JP2001161364A - Pna skeleton telomere recognition probe and method for determining telomere length - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly and accurately determine the length of a telomere using a small amount of DNA so as to easily assess the extent of skin aging. SOLUTION: The subject method comprises determining the length of a telomere by electrophoretic separation of a telomere DNA obtained from cells and detecting the telomere DNA using a PNA skeleton telomere recognition probe; wherein the probe to be used is such as to have a PNA skeleton, have a base sequence of N-terminal-(CCCTAA)n-C-terminal or N-terminal-(TTAGGG)n- C-terminal (n is 3-5) and be labeled with a radioisotope, fluorescent compound, chemiluminescent compound or enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for determining the length of a DNA fragment containing a telomere region (ie, telomere length) by using a specific PN.
Regarding the method of measuring using a probe of A skeleton, in particular,
The present invention relates to a method for measuring the telomere length of skin epidermal cells and further evaluating the degree of skin aging based on the obtained telomere length of skin epidermal cells.

[0002]

BACKGROUND ART Physiological functions of living organisms decrease with aging, and aging occurs. Similarly, aging occurs in individual cells constituting a living body in a process of changing the traits of the cells, regressing the functions, stopping the proliferation, and death of the cells.

[0003] With respect to such cell aging, in normal human cells, the telomere located at the end of the chromosome is shortened when the cells are passaged repeatedly, and this shortening is related to the life span of the cell (Harley). et al. 1990; Allsop et al. 199
2) It has also been reported that cells collected from older humans tend to have a shorter telomere length and a smaller number of cell divisions (lifetime) during culture (Hastie et al.) . 1990; Lindsey et al. 199
1). In addition, it has been reported that the telomere length of skin cells almost decreases with age, but reflects the degree of cell damage that the skin has suffered in the past (Seii).
chi Y. et al., Fragrance Journal, (1999), 27 , 69-7
Five). For this reason, the telomere length of skin cells has attracted attention as a new index for objectively evaluating the true degree of skin aging (skin age) that is not affected by physiological conditions or climatic conditions at the time of measurement. Therefore, there is an increasing demand for simple and quick measurement of telomere length in the field of beauty as well as in medicine.

[0004]

The conventional technique for measuring the telomere length of skin epidermal cells is based on a large-scale method requiring one million or more skin epidermal cells. Specifically, skin tissue is excised by surgical operation or biopsy, DNA is extracted from the excised skin tissue, fragmented with a restriction enzyme, and a radioisotope (RI) -labeled probe is hybridized thereto, and densitized. Detect autoradiograms by metrology. In addition to requiring a significant amount of more than 1 million cells, this method can only be performed at a special radioactive safety management facility approved by the Science and Technology Agency to use RI as a label. Due to restrictions, it takes time and effort.

Accordingly, it is an object of the present invention to measure telomere length simply and quickly and with high sensitivity from a small amount of skin epidermal cells, and to easily evaluate the degree of skin aging.

[0006]

Means for Solving the Problems The present inventors have determined that a specific P
By using a probe having an NA skeleton, it has been found that telomere DNA can be detected extremely easily and with high sensitivity and telomere length can be measured, and the present invention has been completed.

[0007] That is, the present invention relates to telomere D obtained from cells.
A probe PNA backbone NA hybridizes, N-terminal - (CCCTAA) _n -C-terminus or N-terminus - has (TTAGGG) nucleotide sequence of _n -C-terminal (n = 3~5), radioisotope Provided is a PNA backbone telomere recognition probe that is labeled with a body, a fluorescent compound, a chemiluminescent compound, or an enzyme.

[0008] Further, telomere DNA obtained from cells is electrophoretically separated, and the telomere DNA electrophoretically separated is subjected to the above-mentioned PNA.
Provided is a method for measuring telomere length, which comprises detecting the telomere length using a skeletal telomere recognition probe and determining the telomere length.

Further, the present invention provides a method for evaluating the degree of skin aging, in which the telomere length of skin epidermal cells is determined by the above-described method for measuring telomere length, and the aging degree of the skin is evaluated based on the obtained telomere length.

[0010] The present invention also provides a telomere length measurement kit including the above-described PNA skeleton telomere recognition probe and an instrument for collecting skin epidermis debris.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

The PNA skeleton telomere recognition probe of the present invention, which has a PNA skeleton and has a specific nucleotide sequence, can hybridize stably with telomere DNA obtained from skin epidermal cells, dermal fibroblasts and the like. It is.

Here, PNA is an abbreviation of Peptide Nucleic Acid or Polyamide Nucleic Acid, and the PNA skeleton structure is represented by the following formula, in which the sugar-phosphate skeleton formed by the phosphodiester bond of nucleic acid is N- (2- Refers to the structure of aminoethyl) glycine derivative substituted by a peptide bond chain.

[0014]

Embedded image

In PNA, A, which constitutes a nucleic acid,
T, G and C bases are linked to the skeletal structure via methylene carbonyl. In PNA, similarly to the peptide, the left side is represented as the N-terminal and the right side is represented as the C-terminal,
This N-terminus corresponds to the 5 'terminus of the nucleic acid, and the C-terminus corresponds to the 3' terminus.

[0016] The PNA skeleton telomere recognition probe used in the present invention is one having an N-terminal- (CCCTAA) _n -C-terminal or N-terminal- (TTAGGG) _n -C-terminal base sequence having a repetition number n of 3 to 5. Is preferred. By using such a PNA skeleton telomere recognition probe, the accuracy, sensitivity, and operability of telomere detection can be improved.

The base sequence is N-terminal- (CCCTA
A) n-C-terminal or N-terminal- (TTTAGGG) n-
When using two CNA-terminal PNA skeleton telomere recognition probes, it is preferable to use either one of these two probes. In particular, N-terminal- (CCCTA
A) The ₃ -C terminus is more preferred.

A method for producing a PNA skeleton telomere recognition probe having such a nucleotide sequence is described in PENielsen et al. (Nielsen et al. Science, (1991), 254 , 1497-15).
00; Egholm et al. J Am Chem Soc., (1992), 114 , 189
5-1897; Egholm et al. Nature, (1993), 365 , 566-56
8) can be.

The PNA backbone telomere recognition probe can have various labeling moieties. For example, since the PNA skeleton is a derivative of an amino acid, there is great flexibility in radiolabeling, and by using a radioisotope as a label, a PNA skeleton probe having high radioactivity and a high labeling rate can be obtained. That is, [γ- ³² P] ATP and T
The specific activity of a conventional labeled probe (Harley CB et al.) Labeled at the 5 'end with 4 polynucleotide kinase or the like is 1 to 3 × 10 ⁶ counts per minute / pmol, whereas the specific activity is 5 The PNA scaffold probe has a specific activity of 2 to 7 × 10 ⁷ counts per minute / pmo.
l, DNA extracted from a small amount of skin epidermis debris
Can accurately measure the telomere length.

A specific example of a radioactive label is "Kemptide"
PNA containing a peptide is phosphorylated by a kinase, and thus includes [γ- ³² P] ATP. Also,
A tyrosine having [ ¹²⁵ I] at the N-terminus can be bound to form a label. In addition, PNA binds lysine (amino group) or cysteine (thiol group) to its terminal,
Labeling can be performed with [ ³ H], [ ³² P], [ ¹²⁵ I], [ ¹⁴ C], etc. by using a standard radioactive label introduction method.

A non-radioactive label can be used for the label portion of the PNA skeleton telomere recognition probe. By using a non-radiolabeled probe, operation at a facility other than the radioactivity safety management facility becomes possible, and detection of telomere DNA, measurement of telomere length, or evaluation of skin aging can be performed easily.

As the non-radioactive label, a fluorescent compound, a chemiluminescent compound and the like can be used. In PNA, a label can be introduced at two or more sites instead of a single site such as the 5′-end label of a conventional labeled probe made of a nucleic acid, so that the fluorescence intensity or the emission intensity can be increased. It is preferred.

Here, fluorescein isothiocyanate (FITC), red tetramethylrhodamine isothiocyanate (TRITC) and the like can be used as the fluorescent compound, and horseradish peroxidase (HRP), Alkaline phosphatase (AP), luciferase and the like can be used.

In the probe for telomere recognition of the PNA skeleton of the present invention, the PNA skeleton may be labeled with an enzyme-labeled nucleotide. Thereby, a small amount of telomere D
NA can be detected with high sensitivity. As the enzyme-labeled nucleotides, biotin labeled dCTP, digoxigenin-labeled dUTP, FITC-labeled dGTP, fluorescence-labeled nucleotide such as TRITC-labeled dCTP, ^[alpha-3 H]
dNTP, [γ- ³² P] dNTP or [α- ¹⁴ C]
Examples include radiolabeled nucleotides such as dNTP, or horseradish peroxidase-labeled nucleotides, luciferase-labeled nucleotides, and alkaline phosphatase-labeled nucleotides.

In the above-described labeled probe, when the labeled portion is made of a fluorescent compound, it is preferable to use a sensitizer at the time of detecting the probe, since detection with higher sensitivity is possible. For example, biotin-labeled dCTP as a labeling probe
In the case where is used, streptavidin-conjugated horseradish peroxidase (HRP) is bound to a labeled probe, and then the use of hydrogen peroxide and a sensitizer increases the fluorescence intensity. As the sensitizer, fluorescein-tyramide, biotin, florestin and the like can be suitably used.

In the method for measuring telomere length of the present invention, a sample containing telomere DNA is prepared from skin epidermal cells, dermal fibroblasts and the like, and telomere DNA is obtained from the sample by Southern blotting and other various electrophoresis methods. It is performed by electrophoretically separating the telomere DNA and detecting the separated telomere DNA using a PNA skeleton telomere recognition probe. For example, a DNA having a known length is used as a marker, and a restriction enzyme-treated product of DNA extracted from skin epidermis cells and a marker are simultaneously electrophoretically separated, and then a telomere DNA is detected using a PNA skeleton telomere recognition probe, and the marker is detected. The telomere length is determined by measuring the telomere DNA migration distance using the migration distance as a standard.

The method for obtaining a sample containing telomere DNA from skin epidermal cells, dermal fibroblasts and the like is not particularly limited. For example, a scrap of skin epidermis is collected by scraping the skin epidermis, peeling of the skin epidermis using an adhesive tape, etc., and DNA is extracted from the skin epidermis according to a conventional method.
The NA may be treated with a restriction enzyme to cleave the DNA into a DNA fragment containing a telomere region.

The method for evaluating the degree of skin aging according to the present invention comprises determining the telomere length of skin epidermal cells obtained according to the method for measuring telomere length according to the present invention described above, and determining the degree of skin aging based on the obtained telomere length. It is a method of evaluating. More specifically, for example, in advance, skin epidermis debris at a predetermined site is collected from a plurality of subjects of various ages to determine the telomere length, and the relationship between age and telomere length is determined, and the relationship is determined. Based on the above, the degree of aging of the skin is evaluated from the telomere length of the skin for which the degree of aging is to be evaluated.

When the degree of aging of the cosmetic skin is evaluated by this method, the skin epidermis may be collected on the top of the cheekbone or, as shown in FIG. It is preferable to select the skin or the like at the intersection of the line L1 and the horizontal line L2 at the upper end of the nostril (Japanese Patent Application No. 10-15 / 1998).
No. 7773).

As a method for evaluating the degree of aging of the skin,
For the same subject, skin was sampled from a part that is often exposed to sunlight, such as the face, and a part that was less exposed to sunlight, such as the buttocks.From the telomere length of the collected skin, The degree of aging of the skin at the site may be compared.

The telomere length measuring kit of the present invention is a kit used for carrying out the above-described telomere length measuring method, and includes at least the PNA skeleton telomere recognition probe of the present invention and an instrument for collecting skin epidermis debris. .

There is no particular limitation on the skin epidermic debris collecting device as long as the skin epidermic debris can be collected as thin as not to bleed, and a commercially available cellophane adhesive tape or the like can be used. Further, a file-shaped or brush-shaped device (rasp) for collecting skin epidermis debris in which short fibers having a sharp point are implanted vertically and horizontally in a pedestal can be preferably used (Japanese Patent Application No. 10-157773). Book).

In addition, the telomere length measurement kit includes an adhesive tape as an instrument for collecting skin epidermis debris,
A surfactant such as Sodium-N-lauroylsarcosinate used to peel skin epidermal cells from the adhesive surface of the adhesive tape, and for extracting DNA from collected skin epidermal cells
Solusion A of Iso Quick Kit (manufactured by ORCA Research) may be included.

[0034]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below based on embodiments.

Example 1 Alkaline Phosphatase (A
P) Preparation of labeled PNA skeleton telomere recognition probe

An alkaline phosphatase (AP) -labeled PNA skeleton probe was prepared as a PNA skeleton telomere recognition probe using a chemiluminescent compound as a label.

In this case, as the PNA skeleton probe,
The method of PENielsen et al. (Nielsen et al. Science, (199
1), 254 , 1497-1500; Egholm et al. J Am Chem Soc.,
(1992), 114 , 1895-1897; Egholm et al. Nature, (199
3), 365 , 566-568) to prepare (CCCTAA) ₃ .

The synthesized (CCCTAA) ₃ PNA backbone probe was treated with formaldehyde, a bifunctional reagent, and labeled with alkaline phosphatase by chemical direct binding.

The labeling with alkaline phosphatase was performed as follows using AlkPhos Direct (Pharmacia). First, a solution was prepared by adding 80 μL of sterilized water to a 20 μL formaldehyde crosslinking agent solution (work). Sterile water was added to the labeled (CCCTAA) ₃ PNA backbone probe to adjust to 10 ng / μL. 10 μL of the diluted PNA skeleton probe was placed in a microtube.
And the microtube was placed in boiling water and heated for 5 minutes to denature by heat. Thereafter, the microtube was immediately cooled on ice. 10 for cold PNA scaffold probe
μL of reaction buffer was added and mixed. further,
Alkaline phosphatase (Pharmacia) as a labeling reagent
(AlkPhos Direct) (2 μL) was added and mixed. Further, 10 μL of the prepared formaldehyde crosslinking agent solution was added.
The mixture was mixed and reacted at 37 ° C. for 30 minutes to obtain an AP-labeled PNA skeleton telomere recognition probe.

The labeling with alkaline phosphatase was confirmed by reacting with CDP-Star (Pharmacia) to generate chemiluminescence.

Example 2 Preparation of Fluorescein Isothiocyanate (FITC) Labeled PNA Backbone Telomere Recognition Probe

A fluorescein isothiocyanate (FITC) -labeled PNA skeleton probe was prepared as a PNA skeleton telomere recognition probe labeled with a fluorescent compound.

In this case, the PNA skeleton probe was used in Example 1.
(CCCTAA) ₃ was prepared in the same manner as in (1).

To label this PNA skeleton probe with FITC, first, 1.5 mg of FITC was added to DMF15.
Dissolved in μL. In addition, the PNA skeleton probe is 0.5M
Na ₂ CO ₃ / NaHCO ₃ buffer (pH 9.3) 300
dissolved in μL, mixed with the above FITC solution,
The reaction was performed overnight in the dark. The reaction solution was subjected to gel filtration to remove unreacted FITC, and the reaction product was purified by reverse-phase high performance liquid chromatography using a column manufactured by Tosoh. The conditions used for reversed-phase high-performance liquid chromatography were 50 mM Tr
is-HCl (pH 7.4) / 10 mM EDTA, detected by absorption at 260 nm. The absorption spectrum of the collected peak was measured, and the absorption at 260 nm and 495 nm
Of FITC was confirmed.

Example 3 Measurement of Telomere Length Using Alkaline Phosphatase (AP) Labeled PNA Skeleton Probe by Southern Blot Method

(1) Preparation of Samples Twenty subjects aged 0 to 77 years were taken as subjects, and skin scraps were collected from the skin of each subject's face using a rasp.
Using Kit (ORCA Research), DNA was extracted from each skin scraping piece.

The extracted DNA was TE (10 mM Tris-HCl, 1 m
M EDTA, pH 8.0) or dissolved in Reagent 5 of Iso Quick Kit and stored at 4 ° C.

The DNA concentration of the DNA solution extracted from the skin scraping pieces was adjusted using λ DNA (Promega) prepared by adjusting the DNA solution to 100 ng / μL as a marker.
0.35% agarose gel (type H, Nippon Gene)
It was determined by electrophoresis and staining with 2 μg / mL ethidium bromide.

Next, 2 μL of 10 × H buffer (manufactured by Takara Shuzo), DNA of a DNA solution extracted from skin scraping fragments
2 μg equivalent amount and sterilized water were added to a total volume of 19 μL, and finally 6 units / μL of Hi as a restriction enzyme was used.
nf I (Takara Shuzo) was added. This was reacted at 37 ° C. for 3 to 4 hours to obtain a telomeric DNA measurement sample. The sample obtained by this reaction was subjected to electrophoresis immediately after the reaction as follows. Samples not subjected to electrophoresis immediately after the reaction were stored at 4 ° C.

(2) Agarose gel electrophoresis An agarose gel (type I, manufactured by Sigma) was prepared such that the gel concentration in the bridge portion was 1% and the bed portion was 0.8% (Marisol KS-8405, 20 cm). × 14cm). 1 × Boyer buffer was used as the electrophoresis buffer.

As markers, 1 Kb DNA Ladder (GIBCO BRL) adjusted to 0.5 μg / lane and 0.3
λDNA / HindIII digest (Nippon
Gene).

3 μL of a loading buffer was added to each of 20 μL of the DNA sample and the marker, applied to a 0.8% agarose gel prepared as described above, pulled in at 85 volts, and then electrophoresed at 35 volts.

(3) Southern transfer After completion of the electrophoresis, agarose gel was cut out and 2 μg /
The cells were stained with mL of ethidium bromide for 15 minutes, mounted on a UV transilluminator, and photographed with a scale.

After photography, the gel was washed with 0.25M HC
and shaken at room temperature for 15 minutes, and then washed twice with distilled water.

Next, the gel was immersed in a denaturalization solution containing 0.2 M NaOH and 0.6 M NaCl, shaken at room temperature for 25 minutes, and then washed three times with distilled water.

The gel was treated with 0.1 M containing 0.6 M NaCl.
Neutralization of 2M Tris-HCl (pH 7.4) (Neutra
lization), immersed in the solution, shaken at room temperature for 30 minutes, and washed once with distilled water. Thereafter, the plate was immersed again in the neutralization solution and shaken at room temperature for 30 minutes.

In order to prevent air from entering the 3MM filter paper set in the blotting apparatus filled with 6 × SSC, the gel was immersed in 3 × SSC and Hybond ™ N + (Amersha
m), 3MM filter paper soaked in 6 × SSC, a paper towel, a glass plate, and a weight (2 kg) in that order, and blotting was performed overnight.

After the completion of blotting, the membrane filter was immersed in 3 × SSC, lightly drained, and then subjected to UV irradiation.
The location of the well was noted on the transilluminator.

The mixture was sandwiched between filter papers and heated (baking) at 80 ° C. overnight.

(4) DNA hybridization The baked membrane was pre-heated at 55 ° C. using AlkP.
hos Direct hybridization buffer (0.5MN
aCl, containing 12% (w / v) urea) and shaking at 55 ° C for at least 15 minutes to perform prehybridization.

After the pre-hybridization was completed, the membrane filter was put in a shield bag, and the AP-labeled PNA skeleton probe prepared by the method of Example 1 was used.
2 mL of AlkPhos Direct hybridization buffer to which 100 ng was added was added, and sealed so that bubbles did not enter.

Hybridization was carried out by shaking at 55 ° C. overnight.

(5) Washing and Detection After hybridization, the membrane for transblot was preheated at 55 ° C. in a washing buffer (2M
Urea, 0.1% SDS, 50mM sodium phosphate, 150mM NaC
1, containing 10 mM MgCl ₂ ) and shaken at 55 ° C. for 10 minutes. After repeating this operation twice, the membrane was immersed in a washing buffer (containing 0.05 M Tris Base and 0.1 M NaCl), shaken at room temperature for 5 minutes, and this operation was repeated twice.

The membrane is thoroughly drained, and CDP-Star
(Manufactured by Pharmacia) was applied and left for 2 to 5 minutes.

A membrane and a film were set on a hypercassette (manufactured by Amersham) together with Hyperfilm ™ ECL (manufactured by Amersham), left overnight at room temperature, and developed. (6) Data analysis Alignment of the developed film and the membrane The positions of the wells were filled with magic ink. Since the DNA containing the telomere region appears on the membrane as a smear, the peak of the smear density is determined by densitometry (Ultra Sc
an XL Laser Densitometer, manufactured by Pharmacia).

The distance from the well to the peak was defined as the mobility, and the mobility was determined. A calibration curve was prepared from the mobility of the marker, and the length of the DNA containing the telomere region in each sample was determined. As a result, it was confirmed that the DNA containing the telomere region was gradually shortened from 11.7 kb at the age of 0, and almost linearly shortened to the age of 77.

Example 4 Measurement of Telomere Length Using Fluorescein Isothiocyanate (FITC) Labeled PNA Skeleton Probe

(1) Preparation of sample, (2) agarose gel electrophoresis, and (3) Southern transfer were carried out in the same manner as in Example 3.

(4) DNA Hybridization The baked membrane was preheated at 55 ° C. to Denh.
art hybridization buffer (denatured salmon sperm D
(Including NA) and shaken at 55 ° C. for at least 15 minutes to perform prehybridization.

After the pre-hybridization was completed, a membrane filter was placed in the shield bag, and 2 mL of Denhart hybridization buffer containing 100 ng of the FITC-labeled PNA skeleton probe prepared by the method of Example 2 was added to prevent bubbles from entering. Sealed.

The mixture was shaken at 55 ° C. overnight to perform hybridization.

(5) Washing and Detection The hybridized membrane was washed in the same manner as in Example 3, and the membrane was excited with an argon ion laser beam of 488 nm, and the telomere DNA on the membrane hybridized with the FITC-labeled PNA skeleton probe. 530 nm fluorescence generated by Falk at al.,
J. Immunol., (1988), 140 , 2652-2560).

(6) Data Analysis The fluorescence intensity was quantified by dividing the area for each lane of the membrane, using an ARGUS-100 / VIM ultra-high-sensitivity imaging image analyzer manufactured by Hamamatsu Photonics, converting the data into a histogram using an NIH image, and determining the maximum value peak. The half width was determined.

A calibration curve was created based on the distance from the well position (origin) to the peak value (maximum value) of the DNA histogram used as a marker, and the telomeric DNA in each sample was prepared.
The length was determined. As a result, as in Example 3, it was found that telomeric DNA was shortened almost linearly from 0 to 77 years old. The telomere DNA contained in the DNA extracted from the skin scraping debris on the top of the cheekbone of a 25-year-old woman was 9.0 kb.

[0075]

According to the method for measuring telomere length using the PNA skeleton telomere recognition probe of the present invention, a small amount of DN
A can be used to quickly and accurately measure the telomere length, thereby making it possible to measure the degree of skin aging.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a site where a skin epidermis is collected on the face when evaluating the degree of skin aging.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yoshikazu Aman 132-1 Kamiizumi, Oigawa-cho, Shita-gun, Shizuoka Prefecture Inside Sony Corporation Laboratories (72) Miho Sugimoto 132-1 Kamiizumi, Oigawa-machi, Shita-gun, Shizuoka (72) Inventor Mitsuba Shinpichi 1-10-5 Nishihonmachi, Shobara-shi, Hiroshima F-term (reference) 2G045 AA35 AA40 CB09 DA13 FB02 FB07 FB08 FB12 FB13 4B024 AA11 CA05 CA09 HA14 4B063 QA12 QA19 QQ02 QQ42 QR32 QR56 QR58 QS03 QS10 QS14 QS34 QS36 QS39 QX02

Claims (4)

[Claims] 1. A DNA containing a telomere region obtained from a cell.
A probe having a PNA skeleton that hybridizes with a fragment (hereinafter referred to as telomeric DNA),
TAA) _n -C-terminus or N-terminus-(TTAGGG) _n
-A PNA skeleton telomere recognition probe having a C-terminal (n = 3 to 5) base sequence and labeled with a radioisotope, a fluorescent compound, a chemiluminescent compound, or an enzyme. 2. The telomere DNA obtained from a cell is subjected to electrophoretic separation, and the electrophoretically separated telomere DNA is isolated from the Ptelomere DNA according to claim 1.
A method for measuring telomere length, comprising detecting using a NA skeleton telomere recognition probe and determining the length of telomere DNA (hereinafter referred to as telomere length). 3. A method for evaluating the degree of skin aging, comprising determining the telomere length of skin epidermal cells by the method according to claim 2, and evaluating the degree of skin aging based on the obtained telomere length. 4. A telomere length measurement kit comprising the PNA skeleton telomere recognition probe according to claim 1 and an instrument for collecting skin epidermis debris.