U.S. patent application number 12/067710 was filed with the patent office on 2009-12-03 for method for determination of the length of the g-tail sequence and kit for the method.
This patent application is currently assigned to HIROSHIMA UNIVERSITY. Invention is credited to Toshinori Ide, Hidetoshi Tahara.
Application Number | 20090298062 12/067710 |
Document ID | / |
Family ID | 37888940 |
Filed Date | 2009-12-03 |
United States Patent
Application |
20090298062 |
Kind Code |
A1 |
Tahara; Hidetoshi ; et
al. |
December 3, 2009 |
METHOD FOR DETERMINATION OF THE LENGTH OF THE G-TAIL SEQUENCE AND
KIT FOR THE METHOD
Abstract
A method of measuring the length of a G tail sequence,
characterized by hybridizing the G tail of an nondenatured
chromosomal DNA in a sample with a labeled DNA probe having a
sequence complementary to the telomere repeat sequence, measuring
chemiluminescence from the hybridized DNA probe, and determining
the length of the G tail sequence from the measured value, and a
kit used for use in such a method.
Inventors: |
Tahara; Hidetoshi;
(Hiroshima, JP) ; Ide; Toshinori; (Hiroshima,
JP) |
Correspondence
Address: |
FAY SHARPE LLP
1228 Euclid Avenue, 5th Floor, The Halle Building
Cleveland
OH
44115
US
|
Assignee: |
HIROSHIMA UNIVERSITY
Hiroshima
JP
FUJIREBIO INC.
Tokyo
JP
|
Family ID: |
37888940 |
Appl. No.: |
12/067710 |
Filed: |
September 21, 2006 |
PCT Filed: |
September 21, 2006 |
PCT NO: |
PCT/JP2006/318783 |
371 Date: |
March 27, 2009 |
Current U.S.
Class: |
435/6.1 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12Q 1/6827 20130101; C12Q 2525/151 20130101; C12Q 2545/114
20130101; C12Q 1/6841 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 21, 2005 |
JP |
2005-274523 |
Claims
1. A method of measuring the length of a G tail sequence,
comprising hydrizing a G tail of an nondenatured chromosomal DNA in
a sample with a labeled DNA probe having a sequence complementary
to a telomere repeat sequence, measuring chemiluminescence from the
hybridized DNA probe, and determining the length of the G tail
sequence from the measured value.
2. The method according to claim 1, wherein the sample is a cell
pellet of blood, a cultured cell, a fresh tissue, a cryopreserved
tissue or a formalin-fixed tissue.
3. The method according to claim 1 or 2, wherein the fact that the
chemiluminescence is based on the hybridization of the labeled DNA
probe with the G tail sequence is confirmed by using an
exonuclease.
4. The method according to claim 3, wherein the exonuclease is an
exonuclease I.
5. The method according to any one of claims 1 to 4, wherein the
label is an acridinium ester, luminol, isoluminol, pyrogallol,
protohemin, aminobutylethyl-n-isoluminol or
aminohexylethyl-n-ethyl-isoluminol.
6. The method according to any one of claims 1 to 5, wherein the
sequence complementary to the telomere repeat sequence is a base
sequence represented by (CCCTAA).sub.n (n is an integer of 1 to
10).
7. A kit for measuring the length of a G tail sequence, comprising
at least a labeled DNA probe having a sequence complementary to an
nondenatured telomere repeat sequence, a cytolytic solution, and a
hydrolytic reagent.
8. The kit according to claim 7, further comprising an exonuclease
as a confirming agent.
9. The kit according to claim 7 or 8, wherein the label is an
acridinium ester, luminol, isoluminol, pyrogallol, protohemin,
aminobutylethyl-n-isoluminol or
aminohexylethyl-n-ethyl-isoluminol.
10. The kit according to any one of claims 7 to 9, wherein the
sequence complementary to the telomere repeat sequence is a base
sequence represented by (CCCTAA).sub.n (n is an integer of 1 to
10).
11. The method according to any one of claims 1 to 7, wherein the
sample is a human- or mouse-derived sample.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of measuring the
length of a G tail sequence and a kit for use in the method.
BACKGROUND ART
[0002] Human chromosomal DNA have double-stranded DNA with repeated
sequences of 5'-TTAGGG-3' at the terminus, which are called
telomeres. However, the terminal of the telomere has a structure in
which the 3'-terminal is an overhang, and a single-stranded DNA
region of 75 to 300 bases (G tail, hereinafter referred to simply
as G tail). The G tail is normally in a protected state, as a loop
is formed, except during the access of a telomere-elongating enzyme
telomerase or during the replication of DNA (see, for example,
Nonpatent Document 1).
[0003] A telomere double-stranded region occupying the most of the
telomere is known to be shortened after each cell division and thus
involved in cell aging, but the G tail retains a certain length of
75 to 300 bases even after repeated cell division. Contradictorily,
there are reports showing that a G tail retains a certain length of
75 to 300 bases after the termination of cell division by the
shortening of a telomere double-stranded region after may cell
divisions, i.e. after the limited replicative senescence, and some
other reports showed that a G tail is shortened after the limited
replicative senescence. This is probably because there was no
method of measuring the length of a G tail accurately and
quantitatively, as the G tail is much shorter than the
telomere.
[0004] On the other hand, the recent discovery of a POT1 protein
binding to a G tail but not to a double-stranded telomere DNA, a
PIP1 protein binding to the protein, and the like, has showed that
a telomere G tail has a function completely different from that of
a double-stranded region, i.e., it is involved in direct signaling
of cell death, various cell responses, and the like, as described
below.
[0005] A telomere has telomere-binding proteins binding to the
telomere; TRF1 (Telomere repeat binding factor) and TRF2 are known
as such telomere-binding proteins; and it has been recently found
that cancer cells do not form a G tail loop in the absence of TRF2
and consequently have shortened G tails (see, for example,
Nonpatent Document 2). In such case, what is important is that the
G tail is shortened, although the entire telomere length remains
unchanged and also fused with the chromosome terminal.
[0006] In the case of a normal cell, the elimination of function of
TRF2 in the cell leads to the shortening of the G tail, the
termination of cell proliferation, and consequently to aging (see,
for example, Nonpatent Document 2). In this case too, the entire
telomere length remains unchanged, suggesting that the shortening
of the G tail triggers aging.
[0007] Various proteins such as TRF1 and TRF2 described above, as
well as ATM, NBS1, and MRN are known to be essential for the
formation of a G tail loop. DNA damage-sensitive signals, e.g.,
caused for example by various DNA damaging agents or radiation, do
not trigger the shortening of the telomere, but induce the
shortening of the G tail. This is apparent, since proteins needed
for DNA restoration (such as ATM, NBS1 and MRN) are recruited.
[0008] ATM is a gene responsible for angiectatic diseases, and NBS1
is a gene responsible for Nijmegen syndromes, i.e., a rare
autosomal recessive genetic disease characterized by its high
carcinogenicity, immunodeficiency, chromosomal instability, and
radiosensitivity. Therefore, the recruitment of these genes to the
G tail suggests some relationship of the G tail with the
above-described diseases. Actually, the inhibition of the function
of TRF2 as the adhesive of a G tail loop induces ATM-dependent
apoptosis (see, for example, Nonpatent Document 3).
[0009] It has been found that the anticancer agents
specifically-acting on a G tail lead to the shortening of the G
tail without the shortening of the telomere and consequently to the
death of cancer cells (see, for example, Nonpatent Document 4).
[0010] These results suggest that medicines and stresses causing
DNA damage transmit signals to cells via a G tail, causing various
cell responses.
[0011] In addition, a cancer-inhibiting gene product p53, of which
many variants are observed in many cancers, is known to bind to a G
tail (see, for example, Nonpatent Document 5), evidently indicating
that the change in the G tail is a signal even in diseases
associated with cancers and aging.
[0012] Since then, there are developed methods of measuring the
length of a G tail, including T-OLA
(telomere-oligonucleotide-ligation assay), PENT
(primer-extension/nick translation), 3'-overhang protection assay,
and the like (see, for example, Nonpatent Documents 6 and 7).
[0013] However, as will be described below with reference to Table
1, all these methods demand autoradiography and gel preparation
with a radioactive label (such as .sup.32P), which are troublesome
in handling. Also, electrophoresis demands an elongated period for
phoretic separation. For these reasons, all these methods are
tedious assays demanding at least two days for completion, which
are unfavorable for the real-time monitoring of progress of cancer
and rapid diagnosis of clinical outcome. In addition, these methods
could not be applied easily to the high-throughput screening for
analyzing a great number of samples.
[0014] Further, in conventional hybridization protection assay
(HPA; see, for example, Patent Document 1 and Nonpatent Document
8), which uses a chromosomal DNA after denaturation, the G tail
length, which is approximately 1/100 or less of the entire telomere
length, is within the range of its operation and measurement
errors, and therefore cannot be measured.
[0015] Specifically, because the signal intensity of a G tail
obtained by the method is so low at the noise level, it is
difficult to determine the G tail quantitatively and accurately and
also to identify whether the signal is specific to the G tail.
[0016] Patent Document 1: Japanese Unexamined Patent Publication
No. 2001-95586
Nonpatent Document 1: Griffith J D, Comeau L, Rosenfield S, Stansel
R M, Bianchi A, Moss H and de Lange T., Cell: 97 (1999),
503-14.
[0017] Nonpatent Document 2: van Steensel B, Smogorzewska A and de
Lange T., Cell: 92 (1998), 401-13.
[0018] Nonpatent Document 3: Karlseder J, Broccoli D, Dai Y, Hardy
S and de Lange T., Science: 283 (1999), 1321-5.
[0019] Nonpatent Document 4: Gomez D, Paterski R, Lemarteleur T,
Shin-Ya K, Mergny J L and Riou J F., J. Biol. Chem.: 279 (2004),
41487-94.
[0020] Nonpatent Document 5: Stansel R M, Subramanian D and
Griffith J D. J. Biol. Chem.: 277 (2002), 11625-8.
[0021] Nonpatent Document 6: Chai, W., Shay, J. W. & Wright, W.
E., Mol. Cell Biol.: 25, 2158-2168 (2005).
[0022] Nonpatent Document 7: Saldanha, S. N., Andrews, L. G. &
Tollefsbol, T. O., Eur. J. Biochem.: 270, 389-403 (2003).
[0023] Nonpatent Document 8: Nakamura, Y. et al., Clin. Chem.: 45,
1718-1724 (1999).
DISCLOSURE OF THE INVENTION
[0024] An objective of the present invention is to provide a method
of measuring the length of the sequence of a telomere
single-stranded overhang (hereinafter, referred to simply as G
tail) specifically and rapidly at high sensitivity without tedious
processing operation and denaturation, and a kit for use
therein.
[0025] After intensive studies to solve the above problems, the
inventors of the present invention have found that it is possible
to determine the length of a G tail without the denaturation of a
chromosomal DNA in a sample, by measuring chemiluminescence
intensity through a particular HPA method, especially by measuring
the G tail length quantitatively with a calibration curve drawn by
using G tail oligomer standards.
[0026] The inventors also discovered the fact that the
chemiluminescence is specific to the G tail and can be confirmed by
a exonuclease treatment. That is, it is possible to determine the
length under a condition with a higher ratio in luminescence
intensity between the exonuclease treated and untreated samples,
i.e., higher in a signal/noise ("S/N") ratio. In addition, a sample
concentration condition giving a higher S/N ratio also has been
identified. Based on these findings, the inventors have achieved
the present invention.
[0027] The present invention provides followings:
[0028] 1. A method of measuring the length of a G tail sequence,
comprising hydrizing a G tail of an nondenatured chromosomal DNA in
a sample with a labeled DNA probe having a sequence complementary
to a telomere repeat sequence, measuring chemiluminescence from the
hybridized DNA probe, and determining the length of the G tail
sequence from the measured value.
[0029] 2. The method according to Item 1, wherein the sample is a
cell pellet of blood, a cultured cell, a fresh tissue, a
cryopreserved tissue or a formalin-fixed tissue.
[0030] 3. The method according to Item 1 or 2, wherein the fact
that the chemiluminescence is based on the hybridization of the
labeled DNA probe with the G tail sequence is confirmed by using an
exonuclease.
[0031] 4. The method according to Item 3, wherein the exonuclease
is an exonuclease I.
[0032] 5. The method according to any one of Items 1 to 4, wherein
the label is a acridinium ester, luminol, isoluminol, pyrogallol,
protohemin, aminobutylethyl-n-isoluminol or
aminohexylethyl-n-ethyl-isoluminol.
[0033] 6. The method according to any one of Items 1 to 5, wherein
the sequence complementary to the telomere repeat sequence is a
base sequence represented by (CCCTAA).sub.n (n is an integer of 1
to 10).
[0034] A kit for measuring the length of a G tail sequence,
comprising at least a labeled DNA probe having a sequence
complementary to an nondenatured telomere repeat sequence, a
cytolytic solution, and a hydrolytic reagent.
[0035] 8. The kit according to Item 7, further comprising an
exonuclease as a confirming agent.
[0036] 9. The kit according to Item 7 or 8, wherein the label is an
acridinium ester, luminol, isoluminol, pyrogallol, protohemin,
aminobutylethyl-n-isoluminol or
aminohexylethyl-n-ethyl-isoluminol.
[0037] 10. The kit according to any one of Items 7 to 9, wherein
the sequence complementary to the telomere repeat sequence is a
base sequence represented by (CCCTAA).sub.n (n is an integer of 1
to 10).
[0038] 11. The method according to any one of Items 1 to 7, wherein
the sample is a human- or mouse-derived sample.
[0039] The characteristics and advantages of the present invention
described above and other advantages will be more apparent to those
skilled in the art as described below with reference to attached
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a chart showing the summary of a G tail measuring
method according to the present invention.
[0041] FIG. 2 is a graph showing the results of a dose-response
test between a 29-base AE-labeled G tail HPA probe and a 84-base
single-stranded synthetic G tail.
[0042] FIG. 3 is a graph showing the results of a specificity
confirmation test.
[0043] FIG. 4 is a graph showing the amount of an AE-based
chemiluminescence in each combination.
[0044] FIG. 5 is a graph showing the results obtained by using a
genomic DNA previously treated with ExoI before a G tail assay, and
an untreated sample.
[0045] FIG. 6-1 is a graph showing the change in the amount of
chemiluminescence that is dependent on a T7 exonuclease treatment
period of an nondenatured DNA.
[0046] FIG. 6-2 is a graph in which the rlu value of FIG. 6-1 is
converted to an average G tail length by using the calibration
curve of FIG. 2.
[0047] FIG. 7 is a graph showing the results of a sensitivity limit
confirmation test (in particular, minimum length of a detectable CG
tail).
[0048] FIG. 8 is a plot of the amount of chemiluminescence in an
arbitrary unit.
[0049] FIG. 9 is a graph showing the results when the measuring
method according to the invention is applied directly to a cell
pellet of a SiHa cancer cell line.
[0050] FIG. 10a is a graph showing the results obtained when the
measuring method according to the invention is applied directly to
each cell pellet.
[0051] FIG. 10b is a graph showing the results obtained when, for
comparison and confirmation, the measuring method according to the
invention is applied after an nondenatured genomic DNA is isolated
from each cell pellet.
[0052] FIG. 11a is a graph showing the results of measuring a G
tail by the measuring method according to the invention.
[0053] FIG. 11b is a graph showing the results of measuring the
total telomere length according to the measuring method described
in Japanese Unexamined Patent Publication No. 2001-95586 as a
Comparative Example.
[0054] FIG. 12 is a graph showing the linearity in the measurement
of G tail length by using a mouse genomic DNA.
[0055] FIG. 13 is a graph showing G tail lengths observed in mouse
tissues.
[0056] FIG. 14-1 is a plot showing the linearity in a mouse genomic
DNA quantitative determination test by using an internal standard
probe A1a.
[0057] FIG. 42 is a plot showing the linearity in the mouse genomic
DNA quantitative determination test by using an internal standard
probe A1b.
[0058] FIG. 14-3 is a plot showing the linearity in the mouse
genomic DNA quantitative determination test by using an internal
standard probe A2a.
[0059] FIG. 14-4 is a plot showing the linearity in the mouse
genomic DNA quantitative determination test by using an internal
standard probe A2b.
[0060] FIG. 14-5 is a plot showing the linearity in the mouse
genomic DNA quantitative determination test by using an internal
standard probe B2.sub.--1b.
[0061] FIG. 14-6 is a plot showing the linearity in the mouse
genomic DNA quantitative determination test by using an internal
standard probe B2.sub.--2a.
[0062] FIG. 14-7 is a plot showing the linearity in the mouse
genomic DNA quantitative determination test by using an internal
standard probe B2.sub.--2b.
[0063] FIG. 15 is a graph showing the results of measuring the
responsiveness of an AH-labeled G tail HPA probe and a
single-stranded synthetic G tail on a 96-well plate.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0064] Hereinafter, the present invention will be described in
detail.
[0065] The method of measuring the length of a tail sequence
according to the present invention is a method of determining the
length of the G tail sequence, by hybridizing the G tail with a
plurality of labeled probes complementary to the telomere repeat
sequence, constituting the G tail by using a hybridization
protection assay (HPA) method, and using the amount of
chemiluminescence emitted from a nonradioactively labeled substance
bound to the probes as an indicator.
[0066] In general, the HPA method is a method of using an oligomer
labeled with a nonradioactive labeling substance as a probe and
detecting luminescence from the nonradioactively labeled substance
when the probe is hybridized to a targeted DNA or RNA. The
characteristics of the method is that a labeled substance of a free
probe is selectively hydrolyzed and the labeling substance is
inactivated, instead of a physical separation such as washing
performed for the differentiation of a hybridized probe from a
unhybridized free probe.
[0067] Accordingly, in the present invention, which is based on the
HPA method above, a targeted G tail is detected in a short period
of time, and the length of the G tail sequence is determined by
using the amount of chemiluminescence of the labeled substance as
an indicator without a tedious operation such as the amplification
of the targeted G tail by PCR and the like.
[0068] Hereinafter, the preparation of a cell pellet containing an
nondenatured DNA will be described.
[0069] In the measuring method according to the present invention,
a G tail, which is a single-stranded region in a double-stranded
chromosomal DNA, is targeted, and therefore it is possible to use a
cell pellet containing an nondenatured chromosomal DNA as a
sample.
[0070] In the present invention, the cell pellet used as a sample
is a pellet of cells recovered after the centrifugation of cells or
tissues (e.g., at 1,000 G for 5 minutes).
[0071] The pellet may then be washed with a cold phosphate-buffered
saline (PBS(-)) twice, frozen rapidly in liquid nitrogen, and
stored in a frozen state in liquid nitrogen at low temperature (for
example at -80.degree. C.).
[0072] During use, for example, it may be resuspended in a
hybridization buffer described below, and a suspension solution may
be mixed by pipetting and sheared with a 26G syringe before
use.
[0073] In the present invention, when a cell pellet is used as a
sample, the number of cells in the sample is preferably 1.times.100
to 3.5.times.10.sup.6, more preferably 3.times.10.sup.5 to
7.times.10.sup.5, for the viewpoint of carrying out under a
condition higher in the S/N ratio.
[0074] When an nondenatured chromosomal DNA is used, the amount of
the nondenatured chromosomal DNA used is preferably 0.5 .mu.g to 40
.mu.g, more preferably 1 .mu.g to 20 .mu.g, and particularly
preferably 3 .mu.g to 7 .mu.g.
[0075] The kind of the cell sample is not particularly limited, if
it contains nondenatured chromosomal DNA's, and examples thereof
include blood, cultured cells, and various tissues.
[0076] The tissue may be arbitrarily selected, independent of the
origin of the organ. For example, it may be an tissue in organs
such as a cerebral nerve system, muscle and skeleton system,
digestive tissues, respiration system, hematopoietic system or
lymphatic system. In addition, the tissue may be any tissue, for
example, a fresh tissue (immediately after sampling by biopsy), a
cryopreserved tissue, or a formalin-fixed tissue.
[0077] The measuring method according to the present invention is
useful not only for comparison and evaluation of G tail lengths
among individuals, but also for comparison and evaluation of the G
tail length of the blood or tissue cell among different tissues in
a single individual. For example, the G tail lengths of a liver
cell, cardiac muscle cell, cerebral nerve cell, and the like, can
be compared and evaluated in a single individual.
[0078] Further, the tissue is not limited to normal tissues, and
tissues with various diseases (such as cancer and hepatic disease)
may also be used. For example, cancer-derived tissues include
cancer tissues such as of colon cancer and liver cancer; and cancer
cell lines such as cell lines of cervical duct cancer, colon
cancer, liver cancer, cervical cancer, chronic myelofibrosis,
glioblastoma, breast cancer, and fibrosarcoma; and typical examples
thereof include SiHa, K562, HeLa, U937, U373MG, T98G, A172, MCF-7,
HT-1080, LoVo, WiDr, SW857, and VA-4; and the like.
[0079] As described above in the measuring method according to the
invention, it is not necessary to purify an nondenatured
chromosomal DNA from a cultured cell or a human tissue before use,
because it is possible to use a cell pellet as it is, but a
purified nondenatured chromosomal DNA may be used, as dissolved in
a hybridization buffer described below. The nondenatured
chromosomal DNA may be purified by any method (see, for example,
Tahara H., et al., Oncogene 15 (1997), 1911-1920).
[0080] In the present invention, the hybridization buffer which
dissolves the sample, the probe described below, and the like, is
preferably a buffer which dissolves a cell membrane, a nuclear
membrane, and others, because the cell itself may be used as a
sample. Examples thereof include a lithium succinate buffer
containing a laurylsulfate salt, lithium chloride, EDTA and EGTA,
and the like.
[0081] Hereinafter, the labeled HPA probe for use in the present
invention will be described.
[0082] The labeled HPA probe for use in the present invention is an
oligonucleotide having a base sequence represented by
(CCCTAA).sub.n (n is an integer of 1 to 10) that is labeled with at
least one nonradioactive labeling substance. n is properly selected
according to a desired chromosomal DNA, but preferably 2 to 8, more
preferably 3 to 5.
[0083] The oligonucleotide for use as a probe can be prepared by a
commercially available DNA synthesizer by any DNA-producing method
such as the phosphoamidite method. During chemical synthesis, an
amino linker is preferably introduced for labeling with a
nonradicoactive labeling substance.
[0084] Examples of reagents for the introduction of an amino linker
when the phosphamidite method is used include, for example, the
following linker-introducing reagents 1 to 3.
##STR00001##
[0085] The oligonucleotide containing the introduced amino linker
can be prepared, for example, according to the method described in
Japanese Patent No. 3483829.
[0086] An acridinium ester in the present invention (hereinafter,
referred to simply as AE) is the following compound
4-(2-succinimidyloxycarbonylethyl)phenyl 10-methyl
acridinium-9-carboxylate having a phenyl ester group.
##STR00002##
[0087] With the compound AE, an oligonucleotide which introduced
the amino linker by the reaction of an N-hydroxysuccinimide ester
in the AE with an amino group in the amino linker introduced as
described above, can be labeled. Thus, a labeled HPA probe for use
in the present invention is constructed
[0088] The labeling method with AE and the operation procedure can
be performed, for example, according to the method described in
Japanese Patent No. 3483829.
[0089] The labeling site, for example with AE, can be determined
arbitrarily according to the position of an amino linker introduced
during DNA synthesis (Japanese Unexamined Patent Publication No.
2-502283).
[0090] Such a labeled HPA probe is available, for example, form
Gene Probe Inc., and an example thereof is an AE-labeling G tail
HPA probe (5'-CCCTAACCCTAACC*CTAACCCTAACCCTA-3', SEQ ID No. 1, 29
bases). * indicates the AE labeling site, and, as described above,
an amino group in an amino linker introduced to a oligonucleotide
with the introducing reagent 1, 2 or 3 is labeled by the reaction
with an N-hydroxysuccinimide ester in AE.
[0091] Examples of the nonradicactive labeling substances include,
in addition to the compound AE, lumiol, isoluminol, pyroallol,
protohemin, aminobutylethyl-n-isoluminol and
aminohexylethyl-n-ethyl-isoluminol. The nonradioactive labeling
substance has a substituent forming a chemical bond with the amino
group in the amino linker introduced to the oligonucleotide.
Examples of such a substituent group include, for example, a
N-hydroxysuccinimide ester group.
[0092] Examples thereof are not limited to the labeling substances
above and include, for example, acridine derivatives represented by
the following General Formula (I):
##STR00003##
wherein X represents a halogen atom or a group represented by the
following General Formula (II):
##STR00004##
wherein X.sup.1 represents a nitrogen, phosphorus, boron or arsenic
atom; R.sup.1 represents an alkoxy or aryloxy group or a
substituted or unsubstituted alkyl alkenyl or aryl group; and
R.sup.2 represents a hydrogen atom, an alkoxy or aryloxy group, a
substituted or unsubstituted alkyl, alkenyl or aryl group, or a
group represented by the following General Formula (III):
--X.sup.2--R.sup.2 (III)
wherein X.sup.2 represents an oxygen or sulfur atom; and R.sup.2 is
the same as that above; Y represents an oxygen or sulfur atom or a
NH group; R.sup.3 represents a hydrogen atom, an amino, hydroxy,
thiol, carboxylic acid, halogen, nitro, alkoxy or aryloxy group, or
a substituted or unsubstituted acetyl, alkyl, alkenyl or aryl
group; R.sup.4 represents a substituted or unsubstituted alkyl,
alkenyl or aryl group; and at least one group of R.sup.1, R.sup.2,
R.sup.3 and R.sup.4 contains a reactive site forming a chemical
bond with the amino linker.
[0093] Here, examples of the halogen include, for example, a
fluorine, chlorine, bromine, iodine or astatine atom. The alkyl
groups are those having 1 to 20 carbon atoms, preferably 1 to 5
carbon atoms, such as methyl, ethyl, propyl, butyl, amyl and the
like. The alkenyl groups are those having 1 to 10 carbon atoms,
preferably having 1 to 5 carbon atoms, such as example vinyl, allyl
and the like. The aryl groups are, for example, phenyl, tolyl,
naphthyl, xylyl and the like. The alkoxy groups are, for example,
those having 1 to 10 carbon atoms, preferably having 1 to 5 carbon
atoms, such as methoxy and ethoxy; and the aryloxy groups are, for
example, phenoxy, naphthoxy and the like.
[0094] The method of measuring the length of a G tail sequence
according to the present invention will be described with reference
to FIG. 1.
[0095] FIG. 1 is a chart showing the summary of the G tail
measuring method according to the present invention, in which
reference numeral 1 represents a G tail; reference numeral 2
represents a telomere G-strand; reference numeral 3 represents a
telomere C-strand; reference numeral 4 represents a double-stranded
telomere region, reference numeral 5 represents a labeled (HPA)
probe, reference numeral 6 represents the compound AE, reference
numeral 7 represents an unhybridized probe; and reference numeral 8
represents a probe inactivated by hydrolysis. The G tail 1 is
located at the terminal of the G-strand of the telomere
double-stranded region 4 consisting of chromosomal DNA terminal of
the telomere G-strand 2 and C-strand 3.
[0096] The AE-labeled probe 5 for use in the present invention is
labeled with the AE 6 and has a sequence complementary to a repeat
sequence in the G tail, and the probes in the number corresponding
to the repetition number of the repeat sequences are bound thereto
by hybridization, as shown in FIG. 1(a).
[0097] The hybridization between the AE-labeled probe 5 and the G
tail 1 is specifically performed by adding a hybridization solution
containing the AE 6--labeled probe to a cell pellet and then
incubating the mixture, for example, at 60 to 65.degree. C. for 5
to 30 minutes (heated incubation).
[0098] The AE-labeled probe 5 hybridized with the G tail 1 has the
AE stabilized; the ester bond of the AE remains protected even
after hydrolysis for a certain period in FIG. 1(b); the addition of
an alkali and hydrogen peroxide leads to chemiluminescence; and
thus, it is possible to determine the length of the G tail 1 by
measuring the amount of luminescence quantitatively, as shown in
FIG. 1(c).
[0099] On the other hand, in the probe 7, which remains
unhybridized with the G tail 1, the AE is not stabilized. When the
hydrolysis of (b) is performed in that state, the ester bond of the
AE is hydrolyzed, giving an inactivated probe 8 that shows no
chemiluminescence, thus prohibiting the detection of the
inactivated probe 8.
[0100] For the elimination of chemiluminescence due to unreacted
probe, specifically during the hydrolysis (b), a hydrolysis reagent
is added, and the mixture is incubated at 60.degree. C. for 5 to 10
minutes. The measurement of chemiluminescence of the AE in FIG.
1(c) after incubation is carried out by using a luminometer (for
example, Leader I (trade name, manufactured by Gene Probe Inc.)).
In particular, the use of a 96-well luminometer is preferable for
high-throughput screening by using the measuring method according
to the invention.
[0101] In the measuring method according to the invention, it is
preferable to confirm that the chemiluminescence is C tail-specific
by eliminating a G tail sequence selectively by treating a DNA
sample with an exonuclease I (ExoI) in such a manner that
exonuclease removes a single-stranded nucleotide in the 3' to 5'
direction.
[0102] In addition, the ratio of the signal of the
ExoI-unpretreated sample to that of the ExoI-treated sample may be
calculated as an S/N ratio. Thus, it is possible to measure the G
tail length specifically, even if there are some contaminants
present in the sample.
[0103] Further, it is also possible to confirm that the
chemiluminescence is G tail sequence-specific, by treating the G
tail with T7 exonuclease, removing the telomere C-strand 3 in the
5' to 3' direction, and elongating the G tail on the telomere
G-strand 2.
[0104] In the method according to the present invention described
above, for example, if the G tail has sequences obtained by
repeating a base unit sequence (5'-TTAGGG-3') 24 times and the
probe is [5'-(CCCTAA).sub.4-3'] in which a (5'-CCCTAA-3') sequence
is repeated four time, theoretically, six probes hybridize to the G
tail. Accordingly, labels corresponding to six probes is detected.
It is possible to calculate the length of the G tail sequence, by
previously drawing a calibration curve by determining the label
intensity when the probe is hybridized with standard DNA reagents
of known lengths.
[0105] It is also possible determine the length of the G tail by
measuring the chemiluminescence intensity quantitatively, when an
acridine derivative other than AE or another nonradioactive
labeling substance (e.g., luminol, isoluminol, pyrogallol,
protohemin, aminobutylethyl-n-isoluminol, or
aminohexylethyl-n-ethyl-isoluminol) is used as the label.
Specifically, a probe labeled with the acridine derivative or
another nonradioactive labeling substance is allowed to react with
the cell pellet in a suitable amount; after the reaction, the
mixture is processed, for example, by hydrolysis; and then, the
amount of chemiluminescence is determined quantitatively.
[0106] Hereinafter, the kit for measuring the length of a G tail
sequence according to the present invention will be described.
[0107] The kit according to the present invention is a set
containing at least a labeled DNA probe having a sequence
complementary to an nondenatured telomere repeat sequence (e.g.,
(CCCTAA).sub.n (n is an integer of 1 to 10)), a cytolytic solution,
and a hydrolytic reagent. Examples of such labeled DNA probes
include those described above as labeled HPA probes, and the
specific examples and the favorable range thereof are the same as
those above.
[0108] Examples of the cytolytic solution include, for example, a
lithium succinate buffer containing a laurylsulfate salt, lithium
chloride, and EDTA and EGTA. Examples of the hydrolytic reagent
include, for example, a tetrasodium borate buffer containing Triton
X-100.
[0109] The kit according to the present invention preferably
contains a standard for the formation of a G tail length
calibration curve (G tail sequence preferably having 20 bases or
more, more preferably 30 to 100 bases).
[0110] The kit more preferably contains a standard for the
formation of a calibration curve for the normalization of a
chromosomal DNA amount (synthetic Alu-sequence DNA preferably
having 20 bases or more, more preferably 30 to 100 bases) and an
Alu-HPA probe for the normalization of the chromosomal DNA amount
(e.g., 5'-TGTAATCCCA*GCACTTTGGGAGGC-3'; *: AE-labeling site, SEQ ID
No. 2).
[0111] The kit still more preferably contains an exonuclease (e.g.,
ExoI, T7 exonuclease, or the like, preferably ExoI) as a confirming
agent.
[0112] The kit may contain additionally, for example, a purified
chromosomal DNA of any cancer cell as a positive control DNA.
[0113] The Alu sequence is a base sequence represented by
5'-GCCTCCCAAAGTGCTGGGATTACA-3' (SEQ ID No. 3), the amount of which
in the chromosomal DNA is known to be constant also in cultured
cell (J. D. Watson Ed., Molecular Biology of the Gene, p. 668). It
is possible to determine the amount of the G tail sequence in a
certain amount of the chromosomal DNAs, by measuring the amount of
the Alu sequence added as an internal standard to each sample and
determining the rate of the G tail sequence and the Alu sequence
during the measurement or the G tail. In this way, it is possible
to calculate the average length of the G tail sequence.
TABLE-US-00001 TABLE 1 Comparison of the measuring method of the
present invention with traditional measuring methods Detection
Direct range Detection measurement Distribution of Method (nt)
Period RI.sup.(a) of cells Electrophoresis G-tail length High
throughput T-OLA 24-650 2 days Necessary Not possible Necessary
Possible Not possible PENT 130-210 2 days Necessary Not possible
Necessary Possible Not possible electrom 225-650 2 days Necessary
Not possible Necessary Possible Not possible microscopy 3'-overhang
45-384 2 days Necessary Not possible Necessary Not possible Not
possible protection assay Measuring 20-1600< 40 min. Unnecessary
Possible Unnecessary Not possible applicable.sup.(b) method of the
present invention .sup.(a)RI: Radioisotope, .sup.(b)96-multi-plate
screening
[0114] Hereinafter, the measuring method according to the invention
and the traditional method will be compared with reference to Table
1.
[0115] As apparent from Table i, the traditional methods demand a
radioactive label (RI) and gel preparation, which are troublesome
in handling and require electrophoretic separation which demands an
elongated period. Therefore, these methods are tedious assays
demanding at least two days for completion, and cells can not be
used without processing for measurement. In addition, these
traditional methods may be applied to high-throughput screening for
analyzing a great number of samples.
[0116] On the other hand, the method according to the present
invention, which does not used radioactive materials, does not
demand any special waste-processing facility or an electrophoretic
device or the like for the separation of the reactive products and
the unreacted radioactive materials. It is also possible to measure
short length G tails of up to 20 nucleotides specifically,
quantitatively and at high sensitivity, by using a single container
(e.g., test tube), within a short period (about 40 minutes or less)
from the collection of the tissues. In addition, it is possible to
determine the length of the G tail reproducibly, as the measurement
results are less dispersed and to handle a great number of samples
easily. Furthermore, the measuring method according to the present
invention allows the measurement not only of nondenatured
chromosomal DNAs but also of cells directly, and is thus applicable
to the high-throughput screening for analyzing a great number of
samples.
[0117] A genomic DNA may be collected not in an intact (uncleaved)
state, and thus, it is possible to determine the G tail length of
cultured cells, fresh tissues, and others, as well as tissues
stored for a long period (e.g., formalin-fixed tissues).
Furthermore, by the method according to the present invention, it
is possible to obtain detection results high in sensitivity than
those obtained by conventional detection methods. Specifically, the
sensitivity with a purified DNA is about 1000 times higher than
that of the Southern method, and the method of the present
invention allows analysis of several (ng) of a genomic DNA.
[0118] Hereinafter, the advantageous effects of the present
invention will be described. According to the method of measuring
the length of a G tail sequence according to the present invention,
it is possible to measure a short-length G tail of up to 20
nucleotides specifically and quantitatively at high sensitivity,
only in 3 steps without the denaturation of a telomere or any
tedious processing.
[0119] In addition, the measuring method according to the present
invention allows analysis not only of nondenatured chromosomal DNAs
but also of cells directly as samples and thus, acceleration of
analysis, and is applicable, for example, to the high-throughput
screening for analyzing a great number of samples.
[0120] Furthermore, it is possible to analyze a small amount of
cells of 5.times.10.sup.5 cells or less directly; the chromosomal
DNA is not lost during sample preparation; and thus, the method of
the present invention is useful in the cases where the number of
the cells obtained is limited, for example, during blood test or
analysis of clinical samples such as biopsy and urine for cancer
cell analysis.
[0121] With the kit for measuring the length of a G tail sequence
according to the present invention, it is possible to measure the
length of the G tail sequence in a sample within 40 minutes by
using only a single container (e.g., test tube).
[0122] The measuring method according to the present invention can
be used clinically for patients with various diseases associated
with cancer and aging, which may proceed as a result of G tail
loss.
[0123] The measuring method according to the invention is useful in
basic research on cancer, aging, and the biological impacts caused
by telomere abnormality.
EXAMPLES
[0124] Hereinafter, the present invention will be described in
detail with reference to Examples, but it should be understood that
the present invention is not restricted thereby.
Example 1
<1-1> Test for Confirming Dose-Response of Synthetic
Single-Stranded G Tail
[0125] For the confirmation of dose-response relationship by the
measuring method according to the invention, the following
hybridization buffer diluents containing a 84-base single-stranded
synthetic G tail, 5'-(TTAGGG).sub.14-3' (manufactured by Prorigo)
at various concentrations and an AE-labeled G tail HPA probe
(5'-CCCTAACCCTAACC*CTAACCCTAACCCTA-3', SEQ ID No. 1, *: AE-labeling
site, 29 bases) having a amount of chemiluminescence of
3.times.10.sup.7 relative light units (hereinafter, referred to
simply as rlu) were incubated and allowed to hybridize with each
other in 100 .mu.L of the following hybridization buffer at
60.degree. C. for 20 minutes.
[0126] The AE-labeled G tail probe was prepared by labeling, with
AE, an amino linker-introduced oligonucleotide (SEQ ID No. 1)
prepared by using the linker-introducing reagent 3, according to
the method described in Japanese Patent No. 3483829.
TABLE-US-00002 TABLE 2 Composition of hybridization buffer 0.1
mol/L Lithium succinate buffer, pH 4.7 200 g/L Lithium
laurylsulfate 1.2 mol/L Lithium chloride 20 mmol/L EDTA
[0127] Here, EDTA represents ethylenediaminetetraacetic acid, while
EGTA represents ethylene glycol bis(2-aminoethylether)tetraacetic
acid.
<1-2> Hydrolysis of Unhybridized Probe and Detection of
Chemiluminescence
[0128] The hydrolysis of the AE in an unhybridized probe was
performed by charging 300 .mu.L of a hydrolysis buffer (0.6 mol/L
tetrasodium borate buffer containing 50 mL/L of Triton X-100, pH
8.5) into each reaction tube, stirring the mixture vigorously with
a Vortex mixer, and incubating the mixture at 60.degree. C. for 10
minutes. The AE in the hybridized probe was not hydrolyzed under
the above-described condition. These tubes were cooled on ice for 1
minute or more, and the chemiluminescence in each tube was measured
with a luminometer (trade name: Leader 1, manufactured by Gene
Probe Inc.) for 2 seconds.
<1-3> Results
[0129] FIG. 2 is a graph showing the results of the dose-response
test carried out between the 29-base AE-labeled G tail HPA probe
and the single-stranded synthetic G tail (84 bases). As apparent
from FIG. 2, increase in oligonucleotide dosage in the range of
0.05 in to 10 fmol is accompanied by a linear increase of signal
intensity.
<2-1> Specificity Confirmation Test
[0130] It was to confirm whether the 29-base AE-labeled G tail HPA
probe detects the 5'-TTAGGG-3' repeat sequence constituting the G
tail specifically. The following various 84-base G tail
single-stranded DNAs (10 fmol), which are single base substitution
variants of WT (wild type) G tail, were hybridized respectively
with the 29-base AE-labeled G tail HPA probe under a condition
similar to that in <1-1> above:
[0131] WT [5'-(TTAGGG).sub.14-3']; variant G tail oligonucleotide A
[5'-(TTGGGG).sub.14-3']; variant G tail oligonucleotide B
[5'-(TTAAGG).sub.14-3']; variant G tail oligonucleotide C
[5'-(TTCGGG).sub.14-3']; and variant G tail oligonucleotide D
[5'-(TTAGGC).sub.14-3'](all G tail DNAs from Prorigo).
[0132] The hydrolysis of the AE in the unhybridized probe and the
detection of the chemiluminescence were performed under a condition
similar to that in <1-2>.
<2-2> Results
[0133] FIG. 3 is a graph showing the results of the specificity
confirmation test. In FIG. 3, WT represents a wild-type
single-stranded G tail sequence; the variant G tail nucleotide A
represents 5'-(TTGGGG).sub.4-3'; the variant G tail nucleotide B
represents 5'-(TTAAGG).sub.14-3'; the variant G tail nucleotide C
represents 5'-(TTCGGC).sub.14-3'; the variant G tail nucleotide D
represents 5'-(TTAGGC).sub.14-3'; and NC represents a negative
control. NC represents the background signal level in the test. As
apparent from FIG. 3, the HPA probe for use in the present
invention detects the desired mammal G tail sequence specifically
at a high S/N ratio.
<3-1> Test for Confirming Alkali Treatment Resistance of
AE
[0134] A test for confirming an alkali treatment resistance of AE
was performed, to know how many complete nucleotide base pairs are
needed for the hybridization between the G tail and the AE-labeled
G tail HPA probe (29 bases) for the chemiluminescence of the AE. By
using a single-strand G tail (WT) having a base length of 29 bases
similar to the AE-labeled G tail HPA probe above, the following
29-base variant G tail (Mu) containing a point mutation at a
position five bases separated from the probe-labeling site, the
common AE-labeled G tail HPA probe, and the following three kinds
of variant AE-labeled G tail HPA probes (Mut1, Mut2 and Mut3), the
combinations (i), (ii), (iii), (iv) and (v) shown in FIG. 4 were
hybridized under a condition similar to that in <1-1>, and
the amount of chemiluminescence based on that of the AE was
measured. The hydrolysis of the AE in the unhybridized probe and
the detection of the chemiluminescence were performed under a
condition similar to that in <1-2>.
[0135] Mu: 5'-TAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3'(SEQ. ID No. 4,
variant G tail) Mut1: 5'-CCCTAAC*CATAACCCTAACCCTAACCCTA-3' (SEQ ID
No. 5, *: AE-labeling site, underline: point mutation site, 29
bases) Mut2: 5'-CCCTAACCA*TAACCCTAACCCTAACCCTA-3' (SEQ ID No. 6, *:
AE-labeling site, underline: point mutation site, 29 bases) Mut3:
5'-CCCTAACCATAACC*CTAACCCTAACCCTA-3' (SEQ ID No. 7, *: AE-labeling
site, underline: point mutation site, 29 bases)<
3-2> Results
[0136] FIG. 4 is a graph showing the amount of chemiluminescence in
each combination on the basis of that of the AE. As apparent from
FIG. 4, there was almost no chemiluminescence detected in
combinations mismatched at the AE-labeling site (iv) and mismatched
at the position one base separated from the AE-labeling site (iii).
In the cases where the position five bases separated from the
AE-labeling site is mismatched ((ii) and (v)), there was only
slight deterioration in chemiluminescence. The results indicated
that mismatching at a position about six bases separated from the
AE-labeling site had no influence on HPA chemiluminescence.
<4-1> Test for Confirming Nondenatured Genomic DNA Dose
Response and G Tail Specificity (1)
[0137] For the confirmation of nondenatured genomic DNA dose
response by the measuring method according to the invention, SiHa
cancer cell line-derived nondenatured genomic DNA samples in
various amounts (u, 3 .mu.g, 5 .mu.g, 10 .mu.g and 20 .mu.g and the
AE-labeled G tail HPA probe in an amount of 3.times.10.sup.6 rlu
were hybridized with each other. Specifically for detection of the
telomere 3'-tail (G tail) in genomic DNA, the total amount of the
DNA solution in a Falcon 352053 tube (trade name) was adjusted to
100 .mu.L with sterile water or a TE buffer (10 mM Tris/HCl, 1 mM
EDTA, pH 8.0). The AE-labeled G tail HPA probe in an amount of
3.times.10.sup.6 rlu dissolved in 100-.mu.L of the hybridization
buffer was added to the DNA solution, and the mixture was stirred
thoroughly with a Vortex mixer and incubated at 60.degree. C. for
20 minutes. The hydrolysis of the AE in unhybridized probe and the
detection of the chemiluminescence were performed under a condition
similar to that in <1-2>.
<4-2> Isolation of Nondenatured Genomic DNA
[0138] In the present invention, the nondenatured genomic DNA may
not be isolated for use, but the nondenatured genomic DNA used in
the confirmation test <4-1> was previously isolated in the
following manner: The nondenatured genomic DNA used for G tail
length measurement was isolated from each cell line by using the
phenol-chloroform extraction method. Specifically, cells were
centrifuged in an Eppendorf-micro centrifuge tube at 6,000 rpm and
4.degree. C. for 5 minutes, to give a pellet in the microtube. The
pellet was washed once with PBS(-) and resuspended in an extraction
buffer containing 10 mM Tris buffer (pH 7.6), 150 mM NaCl and
NP-40, to a final concentration of 0.5% . After proteinase K
digestion, phenol-chloroform extraction was performed twice. The
genomic DNA as precipitated with ethanol, treated with RNase A, and
then, dissolved in a TE buffer.
<4-3> Exonuclease I Treatment for Confirmation of G tail
Specificity
[0139] The nondenatured genomic DNA was treated with exonuclease I
(ExoI) that removes a single-stranded nucleotide selectively in the
3' to 5' direction, eliminating the G tail sequence selectively,
consequently confirming that the chemiluminescence was G
tail-specific. The exonuclease I treatment of the nondenatured
genomic DNA was performed in the following manner: The genomic DNA
was treated with ExoI (manufactured by New England Biolabs, 0.2
U/.mu.g-DNA) in 1.times. exonuclease buffer (67 mM glycine-KOH (pH
9.5), 6.7 mM MgCl.sub.2, 10 mM 2-mercaptoethanol) at 37.degree. C.
for 2 hours, and the exonuclease was inactivated under heat at
80.degree. C. for 20 minutes before the G tail assay.
<4-4> Results
[0140] FIG. 5 is a graph showing the results obtained by using the
genomic DNA previously treated with ExoI before a G tail assay and
that not treated therewith. It is a graph obtained by plotting the
total genomic DNA amount normalized into arbitrary unit, obtained
by denaturing a 1/20 amount of the nondenatured genomic DNA by the
method described below and hybridizing it with the AE-labeled Alu
probe in an amount of 3.times.10.sup.6 rlu. As apparent form FIG.
5, a linear response was observed at the nondenatured genomic DNA
amount in the range of 1 .mu.g to 20 .mu.g. The results indicate
that 5 .mu.g of nondenatured genomic DNA may be used normally. FIG.
5 indicates that all sample were ExoI-sensitive and the detected
chemiluminescence is specific to the single-stranded G tail, and
the comparison between the ExoI-pretreated graph and
ExoI-unpretreated graph reveals that an extremely large S/N ratio
is obtained in the measurement range.
<5-1> G Tail Specificity Confirmation Test (2) and
Measurement of the Length of a G Tail Sequence
[0141] First, the nondenatured genomic DNA was treated with T7
exonuclease, eliminating the telomere C-strand in the 5' to 3'
direction and elongating the G tail in the telomere G-strand,
consequently revealing that chemiluminescence was G tail-specific.
The SiHa cancer cell line-derived nondenatured genomic DNA (5
.mu.g) was treated with T7 exonuclease in the following manner: The
genome DNA was incubated with T7 exonuclease (manufactured by New
England Biolabs, 1 U/.mu.g-DNA) in 1.times.NE Buffer 4 (50 mM
potassium acetate, 20 mM Tris acetate, mM magnesium acetate, 1 mM
dithiothreitol, pH 7.9) at 25.degree. C. for the period shown in
the Figure. The reaction was terminated by addition of EDTA (pH
8.0) to a final concentration of 25 mM.
<5-2>
[0142] The incubation and hybridization of the AE-labeled G tail
HPA probe and the nondenatured DNA the hybridization buffer for the
detection of the G tail were performed under a condition similar to
that in <4-1> above. The hydrolysis of the AE in the
unhybridized probe and detection of the chemiluminescence were
performed under a condition similar to that in <1-2>.
<5-3> Result
[0143] FIG. 6-1 is a graph showing the change in the amount of
chemiluminescence that is dependent on the T7 exonuclease treatment
period of the nondenatured DNA. The comparison of the ExoI-treated
graph and the ExoI-unpretreated graph shows that an extremely large
S/N ratio can be obtained. FIG. 6-2 is a graph in which the rlu
value of FIG. 6-1 is converted to an average G tail length by using
the calibration curve of FIG. 2. As apparent from FIG. 6-1, the
amount of chemiluminescence increased as the G tail sequence
increased, in the proportion with the period of a T7 exonuclease
treatment for the removal of telomere C-strand in the 5' to 3'
direction.
[0144] As apparent from FIG. 6-2, the SiHa cancer cell line
normally has a G tail sequence having an average length of
approximately 220 nt (nt: number of nucleotides). As apparent from
FIG. 6-2, the T7 exonuclease treatment of the SiHa cancer cell
line-derived nondenatured genomic DNA for a period of 90 seconds
until the increase of chemiluminescence resulted in the generation
of a G tail having an average length of about 1600 nt.
[0145] These results show that the measuring methosod according to
the invention can be used specifically for the measurement of the
length of the G tail sequence.
<6-1> Test for Confirming the Sensitivity Limit of the Method
According to the Present Invention
[0146] The sensitivity limit of the measuring method according to
the invention, in particular the minimum G tail length detectable,
was determined, by using synthetic telomere terminal constructs (T7
TEL_Gt10, Gt20, Gt26, Gt43 and Gt62) having 10 nt, 20 nt, 26 nt, 43
nt and 62 nt G tails respectively. Synthetic telomere terminal
constructs in amounts of 0.5, 1.0, 5.0 and 10 fmol were used in
respective tests (measurement: twice). Synthetic telomere terminal
constructs were prepared respectively by annealing DNA of SEQ ID
No. 8 and DNAs of SEQ ID Nos. 9 to 13 (all, manufactured by
Prorigo) and purifying the DNAs by gel electrophoresis.
TABLE-US-00003 T7_TEL_Gt10 (SEQ ID No. 9)
5'-TAATACgACTCACTATAgggTTAgggTTAgggTTAgggTT-3' Gt = 10 (SEQ ID No.
8) T7_TEL_Gt20 (SEQ ID No. 10)
5'-TAATACgACTCACTATAgggTTAgggTTAgggTTAgggTTAgggTTAggg-3' Gt = 20
17_TEL_Gt26 (SEQ ID No. 11)
5'-TAATACgACTCACTATAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTAggg-3' Gt =
26 T7_TEL_Gt43 (SEQ ID No. 12)
5'-TAATACgACTCACTATAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTA-
ggg-3' Gt = 43 T7_TEL_Gt62 (SEQ ID No. 13)
5'-TAATACgACTCACTATAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTAgggTTA-
gggTTAgggTTAgggTTAggg-3' Gt = 62 indicates data missing or
illegible when filed
[0147] The incubation and hybridization of the AE-labeled G tail
HPA probe and the nondenatured DNA in the hybridization buffer for
detection of G tail were performed under a condition similar to
that in <4-1> above. The hydrolysis of the AE inunhybridized
probe and detection of the chemiluminescence were performed under a
condition similar to that in <1-2>.
<6-2> Results
[0148] FIG. 7 is a graph showing the results of the sensitivity
limit confirmation test.
[0149] As apparent from FIG. 7, when a HPA probe having a 29-base
length was used, it was possible to detect a 10-nt G tail, which
was smaller than the corresponding 29-base G tail DNA, but there
was linearity obtained only in the G tail length range of 20 nt to
62 nt, indicating that the measuring method according to the
invention can be used for quantitative measurement of G tails
having a length of 20 nt or more.
<7-1> Internal Standard Test (Normalization of Total Genomic
DNA Amount)
[0150] Even when the number of cells is adjusted previously before
the practice of the measuring method according to the invention, it
is possible to normalize the total genomic DNA amount by using the
Alu DNA sequence as internal standard. For determination of the
measured rate of G tail to Alu, the nondenatured DNA used in the
measurement test according to the present invention was denatured
thermally and hybridized by using an Alu-HPA probe. The Alu-HPA
probe used was 5'-TGTAATCCCA*GCACTTTGGGAGGC-3' (*: AE-labeled site,
SEQ ID No. 2). The Alu-HPA probe was prepared by labeling the amino
linker-introduced oiligonucleotide (SEQ ID No. 2) prepared by using
the linker-introducing reagent 3, with AE according to the method
described in Japanese Patent No. 3483829.
<7-2> Results
[0151] FIG. 8 is a plot of the amount of chemiluminescence in an
arbitrary unit (measurement; thrice). As is apparent from FIG. 8, a
linear response was observed in regard to the amount of
chemiluminescence of the Alu DNA sequence at genomic DNA
concentrations in the range of 0.005 .mu.g to 1 .mu.g.
Example 2
<8-1> Measurement of G tail sequence length, directly by
using cell pellet (1)
[0152] The SiHa cancer cell line pellet prepared in the section
<8-2> below for the measurement of the length of the G tail
in cell pellet was resuspended in 100 .mu.L of the hybridization
buffer above, and the suspended solution was mixed by pipetting and
sheared with a 26G syringe before use. The AE-labeled G tail HPA
probe in an amount of 3.times.10.sup.6 rlu and the cell pellet were
incubated and hybridized under a condition similar to that in
<4-1> above. Hydrolysis and chemiluminescence detection of
the AE in the unhybridized probe were performed under a condition
similar to that in <1-2> above. The cell pellet (1/10 volume)
was denatured similarly to <7-1> above and hybridized with
3.times.10.sup.6 rlu of the AE-labeled Alu probe, for normalization
of the total genomic DNA amount.
<8-2> Preparation of Cell Pellet
[0153] A cell pellet of a SiHa cancer cell line was prepared by
collecting cells after the centrifugation of the SiHa cancer cell
line at 1,000 G for 5 minutes, washing the cells with cold PBS(-)
twice, and freezing the cells rapidly in liquid nitrogen. The cell
pellet was stored frozen at -80.degree. C. until use.
<8-3> Results
[0154] FIG. 9 is a graph showing the results when the measuring
method according to the invention is applied directly to a cell
pellet of a SiHa cancer cell line (measurement: twice).
[0155] When the rlu value shown in the graph of FIG. 9 is converted
to the average G tail length by using the calibration curve shown
in FIG. 2 above, the average G tail length of the SiHa cancer cell
line was 220 nt. As apparent from FIG. 9, there was observed
favorable linearity of the measuring method according to the
invention in the cell concentration range of 1.times.10.sup.5 to
3.5.times.10.sup.6 cells, indicating that a cell pellet containing
about 5.times.10.sup.5 cells can favorably be used for G tail
measurement.
<9-1> Measurement of G Tail Sequence Length, Directly by
using Cell Pallet (2)
[0156] The G tail measuring method according to the present
invention was applied directly to various cell pellets (various
TIG-3 human fibroblasts, various SV40-transformed cells and various
SiHa cancer cells) each containing 5.times.10.sup.5 cells; the G
tail length of each cell was determined; and the biological
properties of the cells were evaluated from the difference in G
tail length among the cells. Each cell pellet (5.times.10.sup.5
cells) prepared similarly to <8-2> above was resuspended in
100 .mu.L of the hybridization-buffer, and the suspended solution
was mixed by pipetting and sheared with a 26G syringe before
use.
[0157] The AE-labeled G tail HPA probe in an amount of
3.times.10.sup.6 rlu and the cell pellet were incubated and
hybridized under a condition similar to that in <4-1> above.
Hydrolysis and chemiluminescence detection of the AE in the
unhybridized probe were performed under a condition similar to that
in <1-2> above. The cell pellet (1/10 volume) was denatured
similarly to <7-1> above and hybridized with 3.times.10.sup.6
rlu of an AE-labeled Alu probe, for normalization of the total
genomic DNA amount. For comparison and confirmation, the
nondenatured genomic DNA was isolated from each of the cell pellets
(various TIG-3 human fibroblasts, various SV40-transformed cells
and various SiHa cancer cells) according to a method similar to
that in <4-2>, and the measuring method according to the
invention was applied to the nondenatured genomic DNA of each
cell.
<9-2> Culture of the Cells Used
[0158] The following cultured cells were used in the measuring
method according to the invention.
[0159] Normal human fibroblasts TIG-3, SVts9-3 (SV40-transformed
TIG-3), TIG-3-hTERT (human telomerase reverse transcriptase (hTERT)
cDNA-infected TIG-3), human cervical duct cancer cell line SiHa,
and retrovirus packaging cell line PT67 were cultured in a
Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%
fetal calf serum (manufactured by HyClone).
<9-3> Preparation of SiHa Cell Infected with Dominant
Negative Allelic Gene (TRF2.sup..DELTA.B.DELTA.M)
[0160] Among the cells to which the measuring method according to
the invention was applied, in preparation of the
(TRF2.sup..DELTA.B.DELTA.M)-infected SiHa cell (SiHa dn TRF2), a
pBabe-N-mycTRF2.sup..DELTA.B.DELTA.M retrovirus construct for
preparation of a retrovirus supernatant (obtained from Prof. Titia
de Lange, Rockfeller University and prepared according to the
method described in van Steensel B, Smogorzewska A and de Lange T.,
Cell, 92 (1998), 401-13) was transfected into a PT67 packaging cell
line (manufactured by BD Clontech) by using a FuGene 6 transfection
reagent (manufactured by Roche). After two days, the supernatant
was recovered; polybrene was added to a final concentration of 6
.mu.g/ml; and the mixture was filtered through a 0.22 .mu.m filter
(manufactured by Millipore). The filtered supernatant was used for
infection to the SiHa cancer cell line. On the next day, the medium
was replaced with a fresh puromycin-containing (0.5 .mu.g/ml)
complete medium, and the mixture was cultured for 4 days, to give a
TRF2.sup..DELTA.B.DELTA.M-infected SiHa cell line.
<9-4> Results
[0161] FIG. 10a is a graph showing the results obtained when the
measuring method according to the invention was applied directly to
each cell pellet. Alternatively, FIG. 10b is a graph showing the
results obtained when, for comparison and confirmation, the
measuring method according to the invention was applied after the
nondenatured genomic DNA is isolated from each cell pellet. In FIG.
10b, each right bar shows the result of G tail measurement after
ExoI treatment for confirmation of telomere G tail-specific
chemiluminescence, while each left bar, the result without ExoI
pretreatment. In FIGS. 10a and 10b, "TIG-3(Y)" represents a young
cell having a cell population doubling level (PDL) of 28;
"TIG-3(S)" represents a aged cell having a PDL of 81; "TIG-3-hTERT"
represents a hTERT-introduced cell; "SVts9-3 (50)" represents a
young SV40-transformed cell having a PDL of 50; "SVts9-3 (121)"
represents a cell in the catastrophic state having a PDL of 121;
and "SiHa" represents a control vector-infected SiHa cell; and
"SiHa dn TRF2" represents a dominant negative allelic gene (TRF2,
-infected SiHa cell.
[0162] As apparent from FIGS. 10a and 10b, the data from the cell
pellets are in good agreement with those from the purified genomic
DNA's. In addition, the comparison of the ExoI-treated graph and
ExoI-unpretreated graph reveals that an extremely large S/N ratio
is obtained. The conversion of the ordinate in each graph of FIG.
10a to the average G tail length by using the calibration curve in
FIG. 2 gave the results shown in the following Table 3.
TABLE-US-00004 TABLE 3 Measurement of G tail length Cell SV SiHa
TIG-3 TIG-3 TIG-3- ts9-3 SV ts9-3 dn (Y) (S) hTERT (50) (121) SiHa
TRF2 Average 271 220 265 250 113 220 159 G tail length (nt)
[0163] As apparent from FIG. 10 and Table 3, the average telomere G
tail length was shortened especially in the SV40-transformed crisis
cell, which is in good agreement with the traditional finding that
the human telomere G tail shorten in the crisis stage (see, for
example, Nonpatent Document 6). There was no G tail shortening
observed in the hTERT-expressing TIG-3 cell. In addition, the TRF2
dominant negative allelic gene (TRF2.sup..DELTA.B.DELTA.M) is known
to shortening of the G tail length (see, for example, Nonpatent
Document 2), and the results on an SiHa cell confirmed the expected
shortening of a G tail. The results above showed that the measuring
method according to the invention could be used for direct
measurement of G tail length by using a cell pellet and also for
biological evaluation from the intercellular difference of the
measured G tail length.
Example 3
<10-1> Test for Comparing the Measuring Method According to
the Invention with that Described in Japanese Unexamined Patent
Publication No. 2001-95586
[0164] The dominant negative allelic gene
(TRF2.sup..DELTA.B.DELTA.M) was infected to various cells (HeLa
cancer cell, SiHa cancer cell, MCF-7 cancer cell, MRC-5-hTERT
normal fibroblast, and 90p normal mammary epithelial cell)
according to a method similar to that in <9-3>; The
nondenatured DNA was isolated from each cell (HeLa cancer cell,
SiHa cancer cell, MCF-7 cancer cell, MRC-5-hTERT normal fibroblast,
and 90p normal mammary epithelial cell) according to a method
similar to that in <4-2>; The nondenatured DNA (5 .mu.g) was
used in the G tail measuring method according to the present
invention for measurement of the G tail length; and, as a
Comparative Example, the measuring method described in Japanese
Unexamined Patent Publication No. 2001-95586 was applied to each
denatured DNA (0.5 .mu.g) for measurement of the entire telomere
length. ExoI treatment was performed similarly to the method
described in <4-3> and culture of the cells used, similarly
to the method described in <9-2>.
[0165] FIG. 11a is a graph showing the results of measuring G tail
by the measuring method according to the invention. FIG. 11b is a
graph showing the results of measuring the total telomere length by
the measuring method described in Japanese Unexamined Patent
Publication No. 200-95586 as a Comparative Example. In FIG. 11a,
"C" represents a control; "T" represents a cell treated with a G
tail-shortening medicine telomestatin (5 .mu.m) for 48 hours; +
represents a ExoI-treated cell; and - represents an untreated cell.
Comparison of the ExoI-treated graph with the ExoI-unpretreated
graph reveals that an extremely large S/N ratio is obtained. In
FIG. 11b, "C" represents a control; and "T" represents a cell
previously treated with a G tail-shortening medicine telomestatin
(5 .mu.m) for 48 hours.
[0166] First, FIGS. 11a and 11b will be described briefly. The
values (arbitrary unit) on the ordinate axis were determined as the
ratios of the measured luminescence intensity to the internal
standard Alu sequence by using the same probe and the same
instrument, and thus, FIG. 11a and 11b can be compared and
evaluated. The comparison of FIGS. 11a and 11b shows that the
arbitrary intensities were approximately 2 in FIG. 11a while the
arbitrary intensities about 70 in FIG. 11b, revealing that the
signal intensity in FIG. 11a is only approximately 1/35 of that in
FIG. 11b. Further, the nondenatured DNA was used in an amount of 5
.mu.g in FIG. 11a, compared to 0.5 .mu.g in FIG. 11a, and thus, the
ratio is approximately 1/350 under the same concentration.
[0167] Thus apparently in the measuring method described in Patent
Document 1 (Japanese Unexamined Patent Publication No. 2001-95586),
the G tail length gives signal intensity only at a noise level
within the range of the operation and measurement error,
prohibiting G tail measurement. Then, the values on the ordinate in
FIG. 11a were converted to average G tail lengths by using the
calibration curve shown in FIG. 2 and the values on the ordinate in
FIG. 11b to the entire telomere lengths, to give the following
results shown in Table 4.
TABLE-US-00005 TABLE 4 G tail length and entire telomere length in
various cells Cell MRC-5- HeLa SiHa MCF-7 hTERT 90p C T C T C T C T
C T Average G tail 126 89 220 121 212 162 96 96.5 98 96 length (nt)
Entire telomere 4700 4600 4500 4600 4000 3900 6800 6750 3500 3450
length (nt)
[0168] As apparent from Table 3 and FIG. 11b, because the length of
the G tail, which is 75 to 300 bases, is significantly smaller than
that of the telomere having an entire length of 4 kbp to dozens
kbp, even if the chromosome terminal G tail was shortened forcibly
with telomestatin, for example by about 100 bases, it was not
possible to observe the difference by the traditional telomere HPA
method, i.e., the method described in Patent Document (Japanese
Uexamined Patent Publication No 2001-95586). Alternately as
apparent from Table 3 and FIG. 11a, it was possible to detect that
the G tails of HeLa a cancer cell, SiHa cancer cell, and MCF-7
cancer cell were shortened by dnTRF2 by the measuring method
according to the invention, indicating that it was possible to
detect a difference of 20 bases. In addition, the results show that
the G tails of the MRC-5-hTERT normal fibroblast and 90p normal
mammary epithelial cell were not shortened by dnTRF2.
Example 4
Measurement of G Tail Length by Using Mouse Cultured Cell
<11-1> Isolation of Nondenatured Genomic DNA
[0169] In the present invention, the nondenatured genomic DNA may
not be isolated for use, but the nondenatured genomic DNA used in
the confirmation test <11-2> was previously isolated in the
following manner: The nondenatured genomic DNA used for G tail
length measurement was isolated from each cell line by using
phenol-chloroform extraction method. Specifically, cells were
centrifuged in an Eppendorf micro centrifuge tube at 6,000 rpm and
4.degree. C. for 5 minutes, to give a pellet in the microtube. The
pellet was washed once with PBS(-) and resuspended in an extraction
buffer containing 10 mM Tris buffer (pH 7.6), 150 mM NaCl and
NP-40, to a final concentration of 0.5%. After proteinase K
digestion, phenol-chloroform extraction was performed wice. The
genoic DNA was precipitated with ethancl treated with RNase A, and
vhen, dissolved in a TE buffer.
<11-2> Test for Confirming Nondenatured Genomic DNA Dose
Response and G Tail Specificity
[0170] For confirmation of nondenatured genomic DNA dose response
by the measuring method according to the invention, NIH3T3 mouse
fibroblast-derived nondenatured genomic DNA samples in various
amounts (0.001 .mu.g, 0.003 .mu.g, 0.005 .mu.g, 0.01 .mu.g, 0.03
.mu.g, 0.05 .mu.g, 0.1 .mu.g, 0.3 .mu.g, 0.5 .mu.g, 1 .mu.g, 3
.mu.g, 5 .mu.g and 10 .mu.g) and the AE-labeled G tail HPA probe in
an amount of 3.times.10.sup.6 rlu were hybridized with each other.
Specifically for detection of the telomere 3'-tail (G tail) in a
genomic DNA, the total amount of the DNA solution in a Falcon
352053 tube (trade name) was adjusted to 100 .mu.L with sterile
water or a TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0). The
mixture was heated at 65.degree. C. in a water bath for 5 minutes;
the AE-labeled G tail HPA probe in an amount of 3.times.10.sup.6
rlu dissolved in 100-.mu.L of the hybridization buffer was added to
the DNA solution; and the mixture was stirred thoroughly with a
Vortex mixer and incubated at 60.degree. C. for 20 minutes.
Hydrolysis of the AE in unhybridized probe and detection of the
chemiluminescence were performed under a condition similar to that
in <1-2>.
<11-3> Results
[0171] As apparent from FIG. 12, there was a linear response at the
nondenatured genomic DNA concentration in the range of 0.001 .mu.g
to 10 .mu.g. The results indicate that 0.01 .mu.g of the
nodenatured genomic DNA may ha used normally.
<12-1> Measurement of G Tail Length by Using Mouse Tissue
Genomic DNAs were Isolated from Mouse Tissues of Organs such as
Liver, Kidney, Stomach, Large Intestine, and Thymus, and 1 .mu.g to
5 .mu.g of the Genomic DNAs were Used for G Tail Measurement.
<12-2> isolation of Nondenatured Genomic DNA
[0172] In the present invention, the nondenatured genomic DNA may
not be isolated for use, but the nondenatured genomic DNA used in
the confirmation test <12-1> was previously isolated in the
following manner: The nondenatured genomic DNA used for G tail
length measurement was isolated from each tissue by using
phenol-chloroform extraction method. Specifically, each tissue,
which was stored frozen at -80.degree. C., was thawed, homogenized
immediately, and resuspended in an extraction buffer containing 10
mM of Tris buffer (pH 7.6), 150 mM of NaCl, and NP-40, to a final
concentration of 0.5%. After proteinase K digestion,
phenol-chloroform extraction was performed twice. The genomic DNA
was precipitated with ethanol, treated with RNase A, and then,
dissolved in a TE buffer.
<12-3> Test for Confirming Nondenatured Genomic DNA Dose
Response and G Tail Specificity
[0173] For the confirmation of the nondenatured genomic DNA dose
response by the measuring method according to the invention, 0.5
.mu.g of nondenatured genomic DNAs derived from various mouse
tissues and the AE-labeled G tail HPA probe in a amount of
3.times.10.sup.6 rlu were hybridized with each other. Specifically
for detection of the telomere 3'-tail (G tail) in the genomic DNA,
the total amount of the DNA solution in a Falcon 352053 tube (trade
name) was adjusted to 100 .mu.L with sterile water or a TE buffer
(10 mM Tris/HCl, 1 mM EDTA, pH 8.0). The mixture was heated at
65.degree. C. in a water bath for 5 minutes; the AE-labeled G tail
HPA probe in an amount of 3.times.10.sup.6 rlu dissolved in
100-.mu.L of the hybridization buffer was added to the DNA
solution; and the mixture was stirred thoroughly with a Vortex
mixer and incubated at 60.degree. C. for 20 minutes. Hydrolysis of
the AE in unhybridized probe and detection of the chemiluminescence
were performed under a condition similar to that in
<1-2>.
<12-4> Results
[0174] It was possible to detect G tail signals by the present
measuring method in various tissues of small intestine, large
intestine, testicle, and the like. Relatively longer G tails were
detected in small intestine and testicle, while the G tail in large
intestine was shorter. FIG. 13 shows that all samples were
ExoI-sensitive which confirmed that the detected chemiluminescence
was specific to the single-stranded G tail, while the comparison of
the ExoI-treated graph and the ExoI-unpretreated graph shows that
it is possible to measure the G tail sufficiently also in mouse
tissues.
<13-1> Internal Standard Test by Using Mouse (Normalization
of Total Genomic DNA Amount)
[0175] It is possible to normalize the total moose genomic DNA
amount by using a repeated sequence A1 or B2 present in mouse as an
internal standard, even when the cell number is adjusted before the
measuring method according to the invention is carried out. For
determination of the measurement ratio of the G tail to A1, A2 or
B2, the nondenatured DNA used in the measurement test according to
the present invention was denatured thermally and hybridized by
using an A1, A2 or B2-HPA probe. The A1, A2 or B2-HPA probe used
was A1a probe: 5'-GAA CAG TGT ATA T*C AAT GAG TTA CAA T-3' (SEQ ID
No. 14); A1b probe: 5'-GAA CAG TGT ATA TCA A*T GAG TTA CAA T-3'
(SEQ ID No. 15); A2a probe: 5'-CGT TGG AA* ACG GGA TTT GTA GAA
CA-3' (SEQ ID No. 16); A2b probe: 5'-CGT TGG AAA CGG GA* TTT GTA
GAA CA-3' (SEQ ID No. 17); B21b probe: 5'-GTC TGA AGA CA* GCT ACA
GTG TA-3' (SEQ ID No. 18); B2-2a probe: 5'-CCG ACT G*C TCT TCT GAA
GGT C-3' (SEQ ID No. 19); or B2.sub.--2b probe: 5'-CCG ACT GCT CTT
C*T GAA GGT C-3' (SEQ ID No. 20) (in sequences above, "*"
represents the AE-labeling site).
[0176] The A1, A2 or B1-HPA probe was prepared by labeling the
amino linker-introduced oligonucleotide (SEQ ID No. 2) prepared by
using the linker-introducing reagent 3 according to the method
described in Japanese Patent No. 3483829 with AE.
<13-2> Results
[0177] FIGS. 14-1 to 14-7 are clots of the amount of
chemiluminescence in and arbitrary unit (measurement: thrice). As
apparent from FIGS. 14-1 to 14-7, it was possible obtain linear
response in chemiluminescence by the A1, A2 or B2 DNA sequence in
the genomic DNA concentration range of 0.5 .mu.g to .mu.g.
Example 5
Measurement of G Tail Length by Using a 96-Well Plate
<14-1> Test for Confirming Synthetic Single-Stranded G Tail
Dose-Response Relationship by Using a 96-Multi-Well Plate
[0178] For the confirmation of dose-response relationship by the
measuring method according to the invention, the following
hybridization buffer diluents (30 .mu.L) containing a synthetic
single-stranded G tail, 84 bases, 5'-(TTAGGG).sub.14-3'
(manufactured by Prorigo) at various concentrations and an
AE-labeled G tail HPA probe (5'-CCCTAACCCTAACC*CTAACCCTAACCCTA-3',
SEQ ID No. 1, *AE-labeling site, 29 bases), having a amount of
chemiluminescence of 3.times.10.sup.6 relative light units
(hereinafter, referred to simply as rlu), were incubated and
allowed to hybridize with each other in 30 .mu.L of the following
hybridization buffer described in Example 1 <1-1> at
70.degree. C. for minutes, for example, in Thermomixer Comfort
(manufactured by Eppendorf). The temperature and the period may be
modified according to the apparatus used.
[0179] The AE-labeled G tail probe was prepared by labeling the
amino linker-introduced oligonucleotide (SEQ ID No. 1) prepared by
using the linker-introducing reagent 3, with AE according to the
method described in Japanese Patent No. 3483829.
<14-2> Hydrolysis of Unhybridized Probe and Chemiluminescence
Detection
[0180] The hydrolysis of the AE in the unhybridized probe was
performed by charging 90 .mu.L of a hydrolysis buffer (0.6 mol/L
tetrasodium borate buffer containing 50 mL /L of Triton X-100, pH
8.5) into each reaction tube, stirring the mixture thoroughly, and
incubating the mixture at 70.degree. C. for 25 minutes on a plate
block heater. The AE in hybridized probe was not hydrolyzed under
the condition. These tubes were left at room temperature for about
2 minutes; in a 96-well-plate-compatible luminometer (allowing
simultaneous addition of two reagents, GloMax (trade name) 96
Microplate Luminometer w/Dual Injectors (trade name), manufactured
by Promega), 60 .mu.L of a reader I solution was added thereto; 60
.mu.L of a reader II solution was added additionally after two
seconds; and the chemiluminescence was measure for a measuring
period of 2 seconds.
<14-3> Results
[0181] FIG. 15 is a graph showing the results of a dose-response
test on G tail measurement carried out on a 96-well plate by using
the 29-base AE-labeled G tail HPA probe and the single-stranded
synthetic 84-base G tail. As apparent from FIG. 15, the signal
intensity increased linearly as the increase in oligonucleotide
dosage in the range of 0.05 fmol to 10 fmol.
INDUSTRIAL APPLICABILITY
[0182] The present invention provides a method of measuring the
length of the sequence of the telomere single-stranded tail (G
tail) specifically and rapidly at high sensitivity without tedious
processing operation and denaturation and a kit for use
therein.
[0183] The kit according to the present invention is useful as a
test reagent for patients with various diseases relevant to cancer
and aging, which are regarded as diseases associated with G tail
loss. It is also useful for use in basic physiological research on
diseases associated with aging, cancer, and telomere
abnormality.
[0184] The present invention has been described with reference to
its favorable embodiments, but the description above is not aimed
at restricting the invention in any way unless specified otherwise,
and the invention should be construed widely in the spirit and the
scope of the invention specified by the Claims attached.
Sequence CWU 1
1
20129DNAArtificialG-tail probe 1ccctaaccct aaccctaacc ctaacccta
29224DNAArtificialAlu-probe 2tgtaatccca gcactttggg aggc
24324DNAHomo sapiens 3gcctcccaaa gtgctgggat taca
24429DNAArtificialMu(G-tail) probe 4tagggttagg gttagggtta gggttaggg
29529DNAArtificialMut1 probe 5ccctaaccat aaccctaacc ctaacccta
29629DNAArtificialMut2 probe 6ccctaaccat aaccctaacc ctaacccta
29729DNAArtificialMut3 probe 7ccctaaccat aaccctaacc ctaacccta
29830DNAArtificialC-strand of control for sensitivity test
8ctaaccctaa ccctatagtg agtcgtatta 30940DNAArtificialGt10 control
for sensitivity test 9taatacgact cactataggg ttagggttag ggttagggtt
401050DNAArtificialGt20 control for sensitivity test 10taatacgact
cactataggg ttagggttag ggttagggtt agggttaggg 501156DNAArtificialGt26
control for sensitivity test 11taatacgact cactataggg ttagggttag
ggttagggtt agggttaggg ttaggg 561274DNAArtificialGt43 control for
sensitivity test 12taatacgact cactataggg ttagggttag ggttagggtt
agggttaggg ttagggttag 60ggttagggtt aggg 741392DNAArtificialGt62
control for sensitivity test 13taatacgact cactataggg ttagggttag
ggttagggtt agggttaggg ttagggttag 60ggttagggtt agggttaggg ttagggttag
gg 921427DNAArtificialA1a probe 14gaacagtgta tatcaatgag ttacaat
271527DNAArtificialA1b probe 15gaacagtgta tatcaatgag ttacaat
271625DNAArtificialA2a probe 16cgttggaaac gggatttgta gaaca
251725DNAArtificialA2b probe 17cgttggaaac gggatttgta gaaca
251822DNAArtificialB2_1b probe 18gtctgaagac agctacagtg ta
221921DNAArtificialB2_2a probe 19ccgactgctc ttctgaaggt c
212021DNAArtificialB2_2b probe 20ccgactgctc ttctgaaggt c 21
* * * * *