REVIEW ARTICLE
Assessment of telomere length and factors that contribute
to its stability
Sabita N. Saldanha
1
, Lucy G. Andrews
1
and Trygve O. Tollefsbol
1,2,3
1
Department of Biology,
2
Center for Aging and
3
Comprehensive Cancer Center, University of Alabama at Birmingham,
University of Alabama at Birmingham, AL, USA
Short strands of tandem hexameric repeats known as
telomeres cap the ends of linear chromosomes. These repeats
protect chromosomes from degradation and prevent chro-
mosomal end-joining, a phenomenon that could occur due
to the end-replication problem. Telomeres are maintained by
the activity of the enzyme telomerase. The total number of
telomeric repeats at the terminal end of a chromosome
determines the telomere length, which in addition to its
importance in chromosomal stabilization is a useful indica-
tor of telomerase activity in normal and malignant tissues.
Telomere length stability is one of the important factors that
contribute to the proliferative capacity of many cancer cell
types; therefore, the detection and estimation of telomere
length is extremely important. Until relatively recently,
telomere lengths were analyzed primarily using the standard

stable is the genome.
During normal somatic cell division, the absence of
telomerase results in the erosion of telomeric repeats and
reduction in telomere length. Critically short telomere lengths
correlate with the cessation of cell division, the onset of the
aging process and the genesis of age-related diseases [9,11–
19]. However, in rapidly proliferating cells, such as germline
and tumor cells, telomerase is expressed and stabilizes the
telomere lengths, thereby maintaining the immortal state
[20,21]. Telomeres are important in various cellular processes
and the stability of these structures depends on the activity of
telomerase. Therefore, telomere length is a potential indicator
of telomerase activity and can be used in the prognosis of
disease, including various malignancies [3,22–25].
Any technique employed for disease prognosis must be
accurate, reliable and rapid. Southern blot analysis has been
the standard method of choice in the detection of telo-
mere length. However, the limitations of this method,
which involves a tedious procedure, have stimulated the
Correspondence to T. O. Tollefsbol, Department of Biology, 175A
Campbell Hall, 1300 University Boulevard, University of Alabama
at Birmingham, Birmingham, AL 35294–1170.
Fax: + 1 205 9756097, Tel.: + 1 205 9344573,
E-mail:
Abbreviations: TRF, telomere/terminal restriction fragment; HPA,
hybridization protection assay; FCM, flow cytometery method; FISH,
fluorescent in situ hybridization; Q-FISH, quantitative fluorescent
in situ hybridization; Q-FISH
FCM
, quantitative FISH and flow

replication of the lagging strand leading to attrition of
telomere length with each cell division (Fig. 1). In senescent
cells, telomere lengths are short and the cells lose the
capacity to divide. This is in contrast to about 90% of
tumorigenic cell lines, which are immortal, have only
slightly shortened telomere lengths and express high levels
of telomerase (Fig. 1). Thus, there appears to be a strong
correlation between telomerase reactivation and stabiliza-
tion of the short telomere lengths, which could serve as a
Fig. 1. Influence of telomerase activity and telomere length on the processes of cellular aging, senescence, immortalization and tumorigenesis. The
effects of telomerase expression on telomere length in various cell types are depicted. The broad solid line represents the 3¢ terminal portion of a
chromosome and the narrow solid line, the telomere length. Basal or low levels of telomerase are indicated by single upward arrows, double arrows
indicate an intermediate level of telomerase expression, and elevated levels of telomerase are represented by three upward arrows. (A) In the absence
of telomerase in most normal somatic cells, cellular division is accompanied by the loss of telomeric repeats due to the end replication problem.
(B) Repeated cell division leads to the attrition of telomere length resulting in cells acquiring a presenescent phenotype approaching senescence.
(C) With further telomeric attrition to a critical telomere length, cells approach the senescent stage, M1. Some cells in this phase can escape
senescence and become immortal [100]. However, these cells eventually undergo apoptosis or cell death in the absence of telomerase. (D) Cells in the
M1 phase that do not escape senescence enter the M2 crisis stage (towards cell death). (E) A few rare cells in this phase (M2) may escape crisis and
become immortal with the reactivation of telomerase [100]. (F) During transformation the telomere lengths are stabilized and vary depending on the
cell type. The telomeres of transformed cells are short and in most cases are nearly equal to or less than the length at the M2 threshold stage [100].
They are also much shorter than those of telomerase-positive normal cells [101]. It is the reactivation and up-regulation of telomerase that maintains
the stability of the short telomere lengths. Finally, the transforming events (inactivation of tumor suppressor genes, up-regulation of certain
oncogenes such as ras) along with the up-regulation of telomerase impart an immortal and tumorigenic (benign/malignant) phenotype to the cells.
390 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
prognostic indicator for age-related diseases, including
cancer. Telomere length maintenance, a function of telo-
merase activity, is crucial for cell immortalization and is also
important in tumorigenesis.
Southern blotting, which was once the method of choice
used in the detection of telomere length, measures telomere

TRF values are subject to variation based on the site of
restriction of the subtelomeric region. Another drawback
of this method is that the TRF value that is obtained
represents the measurement of the cell population and
not of an individual chromosome, thereby affecting
interpretation of results. In addition to a low yield of
DNA, the isolation of intact genomic DNA from a
large number of cells (> 10
5
cells) can be difficult to
achieve in some cases.
Most problems encountered with Southern hybridiza-
tion have been eliminated or minimized to some degree
by its combination with other methods [20]. The problem
of genomic fragmentation during the extraction proce-
dure can be overcome if telomere lengths are measured
from whole cells [33]. In this case, the estimated length is
a ratio of the telomere to the centromere, referred to as
the TC ratio. These values can be determined accurately
from as few as 800 whole cells or 9 ng of DNA, thereby
enhancing the sensitivity of the procedure. In addition to
the estimation of TRF values based on band size or
TC ratios, lognormal distributions formulated by mathe-
matical and statistical calculations have proved to be
suitable for the analysis of telomere lengths [34]. Incor-
porating these modifications into the Southern hybrid-
ization procedure has improved the sensitivity of the
method but not its simplicity.
Hybridization protection assay
Unlike Southern blotting, the hybridization protection

faster with the hybridization protection assay (Table 1). In
addition to the ease in quantification by HPA, cell types that
have minute differences in telomere lengths can be distin-
guished easily by the chemiluminescent mode of detection.
Fluorescent
in situ
hybridization
The HPA has reduced most of the limitations encountered
with the standard Southern hybridization technique. How-
ever, the measurement of telomere repeats by HPA includes
all cells and not individual cells or chromosomes [31].
Implementation of techniques such as fluorescence in situ
hybridization (FISH) allows calculation of the telomeric
length based on the number of telomeric repeats [29,35].
Enhanced modifications of FISH such as quantitative FISH
(Q-FISH), quantitative flow cytometry (Q-FISH
FCM
), and
flow cytometry and FISH (flow FISH) have provided a
means for the accurate measurement of telomere length
from individual cells (Table 1) [20,29,36].
The FISH method involves the treatment of cells in a
suitable fixative followed by exposure to a hybridization
mixture containing appropriate amounts of formamide,
blocking reagent and a fluorescent peptide nucleic acid
probe (PNA) that is complementary to the telomeric repeats
[37,38]. Fluorescent labeling of the telomere repeats allows
the direct measurement of the telomere length by a
quantitative method referred to as Q-FISH [26,29]. The
PNA probes have an uncharged glycine backbone that

However, the entire process takes
 30 h.
A high degree of complexity
but the final output is probably
faster due to the additional
refinements of FCM and digital
microscopy with state of the art
computerized softwares.
Better suited for measuring
G-rich overhangs than
telomere lengths per se but
comparatively less complex
than the FISH-derived
improvizations.
Can measure
telomere repeats
from purified,
sheared DNA,
as well as
unpurified DNA
in cell and
tissue lysates
(1000 cells).
Requires intact and pure
DNA. Large numbers of
cells are needed in the
extraction of genomic
DNA.
Requires intact metaphase
spreads.

fluorescent probes provides a
more precise quantitative
estimate of telomere lengths.
The use of two probes, one that
specifically stains DNA and the
other telomeres, aids better visual
assessment. Utilization of a control
cell population provides for an
internal telomere length standard
that allows comparisons of differ-
ent samples with high precision.
Advantages are further
enhanced by the use of digital
microscopy improving the rate
at which the results can be
obtained.
Visual measurement of
telomere length of the
G-rich overhang. Standards
for internal and terminal
telomeric repeats are gener-
ated for quantitation
against which the visualized
lengths are measured.
Wide linear range
and can measure
telomere repeats
in biopsy
specimens as well
as cells in body

chromosome with greater
accuracy from limited number
of cells.
Ranges from less than 90 nt
to about 400 nt. This range
depends on the cell type
and population doubling.
392 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Chemilumines-
cent mode of
detection makes
it safe to use over
long periods of
time.
Safety issues regarding the
use of radioactive probes
may be a concern.
The method is limited in appli-
cability due to the high degree
of technical expertise required.
Peptide nucleic acids are used as
probes and their interaction with
the DNA is more stable producing
stronger fluorescence signals. The
use of two probes enables simulta-
neous visualization of DNA and
telomere repeats with increased
sensitivity and accuracy.
The use of a digital fluorescence
microscope has increased the

Southern blot requires the
use of densitometer and
autoradiogram for reading
the blots that can be
expensive.
The basic equipment required
for FISH is easily obtained in
most laboratories. However,
the type of microscope used
does affect the quality and
accuracy of the results.
In addition to the general equip-
ment utilized in Q-FISH, this
method employs the use of a flow
cytometer. Depending on the
probes used and the type of signal
measured, several types of flow
cytometers are available on the
market.
The type of image accquisition
and data analysis needed would
dictate the type of software to
be used. Image capture may
require dedicated software.
The basic equipment
required for this method is
an electron microscope.
Overall it is a
reasonably good
method

of each telomere is an integrated value of the intensities of the
two fluorescent dyes, and is measured as integrated fluor-
escent intensity (IFI) [36]. The system allows the detection of
average telomere length within a cell, of chromosome-
specific telomere lengths in a suspension of cells, and of the
length of individual telomeres. For the measurement of IFI
values of each telomere, a process called segmentation is
performed. This involves the identification of exact bound-
aries of each telomere in the segmented telomere region.
Thresholding or edge detection methods are employed to
determine the approximate location of the telomere spots
[42,43]. For chromosome segmentation, the IFIs are deter-
mined using the
TFL
-
TELO
program. Detailed features of the
program are described elsewhere [36]. The
TFL
-
TELO
gener-
ated output value corresponds to the fluorescence intensity
of each telomere, which is proportional to the number of
probe molecules that hybridize to the region. Utilizing digital
microscopy, telomere length was assessed from two different
samples, same metaphase samples and random metaphase
samples measured on five different days [36]. The average
mean telomere values measured from day one to day five
indicated by the telomere fluorescent values (TFV) were

Many different fluorescent probes have been utilized to
stain DNA [26,28,31,40] and have contributed greatly to
telomeric analysis. The importance of using the appropriate
probe as well as the right method of fixation has been
discussed elsewhere [20]. In the Q-FISH
FCM
procedure,
fluorescence-labeled PNA probes are employed in the
hybridization process [29,47]. Cells can also be treated with
specific antibodies of interest, which are tagged with a
fluorescent dye. Using the fluorescence-activated cell sorter
(FACS), the cells are sorted based on the intensity of the
fluorescence signal produced [29] and the signals generated
by the respective probes can be detected by different
channels. Telomere length values are calculated as the ratio
of the telomere fluorescence signal (TFS) of the sample to
that of the internal control, normalized to the DNA values
at the G
0
/G
1
phase. The use of an internal control (e.g. 1301
cell line) is important to monitor the accuracy of the
procedure and also to serve as a standard for telomere
length [29]. Normalization of the relative telomere length to
the DNA index of G
0
/G
1
phase compensates for the

minute changes in telomere length from a population of cells
and from individual cells is critical to various aspects of
scientific research. Therefore, in choosing the method for
the detection of telomere length (Table 1), cost considera-
tions as well as time constraints are essential factors that
need to be considered. Flow cytometers have facilitated
analyzing and sorting a large number of cells ranging from
300 cellsÆs
)1
to > 20 000 cellsÆs
)1
, depending upon the type
of flow cytometer used, with high purity and accuracy [48].
In general, flow cytometry sorting and analysis of cells are
based on the staining and intensity of the fluorescent signal.
Thus, the choice of the flow cytometer depends on factors
394 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
such as type of sample (cell/tissue), type of information
required, the number of samples to be analyzed and the
quality [49]. When considering microscopy, the flow
cytometry system parameters such as the type and number
of fluorochromes/probes used, light source, objective,
eyepiece and filters are essential [49].
Due to space limitations, these aspects are not described in
this review. However, these parameters, including technical
aspects, advantages, specifications of the different types of
flow cytometer analyzers and sorters are well described
elsewhere [48,50,51]. Flow cytometers are commercially
available with companies such as Becton Dickinson and
Coulter (Beckman Coulter). FACSCalibur, FACSVantag-

proteins (TBP) or follow the pattern of TBP at the time of
telomere elongation and replication.
Several software applications are commercially available
[48].
CELLQUEST
appears to be the more commonly used
software for data acquisition and analysis.
CELLQUEST
is
user-friendly and is quite versatile in terms of its functions
[29]. Some of these features include user-defined calculations
on the data, management of data acquisition, ability to
export graphics and documents from a variety of formats,
format plots and text objects, and the ability to adjust the
specifications and instrument settings for each tube. The use
of these software programs with the sophisticated flow
cytometers has enabled high purity and accuracy with
greater speed.
Telomeric-oligonucleotide ligation assay
G-rich overhangs are located at the 3¢-end of each
DNA strand of the chromosome and serve as a
substrate for telomerase. Telomere shortening is found
to be directly proportional to the length of the overhang
[55]. The information obtained from the G-rich over-
hang lengths can be used for analyzing drug efficacy
and disease progression as well as other processes. Also,
based on the values obtained, suitable inhibitors may be
designed to increase the rate of the telomere length
attrition process [55]. Analysis of the molecular structure
of the G-rich overhangs is useful as they are suitable

specificity and the products are resolved on a denaturing
polyacrylamide gel. However, the T-OLA assay can be a
time-consuming procedure due to the gel-based length
detection. Also, the safety issue regarding the handling of
radioactive oligonucleotides can be a concern. Though it
has a wide detection range and applicability to many cell
types, its use in large-scale screening of samples is
questionable.
Factors influencing telomere length
Telomeres
Telomeres consist of tandem repeats (of a hexameric
sequence in humans), which are positioned at the
extreme ends of chromosomes. The repeats are mostly
G-rich although some organisms such as certain fungi
and invertebrates have interspersed C-nucleotides. The
G-nucleotides in telomeric repeats vary by species (from
one to eight nucleotides) and are flanked by T/A
nucleotides at the 5¢-end (e.g. 5¢-TTAGGG-3¢ in Homo
sapiens,5¢-TTAGGC-3¢ in Ascaris) [59]. Telomeres
impart stability to the chromosomes by facilitating the
formation of stable structures. The structural unit of
telomeres, termed the G-quartet, resembles a square
where the G residues occupy the four corners and T/A
residues form the variable arms, which can form loops
Ó FEBS 2003 Telomere length detection (Eur. J. Biochem. 270) 395
enclosing the G residues. Stacks of two, three or four
quartets tethered by cations (primarily potassium) can
form dimeric, trimeric or quadruple structures [60,61].
These structures are thermodynamically and kinetically
very stable; hence their contribution to the stability

and their importance
Many investigations have unraveled the importance of
telomerase in maintaining stable telomere lengths [69].
However, in telomerase-negative cells or even in some
species, an alternate mechanism exists that enables main-
taining an average telomere length relative to the species
[69,70]. Nonhomologous end-joining or recombination is
one of the alternative lengthening of telomeres (ALT)
mechanisms believed to maintain stable telomere lengths
and evidence supporting this mechanism has been seen in
smaller eukaryotes and in some cases, even mammals [71].
The expression and activity of telomerase has been known
to be significant in the development of a majority if not all
malignant tumors [71]. However, telomeres are also
important in cancer biology. In chromosomes telomeres
serve as stabilizing caps. Irregularities in telomere replica-
tion or structure may therefore affect the generation of
stable telomere lengths. Given that the end-replication
problem in part causes telomere attrition, abnormal
telomeric synthesis and architecture would further enhance
the rate at which telomere attrition would occur leading to a
destabilized telomere length. The genomic instability created
within the cell due to telomere fusions and formation of
dicentric chromosomes may therefore potentiate the for-
mation of abnormal cellular phenotypes and possibly
trigger the onset of cellular senescence or even apoptosis
[41]. These plausible occurrences necessitate a balance
between telomere replication and telomere length stability.
The rapid pace at which telomere biology has moved has
provided fascinating insights to several factors that contri-

Fibroblasts and lymphocytes 400  1 [56]
396 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Table 3. Telomere and telomerase complex bound proteins.
Protein Component Bound Organism Function Reference(s)
TRF1 (telomere
repeat factor 1)
Binds as homodimers
to double strand (ds)
telomeric repeats.
Mammals May play a role in telomere
replication. When bound to
telomeres, telomerase access to
telomeres is prevented and
thus appears to have a role in
regulating telomere length
through inhibition of telomerase
by its interaction with tankyrase.
[70,71,79,102,103]
TRF2 Binds to ds telomeric
DNA only.
Mammals Although TRF2 binds ds repeats
only, it may have an indirect
role in protecting the G-rich
overhang by recruiting other
TBPs to the G-tails or by
mediating the formation of the
telomeric T–loop.
Prevents chromosome fusion.
Interacts with TRF1 to regulate
telomere length via its interaction

important in telomere maintenance.
Plays a role in telomere end
structure either by assisting in
the formation of G-tails through
the recruitment/regulation of an
exonuclease or by protecting the
G-tails from degradation. Ku may
be involved in clustering of
telomeres and may be involved in
the interaction with the nuclear
envelope.
[70,79,107,108]
pKu70 Homolog of budding
yeast S. cervisiae.
Fission yeast
S. pombe
By its interaction with the
stem-loop structure of telomerase
RNA, may be involved in the
direct recruitment of telomerase.
Absence of this protein results
in telomere fusions and increased
recombination of subtelomeric
sequences, and therefore may
be important in telomere tract
protection from nuclease
and recombinatorial activities.
[79,109]
Rad50/Mre11/Xrs2 A protein complex that
may bind telomeric DNA.

with relaxed specificity and
requires a free 3¢-end
to bind. May either be a
component of telomeric
chromatin or a protein
subunit of telomerase.
Yeast Along with Cdc13p may assist in
the extension of the 3¢-end in vivo
by telomerase.
[79,110,111]
Stn1 Forms a complex
with Cdc13p.
Yeast Negative regulator of telomerase
recruitment.
[79,107,110,112]
Ten 1 Associates with
Stn 1 and Cdc13
Yeast
S. cerevisiae
Protects telomere ends and
regulates telomere length.
[113]
TBP Binds ss 3¢-overhang. Ciliates Protects the chromosome end. [110]
Oxytrichia and
Euplotes
rTP (replication
telomere protein)
Binds telomeric DNA. Ciliates Euplotes Expressed at all times of DNA
replication and may be an
important telomere-bound

Yeast Not absolutely essential
for telomerase activity in vitro.
However, required for telomerase
activity and telomere maintenance
in vivo.
[79]
Rap1p (repressor-
activator protein 1)
Binds duplex ds
telomeric DNA.
Budding yeast Negatively regulates telomerase
elongation via its carboxyl
terminus May be involved in telomere
length homeostasis by a negative
feedback mechanism. May
be a part of the counting mechanism
that measures telomere length.
[69,79,114–116]
Tankyrase Associates with TRF1. Yeast and
Mammals
In vitro tankyrase adds poly
ADP-ribose to TRF1, decreasing
the affinity of TRF1 for telomeric
DNA which may signal telomerase
to elongate the telomeres.
[69,79,117]
398 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Proteins such as telomere repeat factor 1 and 2 (TRF1,
TRF2), Tankyrase 1 and 2 (TANK1, TANK2), Ku70/86
and DNA dependent protein kinases (DNA-PKcs), poly

The network that controls telomere replication, telomere
elongation and telomerase activity remains to be ascer-
tained. The vast numbers of proteins involved intensify the
need for further investigations in telomere research. Cur-
rently there are few proposed plausible mechanisms instru-
mental in telomere length homeostasis. Rap1, a protein that
binds to telomeric DNA, is thought to be a part of the
counting mechanism that measures the telomere length,
which probably signals other proteins and protein com-
plexes to control the regulation of telomere synthesis by
telomerase [79]. Although this has led to the speculation that
a regulatory on/off switch exists that controls telomere
lengthening, there is little experimental evidence to support
this [79]. Telomeres that are critically short are elongated at
a faster rate initially which gradually slows down once
equilibrium is attained [79]. In addition, long telomeres are
extended much more slowly. Therefore, the regulatory
mechanisms governing this lengthening process are more
rheostatic in nature [79]. Proteins involved in DNA damage
check-points, telomeric silencing and telomere positioning
effects are essential to telomere replication but their roles in
telomere and telomere length homeostasis are unclear and
need to be investigated further [79,80].
Importance of telomeres and telomere length
Telomeres extend up to 10–15 kb at each human
chromosome end (Fig. 1) and protect the ends of the
chromosomes. The geometric configuration of telomeres
appears to be of great importance. The quartets are
very resistant to nuclease attack [81] and contribute to
protecting the chromosomal termini. Telomeres are

(Rap 1p- interacting
factor 1 and 2)
Interact with Rap1. Yeast Important for Rap1 functions. [71]
Mlp2 (myosin like
protein 2)
Interacts with YKu70. Yeast Important for Yku70-related
functions.
[79]
MeC3p Interacts with SET
domain proteins Set 1.
Yeast Negatively regulates telomere
positioning and telomere elongation.
[79,80,120]
pot1 (protein on
telomeres 1)
Binds G-rich overhangs. Yeast and
Mammals
Plays an important role in telomere
capping. May protect the G-rich
overhang when the t-loop unfolds.
May prevent telomere elongation
of the 3¢-end within a t-loop.
[121]
Ó FEBS 2003 Telomere length detection (Eur. J. Biochem. 270) 399
the maintenance of stable telomere length although
telomere length can, in some cases, be maintained in
the absence of telomerase by mechanisms referred to as
alternative lengthening of telomeres [90–98]. Other
proposed mechanisms are homologous recombination,
which can involve intertelomeric recombination, T-loop

providing prognostic information that may help guide the
direction of treatment. For these reasons, the methods
utilized in measuring telomere length should be efficient and
expedite the process of determining minute and subtle
changes in telomere length. Unquestionably, telomere
length serves as a marker of telomerase activity, and its
measurement by various methods as described in this review
is therefore necessary and important. Summarizing the
advantages and limitations of the methods described, it is
apparent that the quantitative flow FISH in combination
with digital microscopy will enable efficient large-scale
processing of samples and this combined technique
shows great promise in facilitating analysis of telomere
lengths.
Acknowledgements
We thank Mark Casillas, Dr Mitchell Pate, Dr Nadejda Lopatina, and
Nathaniel Hansen for critical reading of the manuscript. This work was
supported by grants from the National Institute on Aging (1 R03
AG20375 01), the American Cancer Society (IRG-60-001-41), the John
A. Hartford Foundation (South-East Center for Excellence in Geriatric
Medicine), the Leukemia Research Foundation and the UAB Center
for Aging, Comprehensive Cancer Center and Department of Biology.
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