Methods for measuring telomere length

Methods and compositions for the measurement of telomere length have application in medical diagnostic, prognostic, and therapeutic procedures. The methods for measuring telomere length include primer extension-based methods and probe-based methods. The primer extension methods involve elongation of telomeric, linker, and/or subtelomeric based primers under conditions such that the telomere serves as a template for primer extension and that the resultant primer extension products can be compared to standards of known length to provide a measure of telomere length. The probe based methods allow for telomere length measurements using DNA from lysed or whole cells and involve hybridizing an excess of probe to all telomeric repeat sequences in the telomere, measuring the amount of bound probe, and correlating the amount of bound probe measured with telomere length.

BACKGROUND OF THE INVENTION 
1. Field of Invention 
The present invention relates to methods and reagents for the measurement 
of telomere length. The invention has applications in the fields of 
molecular biology, cell culture technology, and medical therapeutics and 
diagnostics technology. 
2. Description of Related Disclosures 
Telomeres are specialized nucleoprotein structures at the ends of 
chromosomes that are important in maintaining chromosome stability and 
function (Blackburn, 350 Nature 569, 1991; all references cited herein are 
incorporated by reference herein). Telomeres function through prevention 
of aberrant recombination and degradation at the ends of the chromosomes 
(Henderson et al., 29 Biochemistry 732, 1990; Bourgain et al., 19 Nucl. 
Acids Res. 1541, 1991), organization of the sub-nuclear architecture 
(Gilson et al., 3 Trends Cell Biol. 128, 1993), and involvement in the 
transcriptional suppression of genes at distal loci (Sen et al., 334 
Nature 410, 1988). Telomeres are typically composed of a tandem repetitive 
array of a short sequence. 
In humans, the telomeres are composed of many kilobases of simple tandem 
5'-TTAGGG repeats (Moyzis et al., 85 Proc. Natl. Acad. Sci. U.S.A. 6622, 
1988). These repeats are arranged such that the G-rich strand runs 5' to 
3' towards the end of the chromosome and sometimes extends beyond the 5' 
end to generate a single-stranded 5'-(TTAGGG).sub.n overhang, where n is 
typically 9 to 35 (but n can be more than 35 or less than 9). During DNA 
synthesis, the termini of the chromosomes are not fully replicated 
(Watson, 239 Nature New Biology 197, 1972) by the action of DNA 
polymerase. Incomplete replication occurs at the 3' end of each of the two 
template strands of the chromosome, because the RNA primer needed to 
initiate synthesis in effect masks the 3' end of the template. The RNA 
primer is degraded after strand synthesis, and, as there are no additional 
sequences beyond the 3' end of the template to which primers can anneal, 
the portion of the template to which the RNA primer hybridized is not 
replicated. In the absence of other enzymes, the chromosome is thus 
shortened with every cell division. This phenomena is referred to as the 
"end-replication problem" and is believed to be a key factor in the onset 
of cellular senescence and aging. 
Evidence for this end-replication problem was provided by demonstrating 
that, in normal human somatic cells (e.g., fibroblasts, endothelial, and 
epithelial cells), telomeres shorten by 50-200 bp with each cell doubling 
(Harley et al., 345 Nature 458, 1990; Allsopp et al., 89 Proc. Natl. Acad. 
Sci. U.S.A. 10114, 1992). As a consequence, all normal human somatic cells 
have a limited capacity to proliferate, a phenomenon that has come to be 
known as the Hayflick limit, after which the cells enter replicative 
senescence. In human fibroblasts, this limit occurs after 50-100 
population doublings, after which the cells remain in a viable but 
quiescent state for many months. See, Goldstein, 249 Science 1129, 1990. 
Cellular immortalization (the acquisition of unlimited replicative 
capacity) is an abnormal escape from cellular senescence. See, Shay et 
al., 196 Exp. Cell Res. 33, 1991. Cells can escape from cellular 
senescence by adding telomeric DNA to the telomeres to overcome the 
end-replication problem. Most eukaryotic species utilize a novel enzyme, 
telomerase, to generate telomeric DNA de novo, thus compensating for, 
rather than avoiding terminal deletions of telomeric repeat sequences. The 
enzyme human telomerase can add 5'-TTAGGG repeats to the 3' end of 
telomeric DNA, thus extending the DNA and preventing telomere shortening. 
Telomerase is a complex of protein components and an integral RNA 
component. The RNA component of the human enzyme contains a short region 
complementary to the human telomeric repeat sequence (Feng et al., 269 
Science 1236, 1995). This complementary sequence allows the telomerase RNA 
to serve as a template for the catalytic extension of the 3' telomeric 
termini (Greider et al., 337 Nature 331, 1989). 
Cycles of elongation and translocation allow human telomerase to extend 
processively the 3' region of chromosomes with 5'-TTAGGG repeats. 
Telomere shortening occurs systematically with each cell division, and 
telomerase activation stabilizes telomeres; therefore, knowledge of the 
telomere length and the presence or lack of telomerase activity can 
provide information about the replicative history and the proliferative 
potential of cells. Harley, 256 Mutation Research 271, 1991, suggests that 
telomeres may act as a mitotic clock. The progressive shortening of 
telomeres can be viewed as the means by which cells count divisions; a 
sufficiently short telomere(s) can signal replicative senescence in normal 
cells (Wright and Shay, 8 Trends Genetics 193, 1992). 
U.S. Pat. No. 5,489,508, issued Feb. 2, 1996; PCT Pub No. 95/13381, 
published May 18, 1995; and PCT Pub. No. 95/13382, published May 18, 1995, 
describe, inter alia methods by which the length of telomeres can be 
measured. 
One approximate measure of telomere length, the length in nucleotides of 
the sum of all telomeric repeat sequences, is the length of a "terminal 
restriction fragment" (TRF). The TRF is defined as the length (or average 
length) of fragments generated by complete digestion of the genomic DNA 
with a restriction enzyme that does not cleave nucleic acids composed 
entirely of tandem arrays of the specific telomeric repeat sequence of 
interest. These large fragments can, depending on the restriction enzyme 
used and the source of the telomeric DNA, comprise both telomeric repeats 
and also "subtelomeric" DNA. Subtelomeric DNA is composed of DNA sequences 
adjacent to the tandem telomeric repeat sequences and generally contains 
telomeric repeat sequences interspersed with variable telomere-like 
sequences (Cross et al., 18 Nucl. Acid Res. 6649, 1990; deLange et al., 10 
Mol. Cell Biol. 518, 1990; Brown et al., 63 Cell 119, 1990). Mean TRF 
length can provide a measure of telomere length of telomeres in a cell or 
a cell population. 
TRF length measurement entails digesting genomic DNA with a restriction 
enzyme, typically one with a four-base recognition sequence (e.g., AluI, 
HinfI, MspI, RsaI, and Sau3A), used individually or in combination. This 
digestion results in the production of short fragments of non-telomeric 
DNA and longer fragments of telomeric DNA. The digested DNA is 
electrophoresed, and a Southern blot is performed by hybridizing the DNA 
to a radiolabeled telomeric probe, such as for human telomeres, 
5'-(TTAGGG).sub.3 or 5'-(CCCTAA).sub.3. The telomeric DNA can then be 
visualized by autoradiography and mean lengths of terminal restriction 
fragments calculated from densitometric scans using computer programs 
known in the art. See, Harley et al., 345 Nature 458, 1990. 
Another method for telomere length measurement (see PCT Pub. No. 95/13882, 
supra) involves the synthesis of DNA complementary to the telomeres of 
genomic DNA. The synthesized DNA can be labeled or unlabeled, and the 
length of this DNA can be determined by gel electrophoresis or other 
techniques known in the art. Alternatively, telomere length can be 
measured by the "anchored terminal primer" method, or by a modified 
Maxam-Gilbert reaction (see PCT Pub. No. 95/13382, supra). These two 
techniques provide for a more direct measurement of telomere length by 
exclusion of "the subtelomeric region" in the analysis. 
Telomere length serves as a biomarker for cell turnover. Thus, information 
on the relative age, proliferative capacity, and other cellular 
characteristics associated with telomere and telomerase status can be 
obtained by measuring telomere length. Measurement of telomere length can 
be used to diagnose and stage cancer and other diseases as well as cell 
senescence. Other applications for telomere length measurement include 
determining the efficacy of treatment with a telomere length modulating 
compound (Feng et al., 269 Science 1236, 1995); discovering agents that 
modulate telomere length, telomerase activity, or the rate of telomere 
loss; and determining the presence of telomerase activity. 
There remains a need for more rapid, reliable, accurate, and efficient 
methods for measuring telomere length so that the full potential of such 
applications can be realized. This invention meets this and other needs. 
SUMMARY OF THE INVENTION 
The present invention provides improved methods for measuring telomere 
length. The methods of the invention can be performed rapidly and provide 
increased sensitivity, efficiency, reliability, and accuracy. Moreover, 
these methods are amenable to automation and high through-put formats and 
provide, in some embodiments, a means to measure the telomere length of an 
individual chromosome, to compare interchromosomal variance in telomere 
length, and to measure telomere length of a specific cell population 
within a mixture of cells. In addition, the methods allow one to sort 
cells and/or chromosomes on the basis of telomere length. The present 
invention provides numerous advantages over the conventional method of 
telomere length measurement. 
In one aspect of the invention, a method for measuring telomere length is 
provided that comprises the steps of: 
(a) covalently attaching an oligonucleotide linker to a telomere for which 
a measure of length is desired; 
(b) contacting a primer comprising a sequence sufficiently complementary to 
said linker to hybridize specifically thereto under conditions such that 
said primer extends to form a primer extension product complementary to 
said telomere; and 
(c) correlating telomere length with primer extension product size, thereby 
providing a measure of telomere length. 
In one embodiment, the method involves replication or amplification of the 
telomere sequences by, for example, "polymerase chain reaction" (PCR) 
amplification. A product defined by extension of two primers, a "forward" 
primer complementary in sequence to the linker covalently bound to the 3' 
end of the telomere and a second primer complementary to a subtelomeric 
region of the chromosome, is exponentially amplified by this method. This 
method provides an accurate and sensitive measurement of the telomere 
length. In a preferred embodiment of this method, a double-stranded 
oligonucleotide linker is used, and prior to the ligation of the linker, 
the chromosomal DNA is treated with a nuclease to generate blunt ends to 
improve ligation. Those of skill in the art will recognize that the use of 
two primers for the extension step provides for exponential amplification 
but that linear amplification, with a single primer, can also be used to 
determine telomere length in accordance with the method of the invention. 
Another embodiment of the primer extension method of the invention provides 
a rapid means for measuring telomeres using only one primer. In this 
method, a primer complementary to the covalently bound linker is extended 
using a polymerase and either (i) only those nucleotides complementary to 
the nucleotides in the telomeric repeat; or (ii) those nucleotides and a 
nucleotide analog known as a chain terminator, such as a 
dideoxynucleotide. One or more of the nucleotides can be labeled. For 
human telomeres, exclusion of dGTP and/or addition of dideoxy GTP (ddGTP) 
nucleotide results in termination of primer extension at the first C 
nucleotide relative to the 3' end of the G-rich strand of the chromosome. 
Denaturation and repeated cycles of primer extension and denaturation 
result in multiple copies of the telomeric region. One then measures the 
size of the extension products to estimate telomere length. For example, 
if one uses a labeled nucleotide, and the label is a radioactive label, 
one can measure telomere length by correlating scintillation counts of 
labeled nucleotide incorporated into primer extension products with 
telomere length. 
In another aspect of the invention, one can optionally dispense with the 
linker altogether. In one alternate embodiment of this method, a 
subtelomeric primer is used as the sole primer, eliminating the need for 
ligating or otherwise covalently attaching a linker to the 3' end of the 
telomere. As noted above, repeated steps of primer extension and 
denaturation generate multiple single-stranded copies of the telomeric 
region. In another alternate embodiment, nucleotide analogs known as chain 
terminators are employed in the primer extension step. This method 
comprises the steps of: 
(a) contacting double-stranded chromosomal DNA in a sample with a primer 
having a sequence sufficiently complementary to a 3' end of a telomere to 
hybridize therewith in the presence of a mixture of nucleotides and a 
dideoxynucleotide under conditions such that said primer extends to form a 
primer extension product terminating with said dideoxynucleotide; and 
(b) correlating telomere length with primer extension product size to 
provide a measure of telomere length. 
In this embodiment, the use of a specific dideoxynucleotide in the primer 
extension step provides a means to replicate only the telomeric portion of 
the chromosome. The dideoxynucleotide selected depends on the telomeric 
repeat sequence and the particular strand of the telomere that will serve 
as the template for primer extension. One selects a dideoxynucleotide that 
will not be incorporated until the primer has been extended past the 
telomeric region. Incorporation of a labeled nucleotide or 
dideoxynucleotide into the extension product, or probe-based 
identification of the extension products on a gel, provides a means to 
determine extension product size, which correlates with telomere length. 
In another aspect of the invention, one can avoid the use of labeled 
nucleotides and gel electrophoresis by employing labeled probes to measure 
telomere length. This method comprises the steps of: 
(a) contacting denatured chromosomal DNA with a labeled probe having a 
sequence complementary to a telomere repeat sequence under conditions such 
that said probe hybridizes specifically to telomeric DNA; 
(b) measuring amount of bound probe; and 
(c) correlating said amount of bound probe measured with telomere length. 
As noted above, this method does not require the use of gels, as in the 
conventional assay for telomere length determination, and is conducive to 
high through-put or automated processes, which is especially useful for 
clinical applications. In a preferred embodiment, the analysis of telomere 
length utilizes imaging techniques that allow for not only intercellular 
and intracellular telomere length determination and comparison, but also 
the separation of cells or chromosomes based on telomere length. 
The methods of the invention are broadly applicable to the measurement of 
telomere length in any sample from any origin. The methods are especially 
useful and applicable to the measurement of telomere length in samples of 
biological material obtained from humans. Such samples will contain cells 
or cellular materials and will typically be obtained from humans for the 
purposes of determining remaining proliferative capacity or lifespan of 
the cells in the sample, diagnosing medical conditions, or identifying 
disease or proliferative states. These and other aspects of the invention 
are described in more detail below, beginning with a brief description of 
the accompanying drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention provides improved methods for measuring telomere 
length. Telomeres are nucleoprotein structures at the ends of chromosomes 
and have been shown to function in chromosomal stabilization, position, 
and replication. Telomeres are also believed to serve as the mitotic clock 
for signaling cellular senescence. Because chromosomes of normal somatic 
cells have been shown to lose about 50-200 nucleotides of telomeric 
sequence per cell division, telomere length measurement provides a means 
for determining the proliferative lifespan of a cell. Numerous diseases 
are characterized by accelerated cell proliferation (hyperproliferative 
states) or decreased proliferative capacity. Therefore, improved telomere 
length measurements help meet the need for improved diagnostic and 
prognostic methodology. 
To facilitate understanding of the invention, the disclosure of the 
invention is organized in sections as follows. First, a definition section 
is provided to define terms and phrases used commonly throughout the 
disclosure. This definition section includes a comprehensive description 
of the types of samples, primers, probes and labels that can be used with 
the invention. The next section describes methods of the invention for 
measuring telomere length. The methods for measuring telomere length are 
divided into two major categories, primer extension based methods and 
probe based methods. The probe based methods section is further subdivided 
to describe telomere length measurements using DNA from lysed cells and 
using whole cells. Then, various applications of the invention are 
described; this description is followed by detailed examples illustrating 
the invention. 
DEFINITIONS 
To assist in the understanding of the invention, the following terms as 
used herein are defined below. 
"Abnormal chromosome" means a chromosome which has undergone a deletion, 
addition, or translocation such that the telomeric region is adjacent to 
chromosomal DNA not normally adjacent to the telomere. 
"Blot" means a DNA-binding filter or substrate, such as nitrocellulose or a 
Silent Monitor.TM. Biodyne B membrane. 
"Branched DNA probe" or "bDNA probe" means a probe designed for branched 
DNA signal amplification (Urdea, 12 BioTech. 926, 1994; U.S. Pat. No: 
5,124,246), which involves amplification of the signal produced upon probe 
hybridization to a target nucleic acid. The bDNA probe is comprised of a 
hybridizing portion complementary to the telomeric repeats (e.g., 
5'-(CCCTAA).sub.n -3' or its permutations, where n comprises 8 or more 
nucleotides in length, preferably 12 to 15 to 20 or more nucleotides in 
length), and so hybridizes with telomeric nucleic acid. The probe further 
comprises a branched region that provides multiple secondary probe binding 
sites. After washing to remove unbound probe, a labeled secondary probe 
specific for the branches of the bDNA is hybridized to the bDNA and is 
detected via the label. The signal increases in direct proportion to the 
secondary probe-accessible-sites on the bDNA molecule; thus a rare 
population of target nucleic acids can be detected by bDNA hybridization. 
Sensitivity can be further enhanced by probing the 
telomeric-repeat-complementary-bDNA with a secondary bDNA probe specific 
for the branches of the primary bDNA probe (and a tertiary probe specific 
for the secondary probe, and so on), thereby presenting more numerous 
hybridization sites for the labeled probe. PNA probes as well as other 
modified nucleic acid robes, can also be used as bDNA probes. 
"Change in telomere length" means that the average or mean telomere length 
of chromosomal DNA in a particular cell population or sample is increased 
or decreased relative to other normal somatic cells in an individual or 
relative to normal somatic cells in other individuals, i.e., those not 
suffering from a disease condition. 
"Label" means a chemical used to facilitate identification and/or 
quantitation of a target substance. Illustrative labels include 
fluorescent (e.g., FITC or rhodamine), phosphorescent, chemiluminescent, 
enzymatic, and radioactive labels, as well as chromophores. Any of a wide 
variety of labeled reagents can be used for purposes of the present 
invention. For instance, one can use one or more labeled nucleoside 
triphosphates, primers, linkers, or probes in the methods of the 
invention. The term label can also refer to a "tag" that can bind 
specifically to a labeled molecule. For instance, one can use biotin as a 
tag and then use avidinylated or streptavidinylated horseradish peroxidase 
(HRP) to bind to the tag, and then use a chromogenic substrate (e.g., 
tetramethylbenzamine) to detect the presence of HRP. In a similar fashion, 
the tag can be an epitope or antigen (e.g., digoxigenin), and an 
enzymatically, fluorescently, or radioactively labeled antibody can be 
used to bind to the tag. For purposes of the present invention, the 
telomeric repeat itself can be a tag. Telomeric repeat binding proteins 
are known in the art and bind to either double-stranded or single-stranded 
telomeric repeats. If the labeling method involves the use of a protein, 
then native or recombinant proteins can be used; typically, such proteins 
would be purified for use and detected by virtue of a label attached to 
the particular protein or an antibody specific for the particular protein. 
"Linker" means a single- or double-stranded oligonucleotide composed of 
nucleotides that is to be ligated to another oligonucleotide or nucleic 
acid. 
"Linker sequence" means the nucleotide sequence of a linker. 
"Long polymerase chain reaction (PCR)" means PCR amplification conditions 
suitable for amplification of a relatively large nucleic acid (see Cheng, 
"Efficient PCR of Long Targets", New Horizons in Gene Amplification 
Technologies: New Techniques and Applications; San Francisco, Calif. 
(1994)); typically, the amplified nucleic acid has a length greater than 
about 200 nucleotides, but the use of the word "long" is not intended to 
limit the length of the nucleic acid that can be amplified. 
"Metaphase spread" refers to a cluster of chromosomes that are derived from 
cells that have been blocked in metaphase as a result of growth in the 
presence of a spindle formation inhibitor, such as colcemid. The cell is 
treated with a hypotonic buffer to cause swelling and burst upon dropping 
on a surface. Upon bursting, the chromosomes are released out of the cell 
and disperse onto the surface in clusters. Typically, the chromosomes are 
spread out on a microscope slide to facilitate visualization and 
microscopic analysis. 
"Modified rate of telomere loss" means an increase or decrease in telomere 
loss over a defined time period (e.g., a year) or biological occurrence 
(e.g., a population doubling) relative to other normal somatic cells in 
that individual, or to normal somatic cells in other individuals, i.e., 
individuals not suffering from a disease condition. 
"Oligonucleotide" means a molecule consisting of covalently linked 
naturally occurring or synthetically constructed nucleotides and/or 
nucleotide analogs. As used in this disclosure, oligonucleotides are 
generally primers, probes, and linkers composed of deoxyribonucleotides. 
However, the oligonucleotides of the invention can also be composed of 
ribonucleotides, modified analogs of ribo- or deoxyribonucleotides (i.e., 
synthetic or non-naturally occurring), or mixtures of any of the same. 
Usually, nucleotide monomers in an oligonucleotide are linked by 
phosphodiester bonds. However, as will be apparent to one in the art, 
alternate linkages can be used, including phosphorothioate, 
phosphorodithioate, phosphoroselenate, phosphorodiselenoate, 
phosphoranilidate, phosphoroamidate, peptide, and the like linkages. For 
example, a peptide nucleic acid (PNA) is an oligonucleotide with peptide 
bonds instead of phosphodiester bonds. Because a PNA has no charge, a PNA 
has a higher binding affinity than a deoxyribonucleic acid. 
"Primer" means an oligonucleotide designed to hybridize to a target nucleic 
acid and then be extended by the addition of nucleotides or an 
oligonucleotide. A primer is typically extended by action of a polymerase 
or ligase. Typically, an oligonucleotide primer will be 8 or more 
nucleotides in length, preferably 12 to 15 to 20 or more nucleotides in 
length. 
"Probe" means an oligonucleotide designed to hybridize specifically with a 
target nucleic acid. Because human telomeres comprise repeats of sequence 
5'-TTAGGG-3', a telomeric probe, unless otherwise indicated, will be 
identical or complementary to a sequence contained within a sequence of 
two or more such repeats, i.e., the probe will comprise a sequence such as 
5'-CCCTAA-3', (for an RNA probe, 5'-CCCUAA-3'), or 5'-CTAACC-3', for 
example. Typically, an oligonucleotide probe will be 8 or more nucleotides 
in length, preferably 12 to 15 to 20 or more nucleotides in length. 
"Proliferative capacity" means the inherent ability of a cell or cells in a 
tissue to divide for a number of divisions (the "Hayflick" limit) under 
normal proliferation conditions. 
"Riboprobe" means a probe comprised of ribonucleotides. A telomeric 
riboprobe can be produced by transcription of multiple, tandem telomere 
repeat sequences in a recombinant host cell, typically E.coli, as 
described in Example 1. 
"Sample" means a composition of matter comprising a cell or cell extract. 
The methods of the present invention can be applied to any type of sample. 
Samples of particular interest include cell samples such as normal or 
diseased tissue samples, e.g., tumor samples, obtained for purposes of 
diagnostic or prognostic analysis. For diagnosis, telomere length 
measurement may be performed on a particular cell type, on all cells in a 
tissue (where various cell types may be present), or on extracts of a cell 
or cells where extract refers to a whole cell extract or a subfraction 
thereof, such as a specific chromosome from a cell. Typically, but not in 
all instances, the sample is treated to render the telomeric DNA in the 
cells more accessible. The preparation of the DNA can be accomplished by 
any of a variety of methods, depending upon the method for measuring 
telomere length to be employed. 
"Senescent state" means that state in which a cell has lost the ability to 
replicate even in the presence of normally appropriate replicative 
signals. 
"Spot" means the area taken up by a specific fluorescent signal in a FISH 
analysis image; spot size corresponds to the amount of probe hybridized to 
a telomere. 
"Subtelomeric DNA" or "Subtelomeric region" means chromosomal DNA located 
immediately adjacent (100 to 500 bp but can be up to 1 kb) to the tandem 
telomeric repeats of the telomeric DNA and generally contains telomeric 
repeat sequences interspersed with imperfect telomeric repeat or other 
variable sequences. 
"Subtelomeric region of an abnormal chromosome" means the chromosomal DNA 
immediately adjacent (100 to 500 bp but up to 1 kb) to a telomere of an 
abnormal chromosome, e.g., the region adjacent to a telomere from a 
diseased cell, such as those found in .alpha.-thalassaemia patients or 
formed by recombination-based chromosome truncation (Farr et al., 88 PNAS 
7006, 1991). 
"Telomeric DNA" or "Telomeric region" means the chromosomal DNA located on 
the ends of a chromosome consisting of a tandem repeat array of a short 
sequence. In humans, the telomere region is composed of 5'-TTAGGG-3' 
repeats and the corresponding complementary sequence. The telomeric 
regions of different organisms differ with respect to telomeric repeat 
sequence. The telomeric repeat sequence of telomeres from a variety of 
organisms, including human, Tetrahymena, fungi, and non-human mammals, are 
known. For instance, Tetrahymena telomeres consist of repeats of sequence 
5'-TTGGGG-3', and the corresponding complementary sequences. Consequently, 
if one is using the present methodologies to determine telomere length of 
telomeres in a sample of human origin, one will employ a probe or primer 
distinct from that employed if the sample is, for example, of fungal 
origin. For convenience, human telomeric region and human telomeric repeat 
sequences are typically referred to herein for illustrative purposes. This 
illustrative use is not intended to limit the invention, and those of 
skill in the art will recognize that the present methods can be used to 
measure telomere length of telomeres from any organism. 
"Terminal restriction fragment" or "TRF" means the length (or average 
length) of restriction fragments that are generated by complete digestion 
of the genomic DNA with one or more restriction enzyme(s) that do(es) not 
cleave nucleic acids composed entirely of tandem arrays of telomeric 
repeat sequences and that comprise both telomeric repeats and subtelomeric 
DNA. 
"3' end of the telomere" means the single-tranded region of the telomere; 
in humans this region is located on the G-rich strand, and is composed of 
a 5'-(TTAGGG).sub.n repeat sequence, where n is typically 9 to 35 (but n 
can be more than 35 or less than 9). 
Primer Extension Based Methods for Measuring Telomere Length 
The present invention provides methods for measuring telomere length. In 
one embodiment, the method comprises the steps of: 
(a) covalently attaching an oligonucleotide linker to a telomere for which 
a measure of length is desired; 
(b) contacting a primer comprising a sequence sufficiently complementary to 
said linker to hybridize specifically thereto under conditions such that 
said primer extends to form a primer extension product complementary to 
said telomere; and 
(c) correlating telomere length with primer extension product size, thereby 
providing a measure of telomere length. 
In one embodiment, this method involves contacting the telomere with a 
linker under conditions in which the linker is ligated by the action of a 
DNA or RNA ligase that can "blunt-end" ligate together two single-stranded 
RNA or DNA molecules. This method can also be employed using a 
double-stranded linker. Alternatively, the linker can be attached 
nucleotide-by-nucleotide by a terminal transferase and specific dNTPs 
(dCTP, dTTP, dGTP or DATP, depending on the sequence to be added). 
Terminal transferase can add multiple monomers of a specified dNTP to the 
3' end of the telomere; the added nucleotides can then serve as a primer 
annealing site. 
An oligonucleotide complementary in sequence to the sequence of the linker 
is used as a primer in the replication step. This oligonucleotide is 
referred to as the "forward primer". In addition to the forward primer, a 
second primer can preferably be used. The telomere sequences are 
replicated or amplified, for example, by PCR amplification with a first 
primer specific for the linker sequence and a second primer specific for a 
subtelomeric region of the chromosome. The primers can be extended by any 
means that requires the presence of the telomeric region for primer 
extension to occur; preferred means are mediated by a template-dependent 
DNA or RNA polymerase, a template dependent DNA or RNA ligase, or a 
combination of the two. Telomeric nucleic acids in a sample are covalently 
bound to a linker, and a primer complementary to the linker and/or a 
subtelomeric primer provide(s) a substrate for either DNA or RNA 
polymerase or DNA ligase to produce primer extension product(s) 
complementary to the telomeric region. 
As noted above, if the primer extension reagent is a DNA polymerase, and a 
second primer is present, one has the requisite components for PCR, a 
process more fully described in U.S. Pat. Nos. 4,683,195; 4,683,202; and 
4,965,188, provided the appropriate buffer and nucleoside triphosphates 
are present in the reaction mixture. Taq polymerase (available form 
Perkin-Elmer) is a preferred polymerase in PCR amplification, although 
other polymerases, especially thermostable polymerases, can be employed. 
Taq polymerase can misincorporate nucleotides in the primer extension. If 
misincorporation presents a problem, and typically, the misincorporation 
is at such a low frequency that no problem is encountered unless the 
amplification products are very long, then another polymerase with a lower 
frequency of misincorporation, i.e., Pfu, Pwo, or Vent can be employed, or 
a mixture of such polymerases, i.e., Taq polymerase/Vent DNA polymerase in 
a ratio range of 100:1 to 10:1, can be employed. The appropriate selection 
of a polymerase or a polymerase mixture can provide optimal extension of 
the primer extension product. 
Once a primer extension product has formed, one can disassociate or 
denature (typically by heat denaturation, but other methods, i.e., enzyme 
or chemical mediated processes, such as helicase mediated denaturation, 
can be used) the primer extension product from the telomeric region. If 
additional primer and primer extension reagent is present in the sample, 
then a new primer/telomere complex can form, leading to the production of 
additional primer extension products. One can repeat the process of primer 
extension and denaturation several to many times, as desired. Typically, 
primer extension and denaturation of extended primer/telomere complexes 
will be performed at least 5, 10, 15, 20, to 30 or more times. Moreover, 
if a second primer complementary to the subtelomeric region of the 
extended primer is present in the reaction mixture, one can increase the 
replication products (both extended primer and the complementary extended 
sequence) dramatically, because the two primers mediate PCR amplification 
of the telomere. 
The PCR cycles are composed of cycle times and temperatures that can vary 
widely, depending upon the sample, detection format, and application. 
Typically, long PCR reaction conditions are employed. The simplest PCR 
cycle comprises a duplex nucleic acid denaturation step followed by a 
primer annealing and extension step. The denaturation step typically 
involves heating at any of a relatively wide range of temperatures for an 
amount of time sufficient to denature but not damage the DNA. In similar 
fashion, the time and temperature of the primer annealing step depend on 
the reaction buffer and primer sequence, concentration, and composition, 
as well as the specificity required by the practitioner. The time and 
temperature of the primer extension step depend upon the type of DNA 
polymerase or ligase employed. Those of skill in the art will recognize 
and understand that the present invention is not limited by the times, 
temperatures, and reaction condition variations in buffer and other 
reaction components that can be employed. 
The second primer used in this embodiment is a primer comprising sequences 
complementary to the subtelomeric region and is designated the 
"subtelomeric primer." FIG. 1 illustrates this method, showing Pi as the 
subtelomeric primer, P2 as the forward primer, and the zigzag line 
indicates the junction between the subtelomeric region and the telomere 
(the figure is not drawn to scale) . In this Figure, the telomere is a 
human telomere and so comprises the human telomeric repeat 5'-TTAGGG-3' 
and complementary repeat sequences. The region depicted by heavy right 
angled slashed lines in FIG. 1 represents the subtelomeric region 
complementary to the subtelomeric primer. Preferably, the subtelomeric 
primer does not comprise a telomeric repeat sequence and so is a 
non-telomeric repeat sequence of the subtelomeric region. Preferably, the 
subtelomeric primer anneals to the subtelomeric region within 50 base 
pairs of the junction with the telomeric region. Extension of these 
primers in multiple cycles of primer annealing and extension amplifies the 
telomeric region by producing multiple replicates of the same. 
If the primer extension product is generated by PCR, then, as noted above, 
one also employs a primer specific for the subtelomeric region. This 
primer is a site specific and, preferably, a chromosome specific primer. 
The subtelomeric primer is complementary to a sequence in the subtelomeric 
region that is present in at least one chromosome, but optionally present 
in two or more, up to and including all chromosomes, of the cell or cell 
population of interest. Subtelomeric regions of many different chromosomes 
are known in the art. Additional subtelomeric regions of normal and 
abnormal chromosomes can be determined using cloning and sequencing 
procedures known in the art. 
Primers specific for a subtelomeric region of a normal chromosome include 
novel primers based on the heretofore unpublished TelBam 8 probe sequence, 
specific for chromosome 7q, such as TEL8-1, 
5'-TGCAATTATTTTACTATCTGTTATCGG-3' (SEQ. ID NO.:1); and TEL8-2, 
5'-TGACCTGTTTTAAAGAGTATGCTCAG-3' (SEQ. ID NO.:2). Nucleic acids comprising 
all or portions of the TelBam 8 probe sequence can be cloned as described 
in Brown et al., 63 Cell 119, 1990. 
Other illustrative subtelomeric primers include XpJCTN, 
5'-CCCTCTGAAAGTGGACCWATCAG-3' (SEQ. ID NO.:3), where XpJCTN is a mixture 
of two primers, in one of which W is A and in the other of which W is T; 
40BPXpJCTN, 5'-CTTTTATTCTCTAATCTGCTCCC-3' (SEQ. ID NO.:4); 400BPXpJCTN, 
5'-TAGGGGTTGTCTCAGGGTCCTA-3' (SEQ. ID NO.:5); REV40BPXpJCTN, 
5'-GGGAGCAGATTAGAGAATAAAAG-3'(SEQ. ID NO.:6); and revXpJCTN, 
5'-CTGATWGGTCCACTTTCAGAGGG-3'(SEQ. ID NO.:7), which are based on sequences 
in the pseudo-autosomal region of the X and Y chromosomes (Baird et al. 14 
The EMBO Journal 5433, 1995) 
By attaching a linker to the 3' end(s) of the chromosome(s), and using a 
forward primer specific for the linker and a primer complementary to the 
subtelomeric region of a chromosome (e.g., a primer incorporating a 
subtelomeric sequence, such as the sequence of the TelBam 8 probe or the 
pseudo-autosomal region of chromosomes X and Y), PCR amplification can 
generate multiple copies or replicates of the telomeric region, which can 
in turn be used to measure telomere length. The PCR primer extension 
products are, for example, separated by size on a gel. If the 
amplification products have been labeled, i.e., by incorporation of a 
labeled nucleotide or hybridization to a labeled probe, then one can use 
size standards to determine telomere length. Direct incorporation of 
labeled nucleotides into the amplification products allows for elimination 
of the steps comprising denaturing the DNA in the gel, neutralizing the 
gel, drying the gel, hybridizing a probe to the gel, removing unbound 
probe from the gel, and exposing the gel, typically undertaken in 
conventional probe-based methods. Relative to conventional methods, which 
can require about a week to complete, the PCR-based method for measuring 
telomere length is quick, accurate, sensitive, and requires significantly 
less sample to perform. 
In a preferred embodiment of this method, the linker is a double-stranded 
oligonucleotide, and the telomeric DNA is treated prior to linker 
attachment to remove or fill in single-stranded regions. In this 
embodiment, as exemplified in Example 4, the DNA is treated with a 
nuclease (i.e., Bal31, Mung bean, or other nuclease) and/or DNA 
polymerase, such as T4 or Pfu (these polymerases possess 3' to 5' 
exonuclease activity in combination with their 5' to 3' polymerase 
activity), to generate blunt-ended, double-stranded telomere ends prior to 
ligation of the double-stranded linker. 
While those of skill in the art will recognize that any double-stranded 
linker can be used, so long as the linker sequence differs from the 
telomeric repeat sequence, a particularly preferred double-stranded linker 
is composed of complementary single-stranded oligonucleotides SLIC-II and 
aSLIC (see Edwards et al., 19 Nucl. Acids Res. 5227, 1991), shown below. 
5'-GGAATTCTGGTCGACGGATCCTGA-3' SLIC-II (SEQ. ID NO.:8) 
3'-CCTTAAGACCAGCTGCCTAGGACT-5' aSLIC (SEQ. ID NO.:9) 
The 5' end of the SLIC-II oligonucleotide can be constructed so as to 
terminate with a 5'-phosphate, whereas the 3' end of this oligonucleotide 
can be constructed with a terminal 2',3'-dideoxyadenosine group to prevent 
the linker from ligating to another linker. Likewise, the complementary 
aSLIC oligonucleotide can be constructed so that the 3' end terminates 
with a 2'3'-dideoxycytidine to prevent ligation to another linker. A 
double-stranded oligonucleotide formed by hybridization of SLIC-II to 
aSLIC so constructed cannot self-ligate. 
The double-stranded oligonucleotide linker formed by SLIC-II hybridizing to 
aSLIC also contains restriction sites to facilitate cloning or other 
applications. This double-stranded oligonucleotide linker can be ligated 
to the 3' end of the blunted telomere using a ligase (i.e., T4 DNA ligase 
or other ligase). A primer complementary to SLIC-II, such as aSLIC, 
5'-CCGTCGACCAGAATTCC-3' (SEQ. ID NO.:10) or 5'-CAGGATCCGTCGACCAG-3'(SEQ. 
ID NO.:11), can be used to generate the desired primer extension product. 
As noted above, the use of a second, subtelomeric primer for extension 
provides a means for PCR amplification of the telomeric region. Separation 
of the PCR amplified primer extension products by size on a gel and 
visualization of the products, for comparison to a standard(s) of known 
length(s), provides the length of the telomere DNA in the sample. 
While the PCR based embodiments of the present invention are quite useful, 
the present method can be practiced using any method of primer extension 
to provide target amplification or with a method that provides for signal 
amplification or both, as described below. Moreover, target amplification 
can be achieved by means other than PCR. These methods include the ligase 
chain reaction (Barany, 88 Proc. Natl. Acad. Sci. U.S.A. 189, 1991), 
nucleic acid sequence-based amplification (Compton, 350 Nature 91, 1991), 
self-sustained sequence replication (Guatelli et al., 87 Proc. Natl. Acad. 
Sci. U.S.A. 1874, 1990), and strand displacement amplification (Walker et 
al., 89 Proc. Natl. Acad. Sci. U.S.A. 392, 1992). While PCR and other 
amplification methods provide for exponential accumulation of primer 
extension products, even linear accumulation of primer extension products 
can provide useful results. Thus, one can use a single primer and merely 
make many copies of the telomeric region from this one primer, as 
described more fully below. 
This invention provides a method to measure telomere length using linear 
amplification. This method exploits the fact that the human telomeric 
repeat sequence lacks guanidine residues in the C-rich strand; however, 
this method is generically applicable to telomeres of any origin that 
comprise repeat sequences that lack one or more nucleotides. In this 
embodiment, a primer complementary to the covalently bound linker is added 
to the genomic telomere DNA in the presence of only three (for human 
telomeres, DATP, dTTP, and dCTP) of the four nucleoside triphosphates. 
These three dNTPs form the complement to the G-rich stand of a human 
telomere. Usually, the primer or at least one of the triphosphates is 
labeled with a detectable label, e.g. a radioisotope or a fluorescent 
molecule, which label is retained upon incorporation into the primer 
extension product. 
The primer is extended by means of a primer extension reagent, e.g., a DNA 
polymerase such as the Klenow fragment of DNA polymerase I, T7 DNA 
polymerase, or Taq DNA polymerase or the Stoffel fragment thereof. 
Exclusion of dGTP results in termination of the primer extension at the 
first C nucleotide of the chromosome. Thus, sequences complementary to the 
primer located outside the telomeric and/or subtelomeric region would not 
serve as templates for primer extension products due to the lack of dGTP. 
Denaturation of the primer extension product from the telomeric DNA 
followed by repeated cycles of primer extension results in the generation 
of multiple copies or replicates of one strand, for human chromosomes, the 
G-rich strands, of the telomeric region. For many purposes, a simple 
measure of the label incorporated suffices to quantitate telomere length, 
although one can also measure the length directly by gel electrophoresis 
and comparison to standards of known length. 
In a preferred embodiment, the annealed primer is extended in the presence 
of a mixture of dideoxy GTP (ddGTP), dCTP, DATP, and dTTP by a polymerase 
(for human telomeres). The method differs from the previous embodiment in 
that, instead of leaving one or more nucleotides out of the reaction 
mixture, one uses a chain-terminating nucleotide(s) in place of the 
otherwise missing nucleotide(s). Polymerization proceeds until the 
polymerase encounters the first cytosine residue (for humans) in the 
subtelomeric region. The enzyme will then incorporate the ddGTP nucleotide 
and further extension will be terminated due to the presence of the ddGTP. 
The primer extension reaction can repeated as many times as desired. The 
length of the extended DNA can be determined by gel electrophoresis and 
comparison to standards, as described above. In addition, the amount of 
DNA synthesized can be determined by measuring the label incorporated into 
the primer extension products or the amount of probe hybridized to the 
primer extension products, which will be directly proportional to telomere 
length. While the manner of determining primer extension size may vary 
depending upon the method of analysis selected, any of the methods 
described can be used. 
In another embodiment of this method, linear extension products generated 
using a subtelomeric primer serve to provide a measure of telomere length. 
This embodiment eliminates the need for ligating or otherwise covalently 
attaching a linker to the 3' end of the telomere. As above, this 
embodiment is ideally suited for linear amplification resulting in 
multiple copies of the telomeric region. 
If the subtelomeric primer selected is not specific or unique to the 
subtelomeric region, then the primer extension products generated from the 
subtelomeric region can be distinguished from those generated from an 
internal chromosomal region by hybridizing with a probe that is specific 
to a telomeric region or a region immediately adjacent to the telomeric 
repeat sequences. Alternatively, if the subtelomeric primer hybridizes to 
multiple sites within the subtelomeric region, then one can hybridize a 
primer specific to a region immediately adjacent to the telomeric repeat 
sequences, and this second subtelomeric primer can be annealed to the 
first primer extension products and extended with DNA polymerase and the 
size of the second primer extension products determined as described above 
to provide a measure of the subtelomeric length in the first primer 
extension products. 
Another embodiment of the invention involving the use of chain-terminating 
synthetic nucleotide(s), such as a dideoxynucleotide, eliminates the need 
to attach a linker covalently to the 3' end of the telomere. This method 
comprises the steps of: 
(a) contacting double-stranded chromosomal DNA in a sample with a primer 
having a sequence sufficiently complementary to the 3' end of a telomere 
to hybridize therewith in the presence of a mixture of nucleotides and a 
dideoxynucleotide under conditions such that said primer extends to form a 
primer extension product terminating with said dideoxynucleotide; and 
(b) correlating telomere length with primer extension product size to 
provide a measure of telomere length. 
As noted above, this method can be used as a variation of the linker-based 
method, where the primer is complementary to a linker added to the 3' end 
of the telomere, but in a preferred embodiment, no linker is required. In 
this embodiment, an oligonucleotide sequence complementary to the 3' 
single-stranded region of the telomere, i.e., 5'-(CCCTAA).sub.4 -3' (SEQ. 
ID NO.:12), is annealed to the telomeric DNA termini. The annealed primer 
is extended in the presence of a mixture of dideoxy GTP (ddGTP), dCTP, 
DATP, and dTTP by a polymerase (for human telomeres). In a preferred 
embodiment, one of the nucleotides is labeled (typically, in any method 
involving incorporation of a labeled nucleotide, especially a 
radioactively labeled nucleotide, only a small fraction of the total 
nucleotide is labeled, and so the labeled nucleotide is referred to as a 
"tracer"). The polymerization will proceed until the polymerase encounters 
the first cytosine residue in the subtelomeric region, as described above. 
The DNA is then denatured and separated by size on a gel. Incorporation of 
a labeled nucleotide provides for easy identification of the length of the 
extension product on a gel and direct correlation of telomere length from 
signal intensity. One can, however, also use a labeled probe to detect 
primer extension products. 
In a more preferred embodiment of this method, the genomic DNA is 
fragmented with a restriction enzyme that cuts DNA, but not telomeric DNA, 
frequently, i.e., for human chromosomes, HinfI or any other restriction 
enzymes with relatively short recognition sequences, and subsequently 
treated with a polymerase (i.e., DNA polymerase I or the Klenow fragment 
thereof, Taq polymerase or the Stoffel fragment thereof) in the presence 
of ddGTP, dCTP, dATP and dTTP, prior to addition of the primer. This 
treatment fills in nicks and gaps in the genomic DNA and blocks any 
potential priming sites in the genomic DNA with a ddGTP chain terminator. 
Pretreatment of the DNA to fill in gaps and nicks can result in increased 
sensitivity and decreased background signal. An oligonucleotide primer 
with a sequence complementary to the 3' single strand region of the 
telomere is then added and annealed to the telomeric DNA termini and 
extended as discussed above. As before, telomere length is determined by 
correlation to the size of the primer extension products. 
The foregoing methods involve the use of a primer and the detection of 
primer extension products to measure telomere length. The following two 
sections describe methods using a probe to measure telomere length. 
Probe Based Methods for Measuring Telomere Length 
In another aspect of the invention, labeled probes are employed to measure 
telomere length. This method comprises the steps of: 
(a) contacting denatured chromosomal DNA with a labeled probe having a 
sequence complementary to a telomere repeat sequence under conditions such 
that said probe hybridizes specifically to telomeric DNA; 
(b) measuring amount of bound probe; and 
(c) correlating said amount of bound probe measured with telomere length. 
In these probe-based methods, the probe is added in excess, so that all or 
substantially all of the telomeric repeats in the telomere are hybridized 
to the probe. Typically, the correlation step involves the use of 
standards of known length or the use of conversion factors to convert the 
amount of bound probe to a measure of telomere length. 
This aspect of the invention provides a method for measuring telomere 
length in which an oligonucleotide probe is hybridized to telomere repeat 
sequences. The amount of probe hybridized is determined and then 
correlated to provide a measure of telomere length. This method can be 
practiced without the gel based size separation step used in other 
methods. Thus, this aspect of the invention provides a rapid, high 
through-put method for measuring telomere length. 
In a preferred embodiment, this method involves preparing DNA extracts of 
cells, incubating the extract with an oligonucleotide probe complementary 
to telomere repeat sequences, and determining amount of probe bound as a 
measure of telomere length. For convenience, the cells can be grown in a 
24, 48, or 96-well microtiter plate. 
To practice the method, the cells from each well are collected and the DNA 
isolated by standard DNA extraction procedures. The DNA extract solution 
can be passed through a DNA-binding filter, such as nitrocellulose or 
Biodyne B membrane, to remove other potentially interfering substances. 
The filter is then contacted with a labeled oligonucleotide probe having a 
sequence complementary to telomere repeat sequences. After unbound probe 
is removed, the amount of probe bound to the filter is then quantified, 
and the amount of probe bound provides a measure of telomere length. This 
method, called the dot-blot method, is exemplified in Example 1 below. As 
with other embodiments, standards of known telomere length can be employed 
to help correlate the signal from bound probe with telomere length. In a 
preferred embodiment, this method is carried out in a 24, 48, or 96-well 
plate and is automated. Preferably, Pall SILENT MONITOR.TM. 96-well test 
plates, having 0.4 .mu.M Biodyne B membrane located at the bottom of each 
well, are used. 
In another embodiment, the invention provides a method of measuring 
telomere length in which the genomic (chromosomal) DNA is bound to a solid 
phase using a modified dot-blot method called the slot-blot. This aspect 
of the invention is illustrated in Example 2. The slot-blot is described 
for a distinct application in Kafatos et al., 7 Nucl. Acid Res. 1541, 
1979, and has been used to determine the relative concentrations of 
nucleic acids in a mixture. A distinct DNA to membrane cross-linking step 
and unique filtration apparatus distinguish this method from the dot-blot 
method described above. In this embodiment of the invention, samples of 
nucleic acid (i.e., genomic DNA) are spotted on and cross-linked to a 
nitrocellulose filter (e.g., Schleicher & Schuell nitrocellulose filter) 
using UV irradiation, and the nucleic acid on the filter is hybridized 
with a labeled oligonucleotide probe. Typically, the genomic DNA is 
sheared or cleaved into smaller fragments prior to binding to the solid 
phase. The genomic DNA is cleaved or sheared enzymatically or 
mechanically, i.e., with restriction endonucleases, sonication, or other 
methods known in the art. In a preferred method, the cleaved DNA is probed 
with a riboprobe. The amount of probe hybridized to the nucleic acid in 
each of the slots is quantitated, and the quantitated amount is correlated 
with telomere length, e.g., by comparing to a standard. 
In another embodiment, one measures the loss of or decrease in bound probe 
observed after treatment of the genomic DNA with a known amount of 
exonuclease that degrades DNA specifically from the ends of the chromosome 
to measure telomere length. A preferred exonuclease is Bal31, an 
exonuclease that digests single- or double-stranded DNA specifically from 
the end of a DNA, such as the end of a telomere. The rate of Bal31 
digestion is about 50 bp/min. Thus, when chromosomal DNA is digested with 
Bal31, DNA internal to the telomeres is digested last while telomeric DNA 
is digested first. The method can be conveniently carried out by spotting 
Bal31 enzyme (i.e., serial dilutions) on a DNA binding membrane, i.e., 
nitrocellulose, located on the bottom of each well of a plate, binding 
genomic DNA to the membrane, incubating under conditions where the 
nuclease enzyme is active for a specific period of time, denaturing the 
enzyme and DNA, and hybridizing the remaining DNA to a telomeric probe, 
i.e., for human DNA, a probe comprising 5'-TTAGGG-3' repeats, under 
hybridizing conditions. The amount of probe hybridization, which should 
decrease with increasing Bal31 concentration or reaction time, can again 
be used to determine the telomere length. Preferably, this method is 
carried out in a multi-well, i.e., 96-well, format and, more preferably, 
is automated. If desired, telomeres of known length or cells comprising 
telomeres of known length can be used as standards. 
Telomere Length Measurement in Whole-Cells 
The methods of the invention can be applied to whole cells, as well as cell 
extracts. In one embodiment, whole cells are attached to a solid support 
or surface; the cells are permeabilized; the cellular DNA is denatured; 
and a labeled telomere probe (or a mixture of labeled probes) is added and 
hybridized to the telomeric repeats in the denatured DNA. In preferred 
embodiments, a fluorescein tag and "anti-fade" agents, as described below, 
are used, and the results of this "fluorescence in situ hybridization" 
(FISH) are analyzed using confocal microscopy. See Trask et al., 91 Proc. 
Natl. Acad. Sci. U.S.A. 9857, 1979, incorporated herein by reference. 
In a preferred embodiment, this method is used to measure telomere lengths 
of chromosomes in a metaphase spread. Chromosomes are stained or labeled 
with a chromosomal dye (e.g., DAPI/DA); the slides are preferably prepared 
using antifade mounting medium (e.g. 9:1 glycerol:PBS containing 0.1% 
p-phenylenediamine buffered to pH 8.0 with 0.5M carbonate/bicarbonate 
buffer). The fluorescent signal is preferably amplified. The results can 
be analyzed with an image generator in conjunction with an inverted 
fluorescence microscope. 
FISH analysis of cells or metaphase spreads of cells can be used for a 
variety of purposes: to determine average relative telomere lengths in 
cells in a tissue sample; to determine the longest telomere length in 
cells in a sample; to detect the presence of certain types of cells, i.e. 
certain stem cells can be identified by their long telomeres; to determine 
the size distribution of telomere lengths in a sample; to determine 
changes in telomere length in a cell population over time or after 
treatment with an agent or exposure to certain conditions; and to detect 
different cell types within a tissue, i.e., cancer cells can be identified 
by their having telomeres of a different length than that observed in 
normal cells, such as cells adjacent to tumor cells. 
Quantitative FISH analysis with confocal microscopy using signal 
integration also allows one to obtain an objective measure of the 
distribution of telomere lengths on different chromosomes and to identify 
chromosomes which have lost a critical amount of telomeric DNA, indicative 
of the presence of aberrant cells. The method requires only relatively 
small samples and allows for direct measurement of telomere length on a 
chromosome-by-chromosome and cell-by-cell basis. The intensity of signal 
from bound probe per chromosome or cell is proportional to the number of 
telomeric repeats, and thus to the telomere length. The method also 
provides a means to investigate telomere heterogeneity in cell or tissue 
samples; such information can be especially useful when combined with 
information regarding the presence and amount of telomerase activity in 
the sample (Harley et al. PCT Pub. No. 95/13381, published May 18, 1995; 
Harley et al. U.S. application Ser. No. 08/631,554, filed Apr. 12, 1996; 
and Harley et al. U.S. application Ser. No. 08/632,662, filed Apr. 15, 
1996). 
Many variations of in situ hybridization can be applied in the methods of 
the invention. For example, a variation of the in situ hybridization 
detection method involves primed in situ labeling ("PRINS"; Koch, J., in 
"Nonradioactive in situ Hybridization Application Manual" (1992), 
Boehringer Mannheim, 31-33). This method involves the use of a primer but 
is discussed in this section for ease of understanding. Detection of 
telomere repeats by PRINS involves using an oligonucleotide primer 
specific for telomere repeats and chain elongation incorporating labeled 
nucleotides. In a typical protocol, a PRINS reaction mixture (10 .mu.l) of 
5% (v/v) glycerol; 10 mM Tris-HCl, pH 8.3; 100 mM KCl; 0.05% (w/v) Tween 
20; 0.75 mM EGTA; 2.5 mM MgCl.sub.2 ; 0.4 .mu.M return primer; 200 .mu.M 
DATP, dGTP, dCTP; 110 .mu.M dTTP; 90 mM labeled dUTP is placed on a fixed, 
permeabilized sample, sealed with a coverslip, anchored with nail polish, 
overlayed with mineral oil, and incubated at 70.degree. C. for 30 minutes 
to 3 hours. After completion of the PRINS, the sample is washed 3 times in 
wash buffer (0.6M NaCl and 0.06M sodium citrate (4.times.SSC); 0.05% Tween 
20) heated to 70.degree. C. for 2 minutes, and the signal observed as 
described above. To reduce the background signals that can arise from 
direct incorporation of fluorescent labels during primer extension, 
indirect detection using unlabeled dNTPs and unlabeled primers can be used 
and the product detected using a labeled probe. 
In an additional preferred embodiment, the method allows one to determine 
cellular DNA content simultaneously with measuring telomere length. 
Cellular DNA content can indicate whether a cell is proliferating or 
senescent. If the cell is proliferating, the chromosomal DNA content can 
increase up to two-fold. Consequently, the signal intensity of the bound 
telomeric probe for a rapidly proliferating cell with short telomeres can 
be equal to or stronger than that of a non-dividing cell with longer 
telomeres. Thus, this method may be useful to normalize the measured 
signal intensity of the telomeric probe with respect to DNA content. The 
method comprises the steps of attaching cells or metaphase spreads to a 
support; denaturing the cellular DNA; contacting the chromosomal DNA with 
a labeled telomeric probe and a DNA specific dye; hybridizing the 
denatured DNA with the probe; and measuring the amount of probe hybridized 
and cellular DNA content simultaneously using flow cytometry. This method 
allows one to determine cell cycle position as well as telomere length. 
The intensity of signal from bound probe per chromosome or cell is 
proportional to the number of telomeric repeats, and thus to the telomere 
length. One advantage of this method is that cells can then be sorted, 
e.g., using a flow cytometer (Coulter EPICS ELITE fluorescence activated 
cell sorter (FACS)), based on telomere length. The instrument can be 
programmed to deflect the cells into specific tubes based upon telomere 
length. 
As noted above, the invention provides for the measurement of the length of 
a telomere of an individual chromosome. Flow cytometry facilitates this 
analysis. A combination of fluorescent dyes (e.g., chromomycin A3, a major 
or minor groove binding dye with relative GC specificity, and bisbenzimide 
33242, a groove binding dye with relative AT-specificity) in conjunction 
with a probe hybridized to the telomeric region, can be used to direct the 
flow cytometer to analyze and sort isolated metaphase chromosomes by 
telomere length. The chromosomes can be labeled to obtain a direct measure 
of the telomere length of an individual chromosome and subsequently sorted 
using a three-laser flow cytometer. The measurement is made by quantifying 
the fluorescent intensity for each individual chromosome using flow 
cytometry analysis. Alternatively, simultaneous three-color staining 
methods, in which the chromosomes are prepared, sorted, and subsequently 
hybridized in solution, can be applied to telomere length analysis of 
individual chromosomes. 
The methods of the invention can be performed rapidly and provide increased 
sensitivity, efficiency, reliability and accuracy. Moreover, these methods 
for telomere length measurement can be employed in a high through-put 
and/or automated process format. These telomere length measurement methods 
can be used for diagnostic, prognostic, and research applications. 
Applications 
Telomere length measurement has useful application in medical diagnostics, 
prognostics, and therapeutics. Such applications include, but are not 
limited to: (I) determination of the proliferative lifespan of cells; (ii) 
identification and analysis of the effectiveness of agents capable of 
extending, maintaining, or reducing telomere length; (iii) diagnosis of 
disease or medical conditions characterized by a different telomere length 
in a patient relative to an individual not having the disease or 
particular medical condition; (iv) prognosis of disease or medical 
conditions as correlated to telomere length; and (v) identification of 
cells, cell types, or cell populations. In general, measurement of 
telomere length provides a powerful means to assess and monitor cellular 
lifespan for a variety of useful purposes. 
The length of the telomeres of the chromosomes in a cell is indicative of 
the proliferative capacity of that cell, and so provides an indicator of 
the health of an individual or organism comprising such cells. Certain 
populations of cells may lose telomeres at a greater rate than the other 
cells within an individual. Rapid and/or extended proliferation of those 
cells may make that cell population age-limited or senescent, with 
negative impact on an individual relying on that cell population for 
health. The diagnostic procedures described herein can be used to indicate 
the potential life span of any cell type, as well as to follow telomere 
loss over time, so that revised estimates of life span can be made over 
time. 
Telomere length measurement can be used to monitor the effectiveness of 
various therapeutics in expanding and/or reducing the proliferative 
lifespan of cells. In one example, cells treated with an oligonucleotide 
comprising telomeric sequences had a reduced rate of telomere loss and an 
increased proliferative capacity of about 10 population doublings. 
Conversely, the treatment of cells with AZT or other small organic 
molecule inhibitors of telomerase can increase telomere loss and reduce 
the proliferative capacity of the treated cells. Telomere length 
measurements facilitate the analysis of the efficacy of such agents on 
cells. See U.S. Pat. No. 5,489,508, issued Feb. 2, 1996. 
Telomere length measurement can also be used to monitor the effectiveness 
of cancer chemotherapeutics during treatment. Telomere length measurement 
provides a means to determine the effectiveness of a telomerase inhibitor 
or other agent (i.e., a retinoid) that represses telomerase expression, 
because telomere length will decrease over time in dividing cancer cells 
in which there is inhibition of telomerase or telomerase expression. 
Measuring the telomere length of chromosomes in tumor cells can provide 
information regarding the proliferative capacity of such cells, both 
before and after administration of telomerase inhibitors or other 
treatments that affect telomere length. In a related application, one can 
measure the telomere length of telomeres in hematopoietic stem cells 
(HSCs) such as CD34+ cells, prior to use in bone marrow transplantation. 
The longer the telomeres, the more likely the cells will successfully 
engraft. 
The methods of the invention are also generally useful in discovering 
agents that modulate telomere length. Cells can be treated with test 
agents (e.g., synthetic compounds, fermentation extracts, nucleic acid 
preparations, and other agents) during culture to determine the effect of 
such test agents on telomere length and telomere maintenance of specific 
chromosomes. 
In diagnostic applications of the invention, telomere length measurement 
can detect a change in telomere length and/or the rate of telomere loss. A 
tissue can have a spectrum of cells of different proliferative capacity. 
Average telomere length for a tissue will be informative of the state of 
the tissue generally. Multiple measurements of telomere length over time 
can be used to determine the rate at which the telomere length changes 
over time. 
In addition, telomere length measurement methods of the invention can be 
used to diagnose the presence of abnormal chromosomes. If one uses a 
primer specific for a known subtelomeric region of an abnormal chromosome, 
such as chromosome 6 from cells of .alpha.-thalassaemia patients, the 
primer based methods of the invention can be used to diagnose the disease 
states associated with such cells. The presence of primer extension 
products is indicative of the presence of the abnormal chromosome 
indicative of the disease state. 
In prognostic applications of the invention, telomere length measurement 
can detect whether a cellular disease, such as cirrhosis of the liver or 
muscular dystrophy, has affected the proliferative capacity of the 
diseased tissue so as to impact the recuperative capacity of the patient. 
In other situations, such as those involving injury to a tissue, as in 
surgery, wounds, burns, and the like, the ability of cells, e.g., 
fibroblasts, to regenerate will be of interest, and telomere length, a 
function of proliferative capacity, provides such information. Similarly, 
in the case of bone loss, osteoarthritis, or other disease requiring 
reformation of bone, the renewal or proliferative capacity of osteoblasts 
and chondrocytes will be of interest, and again, telomere length provides 
an indicator of proliferative capacity. In addition to cellular diseases, 
diseases associated with aging can be diagnosed using the present methods. 
In these applications, telomere length provides an indicator of 
proliferative capacity, because the longer the telomere of a cell, the 
greater the potential replicative capacity that cell possesses. 
A variety of diseases and disease states are amenable to diagnostic and 
prognostic evaluation by telomere length measurement. For example, there 
is a reduction in telomere length and replicative capacity in fibroblasts 
from patients with the accelerated aging syndrome Hutchison-Gilford 
progeria relative to age-matched normal individuals (Allsopp et al., 89 
Proc. Natl. Acad. Sci. U.S.A. 10114, 1992). Accelerated telomeric loss is 
also associated with immunosenescence, such as that occurring prematurely 
in lymphocytes of individuals with Down's Syndrome (DS). DS patients show 
many features of premature aging, and lymphocytes from DS patients lose 
telomeres at three times the rate of age-matched controls (Vaziri et al., 
52 Am. J. Hum. Genet. 661, 1993). Accelerated cellular turnover and 
concomitant telomere loss per cell division correlate with the premature 
aging phenotype, and telomere loss or short telomeres in immune cells is a 
biomarker of immunosenescence. Any disease associated with a higher rate 
of cell turnover or division is amenable to diagnosis and prognosis with 
the present invention. 
For example, atherosclerosis in part results from a higher rate of cell 
turnover in the intimal and medial tissue in areas of atherosclerotic 
plaque relative to the surrounding normal tissue. Cells derived from these 
regions of atherosclerotic plaque undergo more cellular divisions than 
cells from plaque-free areas, in effect rendering the cells in plaque 
areas older and nearer to the end of their maximum replicative lifespan. 
Telomere length serves as a biomarker of cell turnover in tissues involved 
in atherosclerosis. In general, telomere loss in intimal and medial tissue 
underlying an atherosclerotic plaque is greater than that in plaque-free 
regions. 
Formation of atherosclerotic plaques occurs more often in the iliac artery 
than in the iliac vein, and as expected, the decrease in mean TRF length 
in one test was shown to be significantly greater, over the age range, 
20-60 years, for iliac arteries (-100 by/yr, P=0.01) than for iliac veins 
(47 bp/yr, P=0.14). See U.S. Pat. No. 5,489,508, issued Feb. 2, 1996. This 
decrease in mean TRF for plaque regions versus plaque-free regions of 
medial tissue from the same blood vessel is consistent with augmented cell 
turnover of tissue associated with atherosclerotic plaques. These results 
indicate that telomere length is a biomarker for cell turnover and 
proliferative capacity in tissues associated with cardiovascular disease, 
including cells of intimal and medial tissues. 
Telomere length can be used not only as a biomarker for a disease condition 
but also as a prognostic indicator of disease stage. For example, in one 
study, telomere length measurements of CD28-CD8+ cells of HIV-infected 
subjects had significantly shorter TRF lengths than those of uninfected 
controls. In addition, the telomere length measurements of the lymphocyte 
subset of CD28-CD8+ cells compared to CD28+CD8+ cells in HIV-infected 
individuals for all subjects studied were consistently shorter. In fact, 
the mean TRF lengths of CD28-CD8+ cells of the HIV-infected subjects (5-7 
kb) were similar to those observed for centenarians and for senescent 
T-cell cultures. Because loss of telomeric DNA is a marker of cell 
division, telomere shortening in CD8+ cells can be attributed to extensive 
cell division or turnover. Therefore, telomere shortening in the CD8+ 
cells can be ascribed to immune exhaustion that results from 
chronic-immune system activation and as such can be an indicator of HIV 
disease progression. Thus, telomere length in all cells such as CD8+ 
cells, CD4+ cells, and other cells of the immune system can be used for 
prognosis of the course of HIV infection or AIDS. 
After a disease is diagnosed, telomere length measurement can be used to 
determine whether the disease is at an early or late stage of disease 
progression. For leukemia, telomere length is indicative of the time since 
disease onset and the relative rate of abnormal cell proliferation. 
Leukemic cells that have been dividing at increased rates for long periods 
of time have shorter telomeres than normal bone marrow cells. There is a 
progressive decrease in mean TRF length in blood and bone marrow 
leukocytes during the course of chronic lymphoid leukemia (CLL): the 
average TRF lengths in one study of CLL patients were: normal individuals 
(controls) 10.0-16.0 kb; early-stage CLL, 7.9 kb; and late stage CLL, 4.4 
kb (Counter et al., 85 Blood 2315, 1995). 
In chronic myeloid leukemia (CML) patients, there is a wide variation in 
TRF of bone marrow cells, i.e., the TRF is 2.8-12.8 kb. In one study of 44 
CML patients, nine had a mean TRF length within the age-matched normal 
range. The remainder of the patients had short telomeres (average 5.6 kb) 
as compared to those of age-matched normal peripheral blood mononuclear 
cells. Those patients with shorter TRF lengths at the time of diagnosis 
experienced a shorter interval until blast crisis and responded less well 
to treatment than did patients with normal TRF. Telomere length depends 
upon the number of cell divisions and so, as illustrated in chronic CML 
patients, represents a new marker of disease state. CML patients having 
leukocytes with normal TRFs may be at an early stage of the disease and 
thus respond better to therapy. 
In a similar fashion, the mean TRF was much shorter in the leukocytes of 
acute myeloid leukemia (AML) patients than in control leukocytes of bone 
marrow and peripheral blood from normal individuals. In addition, blast 
cells from seven AML patients had shorter telomeres than blood mononuclear 
cells isolated during remission (Yamada et al., 95 J. Clin. Invest. 1117, 
1995). Thus, the presence of leukocytes (or other cells) with short TRF 
length is indicative of late stage or acute phase disease in these 
leukemias. 
Telomere length measurement can also be used to diagnose fertility 
problems. In one study, telomere length was measured in sperm cells from 
both fertile and infertile males. Sperm cells from certain infertile males 
had significantly shorter telomeres than did sperm cells from the fertile 
males. 
The methods of the invention have application in determining the 
proliferative capacity of a tissue as well as individual cells or cell 
types within a tissue. Many tissues regenerate from only a small number of 
stem cells. With in situ hybridization, one can identify and quantitate 
telomere length in such stem cells on an individual as well as collective 
basis. These methods allow one to determine telomere length on a 
chromosome-by-chromosome basis and to evaluate interchromosomal variance 
of telomere length. These measurements are made by quantitating the 
fluorescent intensity of bound probe for each individual cell nucleus or 
chromosome using confocal microscopy or flow cytometry analysis. Flow 
cytometry provides the added benefit of allowing the sorting of cells or 
chromosomes based on telomere length and cell type or chromosome identity 
(i.e., X chromosome separated from the Y and all other chromosomes). These 
sorted cells or chromosomes can then used as desired, i.e., for 
manipulation and/or subsequent therapeutic reintroduction into cell or, 
individual. These and other applications of the invention are further 
elaborated in the Examples below. 
EXAMPLES 
The following examples describe specific aspects of the invention to 
illustrate the invention and to provide a description of the methods for 
those of skill in the art. The examples should not be construed as 
limiting the invention, as the examples merely provide specific 
methodology useful in understanding and practicing the invention. 
EXAMPLE 1 
Dot-Blot Method for Measuring Telomere Length 
This example describes a dot-blot method for measuring telomere length. 
Telomere length is determined by correlating the signal intensity of probe 
bound to the telomere region to telomere length. To facilitate 
understanding of the method, a number of different DNA preparation steps 
that can be used in the process are described. In addition, although 
solution compositions are provided, those of skill in the art will 
recognize that variations of these solutions can readily be made by 
appropriately modifying the concentrations of the various components in, 
as well as the composition of, the solution. 
Two six-well plates of 293 cells seeded at 100,000 cells/well and one 
six-well plate of 293 seeded cells at 75,000 cells/well were washed twice 
with cold phosphate buffered saline solution (1.times.PBS is composed of 
10 mM K.sub.3 PO.sub.4 and 150 mM NaCl) to remove residual growth media. 
Cell membranes were lysed by adding 1.5 ml of extraction buffer 
(extraction buffer is 10 mM Tris, pH 8.0, 0.1M ethylenediaminotetraacetic 
acid (EDTA), pH 8.0, 0.1M NaCl, 0.5% sodium dodecyl sulfate (SDS), and 100 
.mu.g/ml proteinase K) to each well and incubating the samples at 
50.degree. C. for 3-16 hours; an additional aliquot (15 .mu.l) of 10 mg/ml 
proteinase K was typically added after 1 hour. 
After this incubation, the cellular RNA in the sample was degraded by 
adding 15 .mu.l of 500 .mu.g/ml DNase-free RNase to each well and 
incubating the samples for an additional hour at 37.degree. C. The lysed 
cell extracts (referred to below as "DNA stock solutions") were then 
removed from the wells and transferred to tubes. The tubes were then 
heated at 65.degree. C. for 10-20 minutes (at this point, the solutions 
can be snap frozen for later use). Alternate or additional DNA preparation 
steps can be used, e.g., enzymatic digestion (i.e., with proteolytic, 
RNase, or restriction enzymes), phenol extraction, and/or ethanol (EtOH) 
precipitation. 
To demonstrate linearity of signal intensity to DNA concentration, aliquots 
of the DNA stock solutions, 150 .mu.l, 75 .mu.l, 37.5 .mu.l, and 18.75 
.mu.l, were transferred from the tubes and spotted into individual wells 
of 96-well plates (Costar). The DNA was denatured in a solution of 0.4M 
NaOH and 10 mM EDTA, and the contents were transferred to 96-well filter 
plates. The filters were rinsed with 160 .mu.l of 0.4M NaOH, vacuum 
filtered, rinsed with a solution of 0.3M NaCl and 0.03M sodium citrate 
(2.times.SSC) to remove the denaturing solution, and dried. 
.sup.32 P-labeled riboprobe was prepared by combining in a sterile 
Eppendorf.TM. tube 50 .mu.l of 5.times.transcription buffer (purchased 
from Stratagene), 10 .mu.l of HindIII-digested pBLRep4 (1 .mu.g/.mu.l, 
purchased from Stratagene), 10 .mu.l of 10 mM rATP, 10 .mu.l of 10 mM 
rCTP, 10 .mu.l of 10 mM rGTP, 10 .mu.l of 0.75M dithiothreitol (DTT, 
purchased from Stratagene), 50 .mu.l of .sup.32 P-UTP (0.25 mCi), 2 .mu.l 
of T3 RNA Polymerase (purchased from Stratagene), and 98 .mu.l of 
DEPC-treated water and incubating in a 37.degree. C. water bath for 30 
minutes. Plasmid pBLRep4 comprises 100 telomere repeat sequences 
(5'-TTAGGG-3') inserted into the EcoRl site of the plasmid 
pBluescriptIISK+ (Stratagene). After incubation, the tube was pulse spun 
in a Pico Fuge.TM. (Stratagene) and then 10 .mu.l of RNase free DNase I 
(Boehringer Mannheim) was added, followed by incubation at 37.degree. C. 
for 15 minutes. To the resultant mixture was added 260 .mu.l of 
phenol/chloroform/isoamyl alcohol (PCIA, 26:25:1 ratio); the solution was 
vortexed and then certrifuged in a Pico Fuge.TM. (Stratagene) for 
approximately 4 minutes. After centrifugation, the top aqueous layer is 
transferred into another sterile, Eppendorf tube into which is also added 
approximately 26 .mu.l of 3M sodium acetate. The reaction is mixed and 
then approximately 650 .mu.l of 200 proof ethanol is added with mixing. 
The probe is precipitated out by maintaining the temperature of the tube 
at -20.degree. C. for at least 30 minutes. The tube is then centrifuged 
for 10 minutes in the Pico Fuge.TM. (Stratagene). The supernatant is 
discarded and the pellet is resuspended in 100 .mu.l of 1.times.TE buffer 
(1.times.TE buffer is composed of 10 mM Tris and 1 mM EDTA). The total 
volume in the tube is then brought up to 1 ml with 1.times.TE buffer. The 
resuspended probe can be stored at -20.degree. C. until used. 
The DNA on the filters was then hybridized with 50 .mu.l (50 .mu.Ci/.mu.g 
DNA) of the .sup.32 P-UTP labeled riboprobe (comprising repeats of the 
sequence 5'-CCCTAA-3', as discussed above) in hybridization buffer 
(hybridization buffer is composed of 6.times.SSC, 1.times.Denhardt's 
solution, 20 mM sodium phosphate, pH 7.2, and 0.4% SDS) overnight at 
65.degree. C. The filters were washed four to five times in a wash 
solution composed of 1.times.SSC and 0.1% SDS and then exposed for at 
least 1 hour in a PhosphoImager cassette (Molecular Dynamics) and scanned. 
FIG. 2 shows the results presented graphically. The signal intensity 
increased with the amount of DNA, as demonstrated by this graph. 
Known concentrations of DNA samples prepared from various cells (OVCAR 4, 
OVCAR 3, OVCAR 5, OVCAR 8, and SKOV-3) were analyzed with this method to 
measure differences in telomere length. As a comparison, the TRF lengths 
of these cells were also determined using a conventional method. Table 1 
shows that signal intensity generally increased with increased TRF using 
the dot-blot method. 
TABLE 1 
__________________________________________________________________________ 
Dot-Blot Method: Signal Intensity and TRF Length 
Sample 
TRF(kb) 
2 .mu.g DNA 
1 .mu.g DNA 
0.5 .mu.g DNA 
0.25 .mu.g DNA 
0.125 .mu.g DNA 
__________________________________________________________________________ 
OVCAR 5 
2.39 NT 851742 
439777 
211908 104927 
OVCAR 3 
3.55 2701170 
1326068 
608263 
247371 NT 
OVCAR 4 
4.89 3235960 
1600895 
688627 
262075 NT 
OVCAR 8 
7.51 2901921 
1406973 
633572 
267539 NT 
SK-OV-3 
10.69 
5612527 
2880505 
1359104 
512160 NT 
__________________________________________________________________________ 
NT not tested. 
For example, SK-OV-3 cells, which have the greatest TRF of the cells in 
these samples (10.69 kb), also exhibited the strongest signal using the 
dot-blot method at each concentration tested. 
These data show, as predicted, that the TRF length also includes at least a 
portion of the length of the subtelomeric region. Using this method, one 
can calculate the length of the subtelomeric region included in the TRF 
length. This ability to distinguish subtelomeric length versus 
5'-TTAGGG-3' telomeric repeat length has important implications in disease 
therapy and prognosis. Because the signal strength generated by this 
method is a more accurate indicator of replicative capacity than TRF 
length, this method can be used to determine which patients have a better 
prognosis and to determine the required duration of treatments, such as 
telomerase inhibition treatment. For example, as demonstrated in Table 1, 
OVCAR 8 comprises a shorter telomeric repeat region than OVCAR 4; however, 
the TRF of OVCAR 8 is greater, indicating that the TRF length for OVCAR 8 
includes a greater portion of the subtelomeric region than that of OVCAR 
4. Because the telomeric repeat length is indicative of the remaining 
proliferative capacity of the cell, a cancer patient with a tumor composed 
of cells like the OVCAR 8 cells would require a shorter period of 
treatment with a telomerase inhibitor than a patient with a tumor composed 
of cells such as OVCAR 4 cells, even though TRF length analysis would 
suggest otherwise. While the telomere length measurements undertaken in 
this example were for immortal cell lines, this method can also be used 
for mortal cell lines as a measure of replicative capacity. 
EXAMPLE 2 
Slot-Blot Method for Measuring Telomere Length 
This example illustrates use of the slot-blot method for measuring telomere 
length. In this process, the DNA is cross-linked to a solid phase prior to 
hybridization with a probe. 
About 1.5 .mu.g of total genomic DNA from BJ cells (human foreskin 
fibroblast) and S2C cells (human skin fibroblast) at different PDL was 
completely digested overnight with a mixture of restriction enzymes EcoRI 
and HindIII. In addition, 1.25 .mu.g of plasmid pBLRep4 was digested with 
restriction enzyme XbaI and used as a standard control. This amount of the 
plasmid is equivalent to 52.6 pmol of the DNA consisting of 5'-TTAGGG-3' 
repeats. The negative control, 1.5 .mu.g of Lambda phage DNA, was digested 
with restriction enzymes Sau3A and HindIII and used to calculate the 
background signal. 
The digested DNA was extracted with phenol/chloroform, precipitated with 
ethanol, and then resuspended in 100 .mu.l of 1.times.TE buffer 
(1.times.TE buffer is composed of 10 mM Tris and 1 mM EDTA). To this 
solution was added and mixed thoroughly 200 .mu.l of 5.times.SSC. 
A Schleicher & Schuell Minifold II.TM. apparatus was used to prepare the 
filters employed in the slot blot method. The blotting paper was wetted 
with 5.times.SSC, and each slot of the apparatus was rinsed with about 300 
.mu.l of 5.times.SSC. The digested DNA samples were then loaded into 
designated slots. To generate a standard curve on the blot, the digested 
plasmid pBLRep4 was diluted serially, and 0.009375 .mu.g, 0.00625 .mu.g, 
0.003125 .mu.g, and 0.0015625 .mu.g were then loaded onto separate slots. 
All liquid was vacuum filtered through the blot; the DNA was retained on 
the blot. The blot was then removed and placed on a piece of 3 MM Whatman, 
paper (VWR) and air dried for 30 minutes. 
The DNA was denatured by placing the blot on a stack of 3 MM paper soaked 
with a solution of 0.5N NaOH and 1.5M NaCl for 30 minutes. The DNA was 
then neutralized by placing the blot on 3 MM paper soaked with a solution 
of 0.5M Tris (pH 7.4) and 1.5M NaCl for 30 minutes. Following 
neutralization, the DNA was cross-linked to the blot with a UV 
STRATALINKER 1800.TM. device (Stratagene). Then, the DNA was incubated in 
15 ml of prehybridization buffer (prehybridization buffer is composed of 
5.times.SSC, 5.times.Denhardt's solution (1 g of Ficoll (Type 400, 
Pharmacia), 1 g of polyvinylpyrrolidone, 1 g of bovine serum albumin 
(Fraction V, Sigma, and sufficient water to make 100 ml), 0.02M phosphate 
(pH 6.5), 0.1 mg/ml salmon sperm DNA, 0.5% SDS, and 50% formamide) for 2 
hours. This treatment was followed by hybridization of the DNA in 15 ml of 
prehybridization buffer containing .sup.32 P-labeled 
5'-TTAGGGTTAGGGTTAGGG-3'(SEQ. ID NO.:13) probe (1 million cpm/ml), and 
incubation overnight at 37.degree. C. 
After probe hybridization, the blot was washed once with 500 ml of a 
solution composed of 1.times.SSC and 0.1% SDS for 10 minutes at room 
temperature, then washed twice with 500 ml of the same wash solution at 
37.degree. C. for 20 minutes, and then placed in a PhosphoImager.TM. 
detector overnight. The signal intensity of the hybridized probe was then 
analyzed. 
The signal intensity of probe hybridization in cells at different PDL was 
converted to a relative number that reflected the average length of 
telomeres according to the standard curve generated using the pBLRep4 
control. The results, shown in Table 2, demonstrated generally a decrease 
in telomere length with increased PDL. The results correlate well with the 
corresponding TRF lengths and signal strength determined by conventional 
methods (see FIG. 3). 
TABLE 2 
______________________________________ 
Slot-Blot Method: PDL, Signal Intensity ("SI"), and Relative 
Signal Intensity ("RSI") 
Control 
BJ Cells S2C Cells pBLRep4 
PDL SI RSI PDL SI RSI (pmol) SI 
______________________________________ 
27.5 357338 100 30 286761 
100 0.06576 
76976 
28.6 346486 97 39 258953 
90 0.132 155074 
41.2 280452 71 44 262724 
91 0.263 285200 
46.6 293800 81 47 209496 
71 0.394 402212 
57.2 270484 74 53 159030 
52 
69.2 219510 59 62 157456 
52 
72.2 230505 62 69.2 166010 
54 
73.5 194970 53 71.2 150172 
49 
80.0 219628 59 73.2 174636 
58 
88.2 119028 29 
91.4 113223 28 
______________________________________ 
The results in Table 2 show generally that bound probe signal intensity 
decreases with increased PDL. In addition, similar results were obtained 
for BJ cells and S2C cells, and signal intensity increased with increased 
amounts of the control, pBLRep4. The pBLRep4 plasmid results can be used 
to produce a standard curve generated by graphing known amounts of 
telomere repeat sequence versus the corresponding signal intensity 
determined using the slot-blot method. A standard curve generated using 
the data for pBLRep4 in Table 2 shows a linear increase in signal with 
increased amounts of pBLRep4. 
EXAMPLE 3 
Flow Cytometry for Telomere Length Quantitation 
This example illustrates a method for measuring telomere length of the 
chromosomes of an individual cell. The method can be performed 
simultaneously with the analysis of cellular DNA content. This methodology 
also allows one to sort cells or chromosomes based on telomere length. 
Growing cells are harvested by trypsinization and washed in PBS 
(phosphate-buffered saline), as per standard procedures. The washed cells 
are then fixed by adding freshly made, cold (4.degree. C.) 3:1 100% 
anhydrous methanol:glacial acetic acid to the resuspended cell pellet with 
gentle mixing. Cells can be stored at 4.degree. C. prior to analysis. 
Alternatively, if one desires to measure the telomere length of a specific 
chromosome, the cells can be lysed to release intact nuclei, which are 
then fixed by incubation overnight in 2% paraformaldehyde (pH 7.0). Lysing 
the cells is accomplished by suspending the cells in 20 mM NaCl, 10 mM 
MgCl.sub.2, 20 mM Tris (pH 7.2); incubating the cell suspension at 
37.degree. C. for 5 minutes; adding an equal volume of triton X-100 and 
mixing. For convenience, only analysis of intact cells, as opposed to 
nuclei, is described below. Those of skill in the art will recognize that 
this method is equally applicable to the analysis of telomeres of specific 
chromosomes. 
Prior to hybridization, the cells are centrifuged and washed three times 
with PBS. Cells are then treated with RNase A for 20 minutes at 37.degree. 
C. (100 .mu.g/ml in PBS) followed by pepsin treatment (1 mg/ml, pH 2.0) 
for 5 minutes at 37.degree. C. 
The cells are centrifuged and then resuspended in hybridization buffer 
composed of 70% deionized formamide containing FITC-labeled PNA probe, 
sonicated salmon sperm DNA (or other commercially available reagent to 
prevent non-specific probe hybridization), and 10 mM Tris (pH 7.2), at 
room temperature for 2-8 hours. Because all cells fluoresce, a control 
experiment is conducted under the same conditions, except that the PNA 
probe is unlabeled to determine background fluorescence. 
After the hybridization step, the cells are washed to remove unbound probe. 
The cells are washed three times in a solution composed of 70% formamide, 
10 mM Tris (pH 7.2), and 0.05% triton X-100. The cells are then 
resuspended in PBS and analyzed on a flow cytometer. 
Alternatively, a labeled DNA probe can be used in place of the PNA probe. 
If a DNA probe is used, the following procedure is implemented after 
fixing the cells. The DNA is denatured by adding 0.5 ml of a solution 
consisting of 70% deionized formamide and 2.times.SSC to the cell pellet 
(.about.1 million cells) and heating to 70.degree. C. in a water bath for 
2-5 minutes. The cells are then cooled on ice and centrifuged. The cell 
pellet is kept on ice until adding the probe to prevent renaturation of 
the DNA. The labeled DNA probe (i.e., biotin or digoxigenin) is suspended 
in 250 .mu.L of hybridization solution (70% formamide, 2.times.SSC, 
2.times.Denhardt's solution, 10% dextran sulfate, 50 mM Tris, pH 7.5), 
heated at 75.degree.-80.degree. C. for 10 minutes to denature the DNA, and 
cooled on ice. The cooled probe is added to the cell pellet and hybridized 
at 37.degree. C. overnight. The cells are then washed in 50% formamide and 
2.times.SSC at room temperature, collected by centrifugation, then 
resuspended in a solution containing streptavidin-FITC or a FITC-labeled 
anti-digoxigenin antibody. The resultant mixture is incubated at room 
temperature for one hour and the cells are collected by centrifugation and 
washed thoroughly. A control can be performed with steptavidin lacking the 
FITC label or employing a non-labeled antibody in the procedure. 
The cells are analyzed using a flow cytometer. A standard optics and filter 
arrangement for a FITC-generated signal is used (488 nm excitation, 525 nm 
bandpass filter for emission). The signals to be collected include log and 
linear FITC fluorescence (525 nm) and light scatter (0.degree. angle and 
90.degree. angle) as correlated parameters. 
During flow cytometry, the cell passes a laser at a wavelength which 
generates scattered light and fluorescence signals from the cell. The 
photomultiplier tube detects the generated photons, and the signal is 
passed through a digital-to-analog converter. The signal can be amplified. 
The resultant signals can be displayed either linearly or logarithmically. 
Logarithmic displays provide better separation of the peaks, whereas 
linear displays generally provide more sensitivity. 
The cells can also be counterstained with a DNA specific dye such as 
propidium iodide (PI) to measure cellular DNA content simultaneously. If 
counterstaining is used, the same filter set-up as described above is used 
(the PI signal is measured using a 610 nm bandpass filter). This set-up 
will allow determination of cell cycle position and cellular DNA content, 
as well as quantitation of the hybridized probe signal. The intensity of 
signal from bound probe per chromosome or cell is proportional to the 
number of telomeric repeats and to the telomere length. As the signal 
intensity is measured, the instrument can be programmed to deflect the 
cells into specific tubes based upon the signal and the corresponding 
telomere length. 
EXAMPLE 4 
PCR-Based Telomere Measurement 
This example describes a PCR-based method for measuring telomere length. 
The telomeric DNA is first treated with an exonuclease to generate blunt 
ends, and then, a double-stranded linker is attached to the 3' end of the 
telomere. A forward primer complementary to the linker and a subtelomeric 
return primer complementary to the subtelomeric region of chromosome X and 
Y are extended by PCR in the presence of nucleotide triphosphates. The 
long PCR primer extension products are then separated by size on a gel, 
and size standards on the gel are used to determine telomere length. 
Genomic DNA is digested with Bal31 nuclease (4 U/.mu.g DNA, Boehringer 
Mannheim) for 5 minutes at 30.degree. C. to remove modified nucleotides at 
the ends of the telomeres and to blunt-end the DNA. Following the 
digestion, the Bal31 nuclease is inactivated by the addition of 0.2M 
ethylenebis(oxyethylenenitrilo)-tetraacetic acid (EGTA) to a final 
concentration of 15 mM. The DNA is then extracted using phenol/chloroform, 
precipitated with ethanol, and resuspended in 1.times.T4 DNA polymerase 
buffer (13.times.T4 DNA polymerase buffer is composed of 50 mM NaCl, 10 mM 
Tris-HCl, 10 mM MgCl.sub.2, 1 mM dithiothreitol, pH 7.9) to a final DNA 
concentration of 0.1-0.5 .mu.g/.mu.l. To improve blunt-ending efficiency, 
the DNA is treated with T4 DNA polymerase (New England Biolabs) in a 
mixture comprising 0.25 mM dNTPs (0.0625 mM each of DATP, dGTP, dCTP and 
dTTP) for 30 minutes at 37.degree. C. The T4 DNA polymerase is inactivated 
by heating for 15 minutes at 65.degree. C. Alternative nucleases and DNA 
polymerases can be substituted in this reaction, i.e., Mung Bean nuclease 
or Pfu DNA polymerase. In addition, the Bal31 treatment of the DNA can be 
eliminated; and the DNA can be treated directly with T4 DNA polymerase in 
the presence of dNTPs to generate blunt ends. 
The double-stranded linker SLIC-II/aSLIC is prepared by adding equimolar 
amounts of the phosphorylated SLIC-II oligonucleotide, 
5'-GGAATTCTGGTCGACGGATCCTGA-3' (SEQ. ID NO.:8), and the non-phosphorylated 
complementary oligonucleotide aSLIC, 3'-CCTTAAGACCAGCTGCCTAGGACT-5' (SEQ. 
ID NO.:9), at room temperature, heating to 94.degree. C. for 3-4 minutes, 
placing the reaction vessel containing the oligonucleotides in a water 
bath pre-heated to 65.degree.-70.degree. C., and cooling the vessel to 
room temperature. The annealing efficiency is checked by digesting 0.5 
pmol of the prepared linker with EcoRI followed by electrophoretic 
analysis (15% polyacrylamide gel) of the digestion products and an 
untreated linker control. The ratio of the single-stranded, 
double-stranded, and digested double-stranded oligonucleotide bands on the 
gel provides a measure of the efficiency of the annealing process and a 
check that annealing is in the correct register. 
The double-stranded linker is ligated onto the blunt ends of the DNA by 
combining, at 16.degree. C., the blunt-ended DNA (0.1-0.5 .mu.g), the 
phosphorylated linker (SLIC-II/aSLIC, 1 .mu.g), deionized water (17 
.mu.l), 10.times.blunt-end ligation buffer containing 10 mM ATP (2 .mu.l, 
New England Biolabs), and T4 DNA ligase (1 .mu.l, 400 U, New England 
Biolabs). The reaction is allowed to proceed for 6-8 hours and then 
quenched by heat inactivation of the T4 DNA ligase for 15 minutes at 
65.degree. C. As a positive control to determine ligation efficiency, a 
radioactively labeled SLIC-II/aSLIC linker is prepared as above using 
.sup.32 P-labeled SLIC-II and ligated to T4 DNA polymerase-treated, MboI 
digested DNA from BJ cells and/or HinfIlRsaI digested DNA from BJ cells. 
PCR amplification of the telomeric region is accomplished by combining 0.1 
.mu.g of DNA ligated to SLIC II/aSLIC; 5 .mu.l of 2.5 mM dNTPs (0.0625 mM 
each of dATP, dGTP, dCTP and dTTP) solution; 5 .mu.l of 10.times.long PCR 
buffer (20 mM Tris.multidot.HCl (pH 9.0), 150 .mu.g/ml BSA, 3.5 mM 
MgCl.sub.2, and 16 mM (NH.sub.4).sub.2 SO.sub.4); 40 pmol of revXpJCTN 
primer (5'-CTGATWGGTCCACTTTCAGAGGG-3', (SEQ. ID NO.:7)), 1 .mu.l of 
ExTaqTM DNA polymerase (Oncor); and deionized water to a total volume of 
50 .mu.l. The reaction vessel is then transferred to a thermal cycler for 
30 cycles, each cycle comprising incubation temperatures and periods of 
94.degree. C. for 1 min. and 65.degree. C. for 1 min. and a final 
incubation at 72.degree. C. for 10 min. 
The amplified primer extension products are resolved on a 0.5% agarose gel. 
The telomere length is determined by comparison of the primer extension 
products with size standards. 
The reagents employed in the examples are commercially available or can be 
prepared using commercially available instrumentation, methods, or 
reagents known in the art. The foregoing examples illustrate various 
aspects of the invention and practice of the methods of the invention. The 
examples are not intended to provide an exhaustive description of the many 
different embodiments of the invention. Thus, although the foregoing 
invention has been described in some detail by way of illustration and 
example for purposes of clarity of understanding, those of ordinary skill 
in the art will realize readily that many changes and modifications can be 
made thereto without departing from the spirit or scope of the appended 
claims. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 13 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
TGCAATTATTTTACTATCTGTTATCGG27 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
TGACCTGTTTTAAAGAGTATGCTCAG26 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
CCCTCTGAAAGTGGACCWATCAG23 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
CTTTTATTCTCTAATCTGCTCCC23 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
TAGGGGTTGTCTCAGGGTCCTA22 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
GGGAGCAGATTAGAGAATAAAAG23 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
CTGATWGGTCCACTTTCAGAGGG23 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
GGAATTCTGGTCGACGGATCCTGA24 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
TCAGGATCCGTCGACCAGAATTCC24 
(2) INFORMATION FOR SEQ ID NO: 10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 
CCGTCGACCAGAATTCC17 
(2) INFORMATION FOR SEQ ID NO: 11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 
CAGGATCCGTCGACCAG17 
(2) INFORMATION FOR SEQ ID NO: 12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 
CCCTAACCCTAACCCTAACCCTAA24 
(2) INFORMATION FOR SEQ ID NO: 13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 
TTAGGGTTAGGGTTAGGG18 
__________________________________________________________________________