) and
MutS (E. coli) produced such a p h e n ~ t y p e . ~ The
~.~~
proposed mechanism involved slippage of DNA
polymerases on the repeat motif during normal repli-
cation and subsequent correction of the frame shifts by
the mismatch repair complex, dysfunction of this repair
complex predisposing to the R E R + phenotype in a
recessive manner.95 Candidate genes in the region of
linkage on chromosome 2p were screened and the
mismatch repair gene hMSH2, identified by sequence
homology to the yeast MSH2 gene and its protein
product, was shown to be a DNA mismatch binding
p r ~ t e i n . ~Subsequently,
~-~~ additional mismatch repair
genes were shown to be involved in the pathogenesis
of HNPCC, comprising hMLH1, hPMSl, and
h PMS2.99.100Analysis of sporadic tumours belonging to
the HNPCC spectrum (colorectal cancer, endometrial
cancer, and gastric cancer) revealed a significant pro-
portion of cases with multiple replication errors, as in
the HNPCC cases.Io' In contrast, other sporadic
tumours (lung, breast, testis, CNS tumours, and soft
tissue sarcomas) showed MSI as a rare event, usually
only affecting a single microsatellite locus of the many
examined.'01~'02Recent reports show that subsets of
lung cancer (small cell cancer), cases with multiple
primary tumours, and late stages of CML may be
significantly associated with R E R + . I o 3 Io5 The
 MICROSATELLITES AND PCR CENOMIC ANALYSJS
microsatellite instability described in sporadic tumours
is thought to differ in aetiology from that seen in the
HNPCC spectrurn.lo6 What is still unclear is whether
these mutations target sequence repeats specifically or
whether the genomic instability has general effects, non-
specifically activating oncogenes and/or inactivating
tumour suppressor genes. Recent reports indicate a
specific inactivation of the TGF-P receptor gene (a
tumour suppressor) in RER+ colon cancer cell lines,
favouring the former possibility. Io7
Population studies and forensic applications
Population studies reveal that microsatellite alleles
conform to Hardy-Weinberg criteria and segregate in
a Mendelian fashion in fa mi lie^.^^^,^^^ Tri- and tetra-
nucleotide repeats (STRs) have been reported to amplify
more reliably, with fewer artefactual bands during
PCR.3 The de novo mutation rate for tri- and tetrameric
repeats ranged from 2.3 x l o p 5 to 15.9 x in a
five-locus study;Io8this is low enough for concerns about
new size mutations not to be a significant problem in
identity determination, especially with multiple-locus
analysis. Microsatellites, especially tri- and tetranucleo-
tide STRs, are therefore excellent candidates for per-
sonal identification systems in medical and forensic
applications and are admissible as evidence in courts of
law. Using fluorescent technology and PCR, as little as
100 pg of target DNA would suffice for amplification in
an STR system.’I0 STR/microsatellite systems have also
been shown to be more effective in personal identifi-
cation with degraded forensic samples than RFLPs with
or without amplifiable fragment length polymorphisms
(AMPFLPs).”’ A study analysing 14 distinct micro-
satellite loci with multiplexing determined that the prob-
ability of two unrelated individuals matching at all 14
loci less than 1 x 10-’4.112Important considerations
before reaching such conclusions include the need for
allele frequency data for differential racial groups and
population substructures, as well as procedural controls
like multiple stain extractions and serial dilutions of
extracted DNA to identify cross-contamination. l3.I l 4
The recent confirmation of the identity of the Romanov
family from skeletal remains by STR analysis demon-
strates the power of this approach.’l5
A number of markers of genetic diversity have been
utilized to construct a phylogenetic tree of human popu-
lations, in an attempt to reconstruct the history of
human mobility and evolution. Genetic similarity can be
interpreted as evidence of recent shared ancestry, though
it must be recognized that most of the genetic variation
in humans exists between individuals within races.] l 6
The markers that have been used include blood groups,
allozymes, RFLPs, and DNA sequences. Additional
phylogenetic data are now being derived from analysis
of mitochondrial DNA and microsatellites. Micro-
satellite analysis of allele frequencies in populations has
been used to determine ‘genetic distance’ between popu-
lations, the assumption being that genetic distance
between isolated populations increases linearly with time
if population sizes are constant;I17 if population sizes
vary, as is common, these rates will vary. Bowcock
245
et al. 1 1 8 developed a microsatellite-based phylogenetic
tree which showed an initial split between Africans and
all other populations. African populations showed the
highest level of microsatellite variability, supporting
evidence that suggests that African populations are the
oldest. However, these data do not resolve the debate
between the ‘multiregional’ and ‘out of Africa’ hypoth-
eses of human origin and geographical ~ariability.1’~
The question, to paraphrase Brookfield,120 is whether
the genetic divergence between human populations in
Africa, Europe, and Asia is due, at least in part, to the
ancient genetic diversity in ancestral Homo erectus and
H. neanderthalis populations which contributed genes to
the extant populations in these regions (the multi-
regional hypothesis) or whether new immigrants ‘out of
Africa’ completely replaced these earlier populations
without any genetic mixing. In other words, is there a
truly bifurcating phylogenetic tree at all and, if so how
can it be dated, given inconstant evolutionary dates?
Complementary methods, mitochondrial (Mt) DNA
analysis and Y-chromosome sequence analysis, suggest
that modern humans emigrated out of Africa 100 000-
200000 years ago and replaced ancient H. erectus
populations throughout the world.121-123
CONCLUSION
As this brief overview reveals, in the space of a few
years, microsatellite polymorphisms have assumed a
major role in genomic analysis, with applications as
diverse as positional cloning, tumour biology, human
evolution, medical diagnostics, and forensic identifi-
cation. The major factors for this popularity are their
informativeness, ubiquity in the genome, and PCR
typability. The eventual aim of developing a detailed
map of human genetic diversity down to the single
base-pair level will, however, require marker densities
several orders of magnitude higher than current maps.
Di-allelic polymorphisms are estimated to occur once
every 1000 bp in the human genome,52 compared
with microsatellite densities of one every 100 000 bp.
Systems recently developed to type diallelic mutations
(5’ nuclease PCR assays; TaqManm) offer a means of
fulfilling the ultimate goal of the human genome
initiative.124126Seen in that context, microsatellites are
an important step in reaching that brave new world.
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