JP2001095586A - Measurement of telomere size - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of measuring the size of telomeres. SOLUTION: This method of measuring the size of telomeres is characterized by hybridizing a chromosomal DNA extracted from a sample with a labeled DNA probe bearing a sequence complementary to a telomere repetitive sequence, measuring a marker signal in the hybridized DNA probe and determining the length of telomere from the obtained measurement result.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring telomere size and a kit used for the method.

[0002]

2. Description of the Related Art The telomere constituting the chromosome end of eukaryotes is located at the end of the chromosome, and in the case of humans, is characterized by 5'-TTAGGG-3 'consisting of 6 bases repeated several hundred times. Has an array. DNA replication is essential for cells to divide and proliferate, but due to the mechanism of DNA replication, once DNA is replicated,
The terminal telomere of the chromosome shortens with each replication. Therefore, for example, in human somatic cells, cell division stops several times after several cell divisions to prevent cell death due to telomere shortening (finite division life span). It has been speculated that this cessation of cell division may be involved in the progression of human aging. That is, with each repetition of cell division, the telomere sequence becomes shorter, and when this portion disappears, chromosome-to-chromosome linkages that adversely affect cells are induced. In addition, other genetic abnormalities add to the death of the cell (cell aging and death). On the other hand, germ cells and cancer cells express telomerase having an action of elongating telomere DNA, and telomere shortening does not occur by cell division (immortalized cells).

[0003] Analysis of telomere length is indispensable for analysis of human aging and cancer progression. Southern hybridization is a method mainly used at present for analyzing telomere length. That is, after purifying the tissue DNA so as not to cut it as much as possible, cut it at an appropriate position with restriction enzymes, transfer the DNA to a membrane after electrophoresis, hybridize it with a ³² P-labeled telomere probe, and perform autoradiography. And quantifying the resulting image to determine the telomere size.

However, in the above-mentioned Southern hybridization, accurate quantification is difficult, and the operation is complicated (one week to 10 days). For example, the analysis of ³² P-labeled reaction products is still required, and their handling (eg, gel or large volume waste treatment) is not easy. further,
A long time is required for a series of operations such as time for gel preparation, separation (analysis) such as electrophoresis, and exposure (detection) time.

[0005] These are unsuitable for promptly diagnosing the progress and prognosis of cancer, in which real-time analysis is required, and it is also difficult to analyze a large amount of samples.
Therefore, development of another measurement system replacing the electrophoresis and autoradiography has been desired.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for measuring telomere size.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above problems, and as a result, hybridized a telomere repeat sequence and a labeled probe complementary thereto, and carried out a hybridization protection assay ( The present inventors have found that telomere size can be measured quickly and with high sensitivity by detecting a probe using a Hybridization Protection Assay (HPA), thereby completing the present invention.

That is, the present invention hybridizes a chromosomal DNA extracted from a specimen with a labeled DNA probe having a sequence complementary to the telomere repeat sequence, and measures the labeled signal of the hybridized DNA probe. This is a method for measuring telomere size, characterized in that the length of telomere is determined from measurement results obtained. Examples of the sample include blood, cultured cells, fresh tissue, cryopreserved tissue, and formalin-fixed tissue. The label of the probe is acridinium ester, luminol, isoluminol, pyrogallol, protohemin, aminobutylethyl-n-isoluminol or aminohexylethyl-n-
Ethyl-isoluminol.

Further, the present invention relates to (CCCTAA) _n (n is 1 to 20)
Represents an integer. ) Is a kit for measuring telomere size, comprising an oligonucleotide probe having a base sequence represented by the formula (1) and a labeling substance. The labeling substance is the same as described above. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention comprises the steps of: hybridizing a labeled probe complementary to a telomere sequence with a telomere repeat sequence which is present in a sample and whose sequence is repeatedly conserved; The telomere length is measured by using the signal amount of the telomere as an index. According to the present invention, it is only necessary to hybridize a probe directly to a telomere for detection without amplifying the telomere repeat sequence by PCR or the like, and the operation is extremely simple.

1. Preparation of cell extract from specimen The type of specimen is not particularly limited, and examples thereof include blood, cultured cells, and various tissues. The above organization
It can be arbitrarily selected regardless of the origin of the organ. For example, cerebral nervous system, muscle / skeletal system, digestive system, respiratory system,
Tissues derived from organs such as the hematopoietic system and the lymphatic system are included.
In addition, these tissues can be used in any state, such as fresh tissues (immediately after biopsy), cryopreserved tissues, or formalin-fixed tissues.

Further, the above-mentioned tissue is not limited to normal one, and tissues of various diseases (cancer, liver disease, etc.) can be used. For example, as cancer-derived, colorectal cancer, in addition to cancer tissues such as liver cancer, colorectal cancer, liver cancer, cervical cancer, chronic myelogenous leukemia, glioblastoma, breast cancer, fibrosarcoma and other cancer cell lines, such as K562, MKN1, HeLa, U937, U373MG, T98G, A1
72, MCF-7, HT-1080, LoVo, WiDr, SW857, VA-4 and the like.

[0013] Examples of liver diseases include chronic hepatitis, acute hepatitis, cirrhosis and the like. Purification of DNA from cultured cells or human tissues is performed by a known method or a modification of a known method (Tahara H. et al., (1997) Oncogene, 15, 1).
911-1920). The obtained purified DNA was subjected to hybridization buffer (lauryl sulfate, lithium chloride, EDT
A, Lithium succinate buffer containing EGTA).

Preparation of DNA from human liver disease patient tissue is performed by the following method. That is, each tissue sample is frozen in liquid nitrogen, powdered, and an aliquot (2 to 3 mg) is suspended in a hybridization buffer to obtain DNA. The released DNA is disrupted by pipetting.

2. Preparation of Probe In the present invention, an oligonucleotide having a base sequence represented by (CCCTAA) _n (n represents an integer of 1 to 20) is used as a probe. n is appropriately selected according to the target chromosomal DNA, but is preferably 2 to 10, and more preferably 3 to 5. Oligonucleotides used as probes can be synthesized by a usual phosphoramidite method using a commercially available DNA synthesizer. At the time of chemical synthesis, an amino linker for labeling acridinium ester (hereinafter referred to as “AE”) or the like is introduced.

The labeling of the DNA probe, such as AE, is carried out by reacting the AE with the amino linker introduced during DNA synthesis and the N-hydroxysuccinimide ester of AE. AE
Can be freely set depending on the position of the amino linker to be introduced during DNA synthesis.
No. 502283).

3. Hybridization A probe is mixed with the cell extract obtained as described above, and hybridized with a telomere sequence contained in the cell extract. Hybridization is performed on the extracted DNA
Is denatured by heating (for example, 95 ° C.), a hybridization solution containing a labeled DNA probe is added thereto, and the mixture is incubated at, for example, 60 to 65 ° C. for 5 to 20 minutes.

4. Detection For detection of the labeled probe, a Hybridization Protection Assay (HPA) method developed by Gen-Probe can be used (Japanese Patent Application Laid-Open No. 2-503147).

The HPA method is a technique in which an oligomer labeled with a non-radioactive labeling substance is used as a probe, and the luminescence from the non-radioactive labeling substance is detected when the probe hybridizes to DNA or RNA to be detected. It is.
The feature is that instead of performing a physical separation operation such as washing performed in order to distinguish a hybridized probe from a probe that is free without hybridization, a labeling substance of the free probe is selectively used. Hydrolysis may deactivate the labeling substance. Therefore, the operation is simple and the target (nucleic acid or oligonucleotide) can be detected in a short time.

Examples of the non-radioactive labeling substance include, for example, acridinium ester (AE), luminol (Luminol), isoluminol, pyrogallol (Pyrogallol), protohemin (Protohaemin), aminobutylethyl-n-isobutylamine Luminol (Aminobutylethyl-n-
isoluminol) or Aminohexylethyl-n-ethyl-isoluminol. However, the present invention is not limited to the above-mentioned labeling substance.

[0021]

Embedded image (Wherein X is halogen or the following formula II:

[0022]

Embedded image (Wherein X ¹ represents a nitrogen atom, a phosphorus atom, a boron atom or an arsenic atom, R ¹ represents alkoxy or aryloxy, or substituted or unsubstituted alkyl, alkenyl or aryl, R ² represents a hydrogen atom, alkoxy or represents aryloxy, or substituted or unsubstituted alkyl, alkenyl or aryl), or the following formula ^{III:. -X 2 -R 2 (} III) ( wherein, X ² represents an oxygen atom or a sulfur atom, R ² Represents the same as the above.), Y represents an oxygen atom, a sulfur atom or NH, R ³ represents a hydrogen atom, amino,
Hydroxy represents thiol, carboxylic acid, halogen, nitro, alkoxy or aryloxy, or substituted or unsubstituted acetyl, alkyl, alkenyl or aryl, R ⁴ is a substituted or unsubstituted alkyl, alkenyl or aryl, R ¹ , R ² , R ³ or R
^At least one of the four contains a reactive site capable of chemical bonding. ])) Can also be used.

Here, examples of the halogen include fluorine, chlorine, bromine, iodine and astatine.
Examples of the alkyl include those having 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms, such as methyl, ethyl, propyl, butyl, and amyl. Alkenyl includes those having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, such as vinyl and allyl. As aryl, for example, phenyl, tolyl,
Examples include naphthyl and xylyl. Examples of alkoxy include those having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, such as methoxy and ethoxy, and examples of aryloxy include phenoxy and naphthoxy.

Since the probe used in the present invention has a sequence complementary to the telomere repeat sequence, a number of probes hybridize according to the number of telomere repeats (FIG. 1).
A). In FIG. 1, since each probe 1 has a predetermined label 2, the size (length) of the telomere 3 can be measured based on the signal intensity of the label. Detection is
Even when the telomere sequence is interrupted in the middle (interruption of the telomere sequence 4), it can be detected almost in the same manner as a full-length telomere (FIG. 1B).

For example, a sequence (5'-TTAGGG-3 ') which is a basic unit of a telomere repeat sequence is repeated 1,000 times, and a sequence of (5'-CCCTAA-3') is repeated four times as a probe. Things [5 '
Assuming that-(CCCTAA) ₄ -3 '] is used, 250 probes can theoretically hybridize to telomeric DNA. Therefore, labels for 250 probes are detected. The length of the telomere can be converted by preparing a calibration curve by previously obtaining the strength of the label detected when the probe is hybridized to DNA of a known length.

Even if the telomere sequence is not the length of one full length but is interrupted in the middle, the amount of hybridization of the probe is not different from the full length. Therefore, the length of the entire length can be measured regardless of the presence or absence of telomere cleavage. It is assumed that the probe does not hybridize to the region near the cleavage site, but in this case, it is acceptable as a measurement error. Hereinafter, detection using AE and detection using other non-radioactive labeling substances will be described as examples.

Detection using AE A probe labeled with AE is added to a cell extract, and incubated at 60 ° C. for 5 to 30 minutes in order to cause hybridization between the probe and telomeric DNA. Add a hydrolysis reagent to remove chemiluminescence based on unreacted probe, and incubate at 60 ° C for 5-10 minutes. After incubation, a luminometer (eg, Lea)
The AE chemiluminescence is measured using derI).

As shown in FIG. 2, when the AE-labeled probe and the telomeric DNA hybridize, the AE is stabilized, and the ester bond of the AE is protected even after hydrolysis for a certain period of time. The AE can emit light by adding hydrogen oxide, and the target can be detected from the emission (FIG. 2 (1)).

On the other hand, if the probe and the target do not hybridize, AE cannot be stabilized. When hydrolysis is performed in this state, the ester bond of AE undergoes hydrolysis. As a result, no chemiluminescence occurs and no probe can be detected (FIG. 2 (2)).

Detection Using Acridine Derivative or Other Non-Radioactive Labeling Substance An appropriate amount of a probe labeled with the acridine derivative is added to a solution containing telomere and reacted. After the reaction, after performing a treatment such as hydrolysis, chemiluminescence is detected in the same manner as in the detection of AE.

The chemiluminescence of luminol, isoluminol, pyrogallol, protohemin, aminobutylethyl-n-isoluminol, aminohexylethyl-n-ethyl-isoluminol is detected in the same manner as described above or by other methods. can do.

In the method of the present invention, since it is not necessary to collect genomic DNA in an intact (uncut) state, it can be used not only for cultured cells, fresh tissues, but also for long-term stored tissues (for example, formalin-fixed). It becomes possible to measure the telomere size. Further, the method of the present invention can provide a highly sensitive detection result as compared with the conventional detection method. That is, the sensitivity of the purified DNA is about 1000 times that of the Southern method, and several ng of genomic DNA can be measured. Furthermore, results can be obtained in a short time (about 45 minutes or less) after tissue collection.

Furthermore, since no radioactive material is used, no special waste treatment equipment is required, and there is no need to separate the reaction product from the radioactive material that has not been taken up as in the prior art. Furthermore, since there is little variation in the measurement results, the telomere size can be measured with good reproducibility, and a large amount of sample can be easily handled.

5. Kit for measuring telomere size Kit of the present invention is a reagent used in the measurement method of the present invention,
That is, it includes an oligonucleotide having a base sequence represented by (CCCTAA) _n (n represents an integer of 1 to 20) and the labeling substance. The kit of the present invention further includes a hydrolysis reagent (a sodium tetraborate buffer containing Triton X-100), a cell lysis reagent (a lithium succinate buffer containing lauryl sulfate, lithium chloride, EDTA, and EGTA), and a standard product. (Synthetic DNA of telomere sequence and Alu sequence) can also be included.

The Alu sequence is 5'-GCCTCCCAAAGTGCTGGGATTA
It has the nucleotide sequence represented by CA-3 '(SEQ ID NO: 1), and it is known that the amount per genomic DNA is constant in cultured cells and tissues (JD Watson, Molecular Biology of Genes, p668). ). Therefore, when measuring the telomere size, the amount of the Alu sequence is also measured for each sample, and the measurement ratio between the telomere size and the Alu sequence is determined, whereby the amount of the telomere sequence per fixed amount of DNA can be determined. Further, the average telomere length can be calculated.

[0036]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. [Example 1] Measurement of telomere size (1) Preparation of chromosomal DNA Purification of DNA from a frozen sample of cultured cells or human colon was performed as follows.
A known method or a modification of a known method was performed (Tahar
a H. et al., (1997) Oncogene, 15, 1911-1920). The obtained purified DNA was dissolved in 100 μL of a hybridization buffer.

[0037]

(2) Detection by HPA 5 μL of the DNA solution was heat denatured at 95 ° C. for 5 minutes. 100 μL AE
Labeled probe (5'-CCCTAA CCCTAA CCCTAA CTCTGC T
CGAC-3 ′) (SEQ ID NO: 2) was added to the hybridization buffer and incubated at 60 ° C. for 20 minutes. The probe was labeled with AE between the 14th and 15th bases (3 × 10 ⁶ relative light units (rl
u)).

To hydrolyze the unhybridized probe, 300 μL of the hydrolysis solution (50 mL / L Trit
0.6 mol / L sodium tetraborate buffer containing on X-100 (pH 8.5) was added, and the mixture was incubated at 60 ° C. for 10 minutes. Chemiluminescence was measured using a luminometer (Leader I, Gen-Prob
In e), it was measured as the amount of chemiluminescence (rlu; relative light units) (measurement time 2 seconds / tube).

As a control experiment for measurement by the HPA method, Southern blotting was performed. That is, 2 μg of genomic DNA cut with the restriction enzyme HinfI was subjected to electrophoresis on a 7 g / L agarose gel (25 V, 20 hours), and TRF (terminal
restriction fragment) length was determined.

(3) Results The results are shown in FIG. FIG. 3A shows the results of Southern blotting, and FIG. 3B shows the results of measurement by the method of the present invention. In FIG.3A, T and N represent tumors obtained from the same patient, respectively.
DNA and normal tissue DNA, and the numbers described below represent sample numbers. In FIG. 3B, “●” indicates DN from colon tissue.
A, “○” indicates DNA from cultured cells. From the results in FIG. 3, it can be seen that there is a correlation between the TRF length obtained by the Southern method and the chemiluminescence value by the HPA method, except for three points.

Example 2 Measurement of Telomere Size from Tissue of Liver Disease Patient (1) Preparation of DNA Genomic DNA was prepared from the tissue of a human liver disease patient by the following method. Each tissue sample was frozen in liquid nitrogen, powdered, and aliquots (2-3 mg) were suspended in 100 μL of hybridization buffer to obtain genomic DNA. The released genomic DNA was divided by a pipetting operation.

(2) Detection Detection was performed in the same manner as in Example 1. In order to determine the measurement ratio between telomere and Alu, 5′-TGTAATCCCA GCACTTTGGG AGGC-3 ′ (SEQ ID NO: 3) was synthesized as a probe for the Alu sequence, and the AE label of the probe was the 10th base and the 11th base. Went between the th base.

(3) Results The results are shown in FIG. In FIG. 4, “normal” means a telomere derived from a healthy person, “CPH” is inactive chronic hepatitis,
"2A" and "2B" indicate chronic active hepatitis (B is more severe than A), and "LC" indicates telomeres from cirrhosis patients.
“0.01” on the vertical axis corresponds to a telomere length of about 2 kb. From FIG. 4, it can be seen that the more severe the liver disease (inflammation), the greater the regeneration of hepatocytes and the shorter the telomere.

[0045]

According to the present invention, a method for measuring telomere size is provided. The method of the present invention is useful for predicting aging lesions and life span in each tissue, tracking the progress of cancer and prognosis of patients.

[0046]

[Sequence List] SEQUENCE LISTING <110> Ide, Toshinori <120> Method of measuring telomere repeats <130> P99-0540 <140> <141> <160> 3 <170> PatentIn Ver. 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 1 gcctcccaaa gtgctgggat taca 24 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 2 ccctaaccct aaccctaact ctgctcgac 29 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 3 tgtaatccca gcactttggg aggc 24

[0047]

[Sequence List Free Text]

 SEQ ID NO: 1: Synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3: Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of a measurement method of the present invention.

[Explanation of symbols] 1: Probe 2: Label 3: Telomere DNA
4: fragmentation of telomere sequence

FIG. 2 is a diagram showing an outline of an HPA method.

FIG. 3 is a view showing a measurement result of a telomere length.

FIG. 4 is a diagram showing a measurement result of telomere length.

Continued on the front page F term (reference) 2G045 AA40 BA13 BB60 CA25 CA26 CB01 CB02 DA12 DA13 FB02 FB07 FB12 FB13 FB14 GC15 4B024 AA11 AA20 CA09 HA14 4B063 QA01 QA13 QA17 QQ42 QR55 QS34 QX01

Claims (5)

[Claims] 1. A chromosomal DNA extracted from a sample is hybridized with a labeled DNA probe having a sequence complementary to a telomere repeat sequence, and a labeled signal of the hybridized DNA probe is measured. A method for measuring telomere size, comprising determining the length of telomere. 2. The method according to claim 1, wherein the specimen is blood, cultured cells, fresh tissue, cryopreserved tissue or formalin fixed tissue. 3. The method according to claim 1, wherein the label is acridinium ester, luminol, isoluminol, pyrogallol, protohemin, aminobutylethyl-n-isoluminol or aminohexylethyl-n-ethyl-isoluminol. Method. 4. (CCCTAA) _n (n represents an integer of 1 to 20)
A kit for measuring telomere size, comprising an oligonucleotide having a base sequence represented by: and a labeling substance. 5. The labeling substance is an acridinium ester,
The kit according to claim 4, which is luminol, isoluminol, pyrogallol, protohemin, aminobutylethyl-n-isoluminol or aminohexylethyl-n-ethyl-isoluminol.