JPWO2013054666A1 - Primer for telomere sequence amplification - Google Patents

Abstract

An object of the present invention is to provide a primer for amplifying a telomere sequence. The present invention provides a telomere sequence amplification primer represented by the following formula (1) or (2). (1) L1- (R1) n1 In the formula, R1 which is a repeating unit represents a base sequence represented by GTAGGG, n1 is 5 or 6, and when n1 is 5, L1 consists of 16 or less bases Represents a linker, and when n1 is 6, L1 represents a linker composed of 8 or less bases. (2) L2- (R2) n2 (X) In the formula, R2 which is a repeating unit represents a base sequence represented by GTAGGG, and X represents a deletion of 1 or 2 bases from the base sequence represented by GTAGGG Or a substituted sequence, n2 5 or 6, when n2 is 5, L2 represents a linker consisting of 4 to 8 bases, and when n2 is 6, L2 is a linker consisting of 8 bases Represents.

Description

  The present invention relates to a primer for telomere sequence amplification. The present invention also relates to a method for amplifying a telomere sequence using the primer.

A telomere is a protective structure at the end of a chromosome and has a repeating sequence of TTAGGG. In normal cells, telomeres are gradually shortened at each cell division, and are known to shorten with age.
In order to detect telomeres, it is necessary to amplify the repeated sequences and detect them. As a method for detecting the presence of a target nucleic acid by selective amplification of the target nucleic acid, a polymerase chain reaction (PCR) using an appropriate primer is known. However, when telomere, which is a repetitive sequence, is amplified by PCR, the primers hybridize to form a primer dimer, and it was considered difficult to selectively amplify the telomere by PCR. .

As a method for amplifying and quantifying telomere sequences by PCR, there are methods reported by Cawthon et al. (International Publication No. 2003/064615 and “Richard M. Cawthon, telomere measurement by quantitative PCR (Telomere measurement by quantitative PCR), Nucleic Acids Research, Vol. 30, No. 10, e47, 2002 ”). In the Cason method, at least one nucleotide of the first primer is mutated to form a mismatch with the 3 ′ terminal nucleotide residue of the second primer and the second primer A primer set consisting of the following primers was used. In the Corson method, the amount of telomeres was quantified by a relative quantification method.
Originally, when telomere, which is a repetitive sequence, is amplified by PCR and detected by electrophoresis, the amplified telomere sequence should be detected in a clean smear form, but in Corson's method, the telomere sequence is clean. It was not detected in a smear shape, but was detected in a shape close to a ladder shape. This is presumably because the PCR conditions such as primers used in the Corson method were not sufficient to amplify the telomere sequence with sufficient amplification efficiency and specificity. Therefore, it can be said that the method disclosed by Corson et al. Cannot be widely used for quantification of telomeres.

Karlsson et al. Reported a method similar to the method of Corson ("Andreas O. Karlsson et al., Telomeric repeat analysis), with the exception that some of the primers used in the above Corson method were modified. (See Estimating human age in forensic sample by analysis of telomere repeats, Forensic Science International: Genetics Supplement Series 1: pp. 569-571, 2008)).
However, it does not achieve significantly higher telomeric amplification efficiency and specificity than the Corson method. Moreover, it cannot be said that the method disclosed by Carlson et al. Can be widely used for quantification of telomeres.

  The present invention provides a primer capable of amplifying a telomere sequence with high amplification efficiency, a method for amplifying a telomere sequence with high amplification efficiency, and a method for quantifying telomere per test diploid cell. Objective.

As a result of extensive studies, it has been found that the above object can be achieved by using a primer having a specific base sequence. The present invention has been completed based on these novel findings.
That is, the present invention provides a telomere sequence amplification primer represented by the following formula (1) or (2).
Equation _{(1) L 1 - (R} 1) n 1
(In the formula, R _{1 as} a repeating unit represents a base sequence represented by GTAGGG, n ₁ represents the number of R ₁ as a repeating unit, 5 or 6, and when n ₁ is 5, L ₁ is A linker composed of 16 or less bases is represented, and when n ₁ is 6, L ₁ represents a linker composed of 8 or less bases.)
Equation _{(2) L 2 - (R} 2) n 2 (X)
(In the formula, R ₂ which is a repeating unit represents a base sequence represented by GTAGGG, and X represents the 5 ′ end side of the repeating unit, between any two adjacent repeating units or 3 ′ of the repeating unit. Represents a sequence in which one or two bases are deleted or substituted from the base sequence represented by GTAGGG, which is bonded to any one of the terminal sides, and n ₂ represents the number of R ₂ as a repeating unit. And when n ₂ is 5, L ₂ represents a linker composed of 4 to 8 bases, and when n ₂ is 6, L ₂ represents a linker composed of 8 bases. )
Further, the present invention provides a telomere sequence amplification in which the primer represented by the above formula (1) or (2) is combined with at least one primer comprising the base sequence represented by any of SEQ ID NOs: 2 to 4. A primer set is provided.
Furthermore, the present invention provides a primer for amplifying a telomere sequence, which is a combination of the primer represented by the above formula (1) or (2) and at least one primer comprising the base sequence represented by any of SEQ ID NOs: 2 to 4. A method for amplifying telomere sequences by performing polymerase chain reaction (PCR) using the set is provided.

Furthermore, the present invention is a method for quantifying telomere per test diploid cell, wherein (a) a synthetic telomeric oligomer that is a repetitive sequence of a base sequence consisting of TTAGGG is used by real-time PCR, Creating a calibration curve showing the relationship between telomere amount and Ct (threshold cycle) value,
(B) subjecting a cell population sample of test diploid cells to real-time PCR under the same conditions as in step (a) to obtain a Ct value;
(C) applying the Ct value of step (b) to the calibration curve of step (a) to obtain the telomere amount (X) in the cell population sample; and
(D) Applying X in step (c) to the following formula to obtain the amount of telomeres (Y) per test diploid cell:
Y = X / Z
Z: including the number of cells contained in the cell population sample obtained using a single copy gene present in the test diploid cell as an index,
The real-time PCR of steps (a) and (b) is performed by using the primer represented by the above formula (1) or (2) and at least one primer comprising the base sequence represented by any one of SEQ ID NOs: 2 to 4. And a telomere sequence amplification primer set combined with the above.

According to the present invention, a telomere sequence can be accurately amplified with high amplification efficiency. This is a significant effect that cannot be predicted by those skilled in the art. This makes it possible to quantify the amount of telomere for a small amount of sample and to detect a slight difference in telomere amount.
Moreover, since the absolute value of the amount of telomeres can be obtained by adopting the method for quantifying telomeres per diploid cell of the present invention, it is possible to directly compare data between different laboratories. become.

Polymerase chain reaction using a primer set for telomere sequence amplification (Yama1F primer) composed of a base sequence represented by SEQ ID NO: 1 and a primer composed of a base sequence represented by SEQ ID NO: 2 (Yama1R primer) ( It is the figure which showed the hybridization with the telomere sequence of each primer at the time of performing PCR. Polymerase chain reaction using a primer set for telomere sequence amplification (Yama1F primer) composed of a base sequence represented by SEQ ID NO: 1 and a primer composed of a base sequence represented by SEQ ID NO: 2 (Yama1R primer) ( It is the figure which showed the hybridization of each primer at the time of performing PCR. FIG. 3 shows fluorescence-amplified PCR amplification products separated by ethidium bromide-containing agarose gel electrophoresis. The positions of 4870 base pairs (bp), 2016 bp, 1360 bp, 1107 bp, 926 bp and 658 bp are shown in order from the top by the PHY marker used in the leftmost lane. Amplified human Placenta telomeric DNA is observed in a smear form of about 2000 bp or less. FIG. 4 is a graph in which the fluorescence intensity observed in FIG. 3 is subjected to image processing analysis using NIH Image 1.58 software and the amplification efficiency is digitized. FIG. 5 shows fluorescence-amplified PCR amplification products separated by ethidium bromide-containing agarose gel electrophoresis. H-Yama1 + B-Yama1 means a combination of Yama1F and Yama1R. H-Caw + B-Yama1 is a primer made by binding the linker sequence of the forward primer used by Cawthon (CawthonF shown in Table 1 below) to the repeat sequence of Yama1F, and the reverse primer used by Corson This means a combination with a primer formed by binding a linker sequence (CawthonR shown in Table 1 below) to a repeating sequence of Yama1R. H-Karl + B-Yama1 is a primer made by binding the linker sequence of the forward primer (KarlssonF shown in Table 1 below) used by Karlsson to the repeat sequence of Yama1F, and the reverse primer used by Carlson. This means a combination with a primer obtained by binding a linker sequence (KarlssonR shown in Table 1 below) to a repeat sequence of Yama1R. H-Yama1 + B-Caw means a combination of a primer formed by binding a Yama1F linker sequence to a CawthonF repeat sequence and a primer formed by binding a Yama1R linker sequence to a CawthonR repeat sequence. H-Yama1 + B-Karl means a combination of a primer formed by binding the linker sequence of Yama1F to the repeated sequence of KarlssonF and a primer formed by binding the linker sequence of Yama1R to the repeated sequence of KarlssonR. Only when a primer having a Yama1F / R repeat sequence is used, the amplified human telomeric DNA is observed in a smear form. FIG. 6 shows fluorescence-amplified PCR amplification products separated by ethidium bromide-containing agarose gel electrophoresis. The marker used in the leftmost lane is φx174. The markers indicate positions of 1353 bp, 1078 bp, 872 bp, 603 bp, and 310 bp in order from the top in the upper half of the figure, and 1353 bp, 1078 bp, 872 bp, 603 bp, and 310 bp in order from the top in the lower half of the figure. The location is indicated. The amplified human telomeric DNA is observed in a smear shape of about 1300 bp or less within a range that can be discriminated with the naked eye with reference to the marker. FIG. 7 left is a diagram showing fluorescence-amplified PCR amplification products separated by ethidium bromide-containing agarose gel electrophoresis. ΛHindIII used in the leftmost lane indicates positions of 23130 bp, 9416 bp, 6557 bp, 4361 bp, 2322 bp and 2027 bp in order from the top. ΦX174 used in the rightmost lane indicates positions of 1353 bp, 1078 bp, 872 bp, 603 bp, and 310 bp in order from the top. The size of the amplified human Placenta telomeric DNA varies depending on the amount of sample. When the sample amount is 80 ng, a smear shape of about 10 kb or less is observed. The right side of FIG. 7 shows the result of Southern blotting. FIG. 8 is a graph of a calibration curve showing the relationship between the telomere amount in the telomere oligomer and the Ct value (telomere calibration curve). FIG. 9 is a graph of a calibration curve showing the relationship between the diploid copy number of the oligomer of the 36B4 gene and the Ct value (36B4 calibration curve). FIG. 10 shows CD34-positive cells containing hematopoietic stem cells obtained from a normal person of a predetermined age (represented by 34+ in the figure) and matured and differentiated granulocytes (represented by Gra in the figure). ) Is a graph comparing the amount of telomeres per cell. 11 (a) to 11 (e) were prepared using a primer (Yama1F (3) to (7)) in which 3 to 7 GTAGGG sequences were repeated on the 3 ′ end side following the linker portion of Yama1F. It is a graph of the calibration curve which shows the relationship between the telomere amount in a telomere oligomer, and Ct value. FIG. 11 (f) is quantified by an absolute quantification method using a primer (Yama1F (3) to (6)) in which 3 to 6 GTAGGG sequences are repeated on the 3 ′ end side following the linker portion of Yama1F. It is the graph which compared the amount of telomeres, respectively, when Yama1F (5) is used as 100%. FIG. 12 is a graph comparing the amount of telomeres quantified by the absolute quantification method using various primers prepared in Example 7, assuming that Yama1F is used as 100%. FIG. 13 is a graph comparing the amount of telomeres quantified by the absolute quantification method using various primers prepared in Example 7, assuming that Yama1F is used as 100%. FIG. 14 is a graph comparing the amount of telomeres quantified by the absolute quantification method using various primers prepared in Example 7, assuming that Yama1F is used as 100%. FIG. 15 is a graph comparing the amounts of telomeres quantified by the absolute quantification method using Yama1F, a primer used by Corson or a primer used by Carlson, assuming that Yama1F is used as 100%. FIG. 16 shows amplification plots obtained by PCR using (a) Primer used by Corson, (b) Primer used by Carlson, or (c) Yama1F. FIG. 17 is a graph comparing telomere lengths per cell obtained from a population of normal persons (male and female) of a predetermined age. FIG. 18 is a graph comparing telomere lengths per cell obtained from a population of normal persons (male) of a predetermined age. FIG. 19 is a graph comparing telomere lengths per cell obtained from a population of normal persons (women) of a predetermined age.

The primer of the present invention is represented by the following formula (1) or (2).
Equation _{(1) L 1 - (R} 1) n 1
(In the formula, R _{1 as} a repeating unit represents a base sequence represented by GTAGGG, n ₁ represents the number of R ₁ as a repeating unit, 5 or 6, and when n ₁ is 5, L ₁ is A linker composed of 16 or less bases is represented, and when n ₁ is 6, L ₁ represents a linker composed of 8 or less bases.)
Equation _{(2) L 2 - (R} 2) n 2 (X)
(In the formula, R ₂ which is a repeating unit represents a base sequence represented by GTAGGG, and X represents the 5 ′ end side of the repeating unit, between any two adjacent repeating units or 3 ′ of the repeating unit. Represents a sequence in which one or two bases are deleted or substituted from the base sequence represented by GTAGGG, which is bonded to any one of the terminal sides, and n ₂ represents the number of R ₂ as a repeating unit. And when n ₂ is 5, L ₂ represents a linker composed of 4 to 8 bases, and when n ₂ is 6, L ₂ represents a linker composed of 8 bases. )
However, it is preferable that L ₁ and L ₂ do not include the base sequence represented by GTAGGG.

The primer consisting of the base sequence represented by the above formula (1) is a linker part in a TTAGGG repetitive sequence in which 5 or 6 GTAGGG sequences in which the first T is changed to G are repeated. When 5 GTAGGG is present on the 5 ′ side, a linker consisting of 0 to 16 bases (A, G, C or T) is bound, and when GTAGGG is 6, 0 to 8 bases A linker composed of (A, G, C or T) is bound thereto. There may be no linker moiety.
In the primer consisting of the base sequence represented by the above formula (1), preferably n ₁ is 5 and L ₁ is 12 or less, for example, 0 to 12, 2 to 10, or 4 to 8 Represents a linker consisting of a base.
In the primer comprising the base sequence represented by the above formula (1), for example, L ₁ is selected from the group consisting of GTTT, GGTTTT, CGGTTTG, GTTTATTA, GTTTGTTG, GGGGAGGA, ATTTATTA and GTTTATTAGTTT, but is not limited thereto. It is not something.
In the primer comprising the base sequence represented by the above formula (1), preferably n ₁ is 6 and L ₁ represents a linker comprising 4 to 8 bases.

The primer consisting of the base sequence represented by the above formula (2) has a linker moiety in a repeating sequence of TTAGGG and a sequence of 5 or 6 GTAGGG sequences in which the first T is changed to G. When 5 GTAGGGs are present, a linker consisting of 4 to 8 bases (A, G, C or T) is bound to the 5 ′ side, and when GTAGGG is 6, 8 bases (A, G, A linker consisting of C or T), and further, the repeating unit represented by any two GTAGGG adjacent to the 5 ′ terminal side of the repeating unit represented by GTAGGG, that is, between the linker part and the repeating sequence. 1 or 2 bases are deleted or substituted from the base sequence represented by GTAGGG at any one position between or 3 ′ end side of the repetitive sequence, that is, 3 ′ end of the primer. It is a combination of sequences.
In the primer comprising the base sequence represented by the above formula (2), preferably, the primer does not include a sequence in which two bases are deleted from the base sequence represented by GTAGGG at the 3 ′ end.
In the primer comprising the base sequence represented by the above formula (2), for example, L ₂ is selected from the group consisting of GTTTATTA, GTTTGTTG and GGGGAGGA, but is not limited thereto.

In the above formula (2), X has a sequence in which 1 or 2 bases are deleted or substituted from the base sequence represented by GTAGGG.
The “deletion” referred to here indicates that any part of the base sequence represented by GTAGGG may be deleted. For example, X is TAGGG, GAGGG, GTGGG, GTAGG, AGGG, TGGG, GGGG, GTGG, GAGG, TAGG or GTAG.
The “substitution” mentioned here (also referred to as “mutation” in the present specification) represents that any part of the base sequence represented by GTAGGG may be substituted. Specifically, the first G of the base sequence represented by GTAGGG can be replaced with A, T or C, and the second T of the base sequence represented by GTAGGG is replaced with A, C or G. The third A of the base sequence represented by GTAGGG can be replaced with T, C or G, and the fourth G of the base sequence represented by GTAGGG can be replaced with A, T or C. The fifth G of the base sequence represented by GTAGGG can be replaced with A, T or C, and the sixth G of the base sequence represented by GTAGGG can be replaced with A, T or C.

More preferably, in the primer of the present invention represented by the above formula (1) or (2), 1 to 5 bases are deleted or substituted in the linker portion from the base sequence represented by GTTTATTA or GTTTATTA. Has an array. Particularly preferably, the primer of the present invention consists of the base sequence represented by SEQ ID NO: 1 below.
5'-GTTTATTAGTAGGGGTAGGGGTAGGGGTAGGGGTAGGG-3 '(SEQ ID NO: 1)
The primer consisting of the base sequence represented by SEQ ID NO: 1 is a 5′-side linker as a linker part in a TTAGGG repetitive sequence in which the first T is changed to G and 5 GTAGGG sequences are repeated. To which a base sequence (GTTTATTA) consisting of 8 bases is added.
The synthesis of the primer of the present invention can be performed based on the technical level of those skilled in the art as of the filing date of the present application. For example, primer synthesis may be outsourced to a synthesis manufacturer such as Sigma-Aldrich Japan.
In the primer of the present invention, the portion that hybridizes to the telomere sequence is a portion of the repetitive sequence. Accordingly, the linker moiety can be mutated as long as the function of the primer of the present invention is not impaired.
The primer of the present invention can hybridize with a repetitive telomere sequence under appropriate hybridization conditions based on the general knowledge of those skilled in the art on the priority date of the present application.

  When the primer represented by the above formula (1) or (2) is used as a forward primer, the reverse primer can hybridize with a strand complementary to the strand to which the primer of the present invention hybridizes; and It is preferable to use a primer that forms a mismatch between the 3 ′ terminal nucleotide residue of the primer of the present invention and the 3 ′ terminal nucleotide residue of the reverse primer.

For example, the primer set of the present invention is a combination of the primer represented by the above formula (1) or (2) and at least one primer consisting of the base sequence represented by any one of SEQ ID NOs: 2 to 4. It is a primer set for telomere sequence amplification. The telomere sequence amplification primer comprising the base sequences represented by SEQ ID NOs: 2 to 4 has the following base sequence.
5'-GGGGCCTAAACCTAAACCTAAACCTAAACCTAAACCTAA-3 '(SEQ ID NO: 2)
5'-GGGGCCTAATCCTAATCCTAATCCTAATCCTAATCCTAA-3 '(SEQ ID NO: 3)
5'-GGGGCCTAAGCCTAAGCCTAAGCCTAAGCCTAAGCCTAA-3 '(SEQ ID NO: 4)

The primer consisting of the base sequence represented by SEQ ID NO: 2 is a linker consisting of a CCCTAA complementary sequence complementary to TTAGGG and a sequence in which the first C is changed to A and the ACCTAA sequence is repeated 5 times. A base sequence consisting of 9 bases (GGGGCCTAA) is added to the 5 ′ side as a part.
The primer consisting of the base sequence represented by SEQ ID NO: 3 is a linker consisting of a CCCTAA complementary sequence complementary to TTAGGG and a sequence consisting of 5 repeats of TCCTAA by changing the first C to T. A base sequence consisting of 9 bases (GGGGCCTAA) is added to the 5 ′ side as a part.
The primer consisting of the base sequence represented by SEQ ID NO: 4 is a linker consisting of a CCCTAA complementary sequence complementary to TTAGGG and a sequence in which the first C is changed to G and the GCCTAA sequence is repeated 5 times. A base sequence consisting of 9 bases (GGGGCCTAA) is added to the 5 ′ side as a part.
In the primer consisting of the base sequences represented by SEQ ID NOs: 2 to 4, the portion that hybridizes to the telomere sequence is a portion of the repetitive sequence. Therefore, the linker moiety described in SEQ ID NOs: 2 to 4 can be mutated as long as the function of the primer of the present invention is not impaired.
The primer comprising the base sequence represented by any one of SEQ ID NOs: 2 to 4 hybridizes with a telomeric sequence that is a repetitive sequence under appropriate hybridization conditions based on general knowledge of those skilled in the art as of the filing date of the present application. Can do.

Polymerase chain using a telomere sequence amplification primer set in which the primer represented by the above formula (1) or (2) and at least one primer comprising the nucleotide sequence represented by any of SEQ ID NOs: 2 to 4 are combined. A reaction (PCR) can be performed.
The telomere sequence amplification primer set in which the primer represented by the above formula (1) or (2) and at least one primer consisting of the base sequence represented by any of SEQ ID NOs: 2 to 4 are combined is as follows: Including embodiments.
A set of a primer represented by the above formula (1) or (2) and a primer comprising the base sequence represented by SEQ ID NO: 2,
A set of a primer represented by the above formula (1) or (2) and a primer comprising the base sequence represented by SEQ ID NO: 3,
A set of a primer represented by the above formula (1) or (2) and a primer comprising the base sequence represented by SEQ ID NO: 4,
A set of a primer represented by the above formula (1) or (2), a primer comprising the base sequence represented by SEQ ID NO: 2 and a primer comprising the base sequence represented by SEQ ID NO: 3,
A set of a primer represented by the above formula (1) or (2), a primer comprising the base sequence represented by SEQ ID NO: 2 and a primer comprising the base sequence represented by SEQ ID NO: 4;
A set of a primer represented by the above formula (1) or (2), a primer comprising the base sequence represented by SEQ ID NO: 3, and a primer comprising the base sequence represented by SEQ ID NO: 4, and the above formula ( A primer represented by 1) or (2), a primer comprising the base sequence represented by SEQ ID NO: 2, a primer comprising the base sequence represented by SEQ ID NO: 3, and a base sequence represented by SEQ ID NO: 4 Set with primer consisting of

  The primer set can also be made into a kit. In addition to the primer set, the kit includes DNA extraction reagents, PCR buffers such as PCR buffers and DNA polymerase, staining agents, electrophoresis gels, etc. In addition, a detection reagent and an instruction manual may be included.

PCR is performed using a telomere sequence amplification primer set in which the primer represented by the above formula (1) or (2) and at least one primer comprising the base sequence represented by any of SEQ ID NOs: 2 to 4 are combined. When performed, the primer represented by the above formula (1) or (2) hybridizes to the first strand of the telomere sequence as a forward primer and comprises at least the base sequence represented by any one of SEQ ID NOs: 2 to 4. One primer hybridizes to the second strand of the telomere sequence as a reverse primer. The telomere sequence can then be extended by the action of DNA polymerase. As an embodiment of the present invention, FIG. 1 shows a case where a set of a primer consisting of the base sequence represented by SEQ ID NO: 1 and a primer consisting of the base sequence represented by SEQ ID NO: 2 is used.
In addition, a telomere sequence amplification primer set in which the primer represented by the above formula (1) or (2) and at least one primer comprising the base sequence represented by any of SEQ ID NOs: 2 to 4 is combined is used. When performing PCR, the primer represented by the above formula (1) or (2) and at least one primer comprising the base sequence represented by any of SEQ ID NOs: 2 to 4 can hybridize with each other. As an embodiment, FIG. 2 shows a case where a set of a primer consisting of the base sequence represented by SEQ ID NO: 1 and a primer consisting of the base sequence represented by SEQ ID NO: 2 is used. The 3 'terminal residue of one primer forms a mismatch with the mutated nucleotide residue of the other primer, thereby preventing primer extension in PCR and preventing primer dimer formation. As a result, telomere sequences can be selectively amplified.
Therefore, PCR is performed using a primer set in which the primer represented by the above formula (1) or (2) and at least one primer comprising the base sequence represented by any of SEQ ID NOs: 2 to 4 are combined. Thus, the telomere sequence can be selectively amplified.

  In general, it is known that the same amplification efficiency can be provided under specific amplification conditions by using a forward primer and a reverse primer having the same Tm value. Therefore, when performing PCR using such a primer set, the concentrations of the forward primer and the reverse primer can be made the same, and it is not necessary to use one primer at a higher concentration than the other primer. There are advantages.

  The primer consisting of the base sequence represented by SEQ ID NO: 1 and the primer consisting of the base sequence represented by SEQ ID NO: 2, 3 or 4 have Tm values close to each other (SEQ ID NO: 75.7 ° C; : 73.0 ° C; SEQ ID NO: 7: 73.2 ° C; SEQ ID NO: 4: 78.5 ° C). Therefore, it has the above advantages. In addition, when PCR is performed using a primer consisting of the base sequence represented by SEQ ID NO: 1 and a primer consisting of the base sequence represented by SEQ ID NO: 2, 3 or 4, there are further advantages over the above advantages. Can bring. That is, by performing PCR using a primer consisting of the base sequence represented by SEQ ID NO: 1 and a primer consisting of the base sequence represented by SEQ ID NO: 2, 3 or 4, significant amplification in the amplification of the telomere sequence Efficiency can be achieved. This is a remarkable effect that cannot be predicted by those skilled in the art at the time of filing this application.

The above-mentioned “primer dimer” means a non-target nucleic acid-dependent primer extension product by a polymerase generated from a primer hybridized to another primer. The presence of a non-target nucleic acid dependent primer extension product is easily performed by performing an amplification reaction in the absence of the target nucleic acid and then detecting the non-target nucleic acid dependent primer extension product using, for example, electrophoresis. Can be evaluated.
In the present specification, the “telomere sequence” refers to a repetitive sequence of the base sequence TTAGGG, and mainly refers to vertebrate telomere sequences, particularly human telomere sequences. A nucleic acid sequence other than a vertebrate is included in the “telomere sequence” in the present specification as long as it has a TTAGGG repeat sequence. The “telomere sequence” includes not only a single-stranded TTAGGG repeat sequence, but also a base sequence complementary to the TTAGGG repeat sequence and a TTAGGG repeat sequence and a complementary strand depending on the usage of the term. A double-stranded DNA sequence consisting of In the present specification, the “telomere sequence” is also simply referred to as “telomere”.

The telomere sequence amplified by the primer of the present invention may be present in various ways. For example, it can be included in all or part of the gene sequence, or in a restriction fragment of plasmid or genomic DNA.
A sample containing a telomere sequence amplified by the primer of the present invention can be obtained from, for example, blood or various tissues as described in International Publication No. 2003/064615. The sample may also contain bodily excretion or bodily excretion such as saliva, urine, feces, cerebrospinal fluid, semen, and milk.
Sources of telomere sequences amplified by the primers of the present invention include all those containing telomere sequences, such as humans and animals. The telomere sequence amplified by the primer of the present invention may be a nucleic acid sequence artificially generated by a chemical or enzymatic process such as a polymerase reaction.

The telomere sequence amplified by the primer of the present invention can be prepared based on the state of the art as of the filing of the present application. For example, it can be prepared by treating a sample containing a telomere sequence with a detergent, sonication, electroporation, a denaturing agent, etc., and extracting DNA from cells or the like. The extracted DNA can be purified based on the technical level at the time of filing the present application, if necessary.
Also, as described in International Publication No. 2003/064615, various additives can be added to a sample containing a telomere sequence to promote optimal hybridization, amplification and detection.

  The primer of the present invention can be contacted with a telomeric sequence so that the forward primer can hybridize to the first strand of the target nucleic acid and the reverse primer can hybridize to the second strand of the target nucleic acid. A variety of hybridization conditions, including high, medium and low stringency conditions can be used to hybridize the primers of the invention with telomeric sequences. Hybridization conditions such as temperature, salt concentration, pH, type and concentration of organic solvent, and type and concentration of chaotropic agent can be appropriately changed based on the technical level at the time of filing this application.

In PCR using the primer of the present invention, the temperature and time of each step of denaturation, annealing, and extension, the type and concentration of DNA polymerase, the composition and pH of 10 X PCR buffer, based on the state of the art at the time of filing this application, Conditions such as dNTP concentration, primer concentration, magnesium chloride concentration, template DNA amount and the like can be appropriately changed.
In PCR using the primer of the present invention, various drugs well known in the art can be used. For example, an agent for increasing the extendibility of polymerase, an agent for stabilizing polymerase, an agent for reducing non-specific hybridization of primers, an agent for increasing the efficiency of DNA replication, etc. Can be used.
PCR using the primers of the present invention can be performed using PCR equipment available in the art.

The PCR procedure is widely known in the art, and the same procedure can be used when the primer of the present invention is used. After DNA is made into a single strand by heat denaturation (eg, 94 ° C. to 96 ° C.), the primer is annealed to the single stranded DNA (eg, 50 ° C. to 65 ° C.), and the complementary strand is made using a heat-resistant DNA polymerase. By repeating the cycle of synthesis (for example, 72 ° C.), for example, 25 to 35 times, only the target gene region can be amplified. In 2-step PCR, for example, after 95 ° C for 10 minutes, 95 ° C for 15 seconds (thermal denaturation) and 60 ° C for 1 minute (annealing and extension) are repeated 40 times to amplify only the target gene region. it can. As PCR reaction conditions, those programmed in a PCR instrument can be used.
The amplification product thus obtained can be detected by, for example, electrophoresis, chromatography, DNA chip (microarray), antigen-antibody reaction or the like. Moreover, the amplification product by PCR can be detected or analyzed as described in WO2003 / 066461 pamphlet.
When the telomere sequence amplified by PCR is detected using means such as electrophoresis, the size and amount of the template DNA can affect how much the telomere sequence is amplified.
The time and temperature of each step usually depends on the polymerase, the length of the target nucleic acid to be amplified and the primer sequence used for amplification.

  Moreover, the amplification product by PCR can be detected and quantified during the amplification reaction by real-time PCR. Real-time PCR is well known in the art, and what equipment and reagents can be used is common general knowledge as of the filing of the present application. For example, the amplification product can be detected and quantified during an amplification reaction by real-time PCR as described in WO2003 / 064615. For example, real-time PCR can be performed using Step One Plus manufactured by Applied Biosystems.

As a method for detecting and quantifying an amplification product using real-time PCR, there are a relative quantification method and an absolute quantification method. The relative quantification method compares the telomere amount of a sample by the ratio to the telomere amount of a known cell. Therefore, when the relative quantification method is adopted, it is usually not possible to directly compare data between different laboratories.
On the other hand, in the absolute quantification method, the amount of telomeres contained in an unknown telomere amount can be quantified as an absolute value by using single copy gene oligomers and telomere oligomers as standards. Can be used to directly compare data between different laboratories.
The above-mentioned “oligomer of single copy gene” means an oligonucleotide containing a base sequence specific to the single copy gene. Similarly, the “oligomer of 36B4 gene” described below means an oligonucleotide containing a base sequence specific to the 36B4 gene.

An absolute quantification method of telomere length using a standard straight line with an oligomer of a 36B4 gene, which is a telomeric oligomer and a single copy gene, has been reported by Nathan et al. (See BioTechniques, 44: 807-809, May 2008). Nathan et al. Used the same primers as those used by Carlson et al.
Real-time PCR is performed using the primer of the present invention to amplify the telomere sequence in the sample, the amount of telomere in the sample is measured by an absolute quantification method, and the amount of telomere in the sample can be quantified as an absolute value. This makes it possible to directly compare data between different laboratories.
In addition, by using DNA obtained from the same person with aging as a standard line of telomere length, it is possible to easily check data comparison and reproducibility between laboratories, and to ensure sufficient accuracy control Is possible.

The telomere quantification method of the present invention is a method for quantifying telomeres per test diploid cell as follows.
A method for quantifying telomeres per test diploid cell, comprising:
(A) For a synthetic telomere oligomer that is a repetitive sequence of a base sequence consisting of TTAGGG, using real-time PCR, a step of creating a calibration curve showing the relationship between the telomere amount and the Ct value,
(B) subjecting a cell population sample of test diploid cells to real-time PCR under the same conditions as in step (a) to obtain a Ct value;
(C) applying the Ct value of step (b) to the calibration curve of step (a) to obtain the telomere amount (X) in the cell population sample; and
(D) Applying X in step (c) to the following formula to obtain the amount of telomeres (Y) per test diploid cell:
Y = X / Z
Z: including the number of cells contained in the cell population sample obtained using a single copy gene present in the test diploid cell as an index,
A method comprising performing the real-time PCR of steps (a) and (b) using the primer set of the present invention.

The “telomere oligomer” in the above step (a) has a base sequence consisting of TTAGGG repeatedly, and has, for example, 10 to 100 TTAGGG repeats. The number of TTAGGG repeats is preferably 10 to 50, and particularly preferably 14 to 28.
In the above step (b), “Applying a cell population sample of test diploid cells to real-time PCR under the same conditions as in step (a)” means that a cell population sample of test diploid cells is subjected to real-time PCR. After processing so that it can be attached, it is meant to be subjected to real-time PCR under the same conditions as in step (a). For example, it means that DNA extracted from a cell population sample of test diploid cells is subjected to real-time PCR under the same conditions as in step (a).
As a single copy gene, for example, the 36B4 gene known as a gene encoding an acidic ribosome phosphorylated protein can be used, but genes other than the 36B4 gene can also be used. For example, albumin, beta globin, etc. can be used.
As a method for calculating the number of cells contained in a cell population sample using a single copy gene present in a test diploid cell as an index, for example, using real-time PCR, the total copy number and Ct value of a single copy gene There is a method for calculating the number of cells contained in a cell population sample by creating a calibration curve indicating the relationship between the cell population samples.

Although the specific example of absolute quantification is shown below, it is not limited to this.
Synthetic oligomers of both telomeres and 36B4 are used. For each synthetic oligomer, the number of molecules is calculated from the molecular weight and the synthesized weight. As the telomere oligomer, not only an oligomer in which TTAGGG is repeated 14 times, but also one in which this is incorporated into a plasmid and stabilized can be used. When the base number of this oligomer is 84 bp and the molecular weight is 26667.2, the weight per molecule is 0.44 × 10 ⁻¹⁹ g (26667.2 / 6.02 × 10 ²³ ). For example, when 60 pg of this oligomer is used, it can be seen that 1.36 × 10 ⁹ (60 × 10 ⁻¹² /0.44×10 ⁻¹⁹ ) oligomers are contained therein. Since one oligomer is 84 bp, the total amount of telomeric sequences can be calculated as 1.14 × 10 ⁸ kb in total (84 × 1.36 × 10 ⁹ bp). Create a dilution series of this oligomer and draw a calibration curve with the Ct (threshold cycle) value. Apply the Ct value of the sample to this calibration curve to obtain the total telomere amount.
Similarly, a calibration curve for a single copy gene is created. When the oligomer of the 36B4 gene (75 bp, MW 22953) is used, the weight per molecule is 3.81 × 10 ⁻²⁰ g (22953.0 / 6.02 × 10 ²³ ), which is calculated in the same manner as for telomeres. For example, in this 36B4 oligomer 500 pg, it can be calculated that 1.31 × ^{10 10} molecules (500 × ^{10 −12} /3.81×10 ⁻²⁰ ) molecules exist. Since the 36B4 gene is present in each of the 12th homologous chromosomes, there are 6.56 × ^{10 9} (1.31 × ¹⁰ 10/2) diploid copy numbers (diploid cell numbers). As in the case of telomeres, if a calibration curve is prepared from the Ct value of the dilution series, the diploid copy number (diploid cell number) in the sample can be obtained. Then, by dividing the telomere amount in the sample by the diploid copy number (diploid cell number), the telomere amount per diploid cell can be obtained. Furthermore, since 92 chromosomal telomeres are considered to exist per cell, dividing by 92 gives the average telomere length of one chromosome as an absolute quantitative value.

The calibration curve for a single copy gene may indicate the relationship between the total copy number of the oligomer of the single copy gene and the Ct value, or the total copy number of the single copy gene is divided by 2 in advance to produce a diploid. The relationship between the copy number (diploid cell number) and the Ct value may be shown.
Usually, the step of calculating the amount of telomeres in the sample and the step of calculating the diploid copy number (diploid cell number) may be performed simultaneously, and either step may be performed first. From the viewpoint of improving accuracy, it is preferable to carry out simultaneously.

By the quantification method of the present invention, the amount of telomeres in a sample can be quantified as an absolute value. This makes it possible to directly compare data between different laboratories, so that the present invention can be used very effectively in disease diagnosis. For example, it finds applications in cancer diagnosis, recurrence prediction, age-related disease diagnosis, cloned organism integrity, genetic disease screening, and the like.
Measurement of telomere amount is very useful in medical diagnosis, prognosis management of diseases, confirmation of the effectiveness of therapeutic agents, and the like. Amplification and quantification of telomere amount is also useful for various diseases associated with changes in telomere length.
It was also known that telomeres are shortened by aging and canceration of cells, but it has been reported that leukocyte telomeres are shortened by stress and lifestyle-related diseases. Therefore, the present invention is useful for use in the auxiliary diagnosis of cancer from biopsy materials, the field of police forensics, the prediction of lifestyle-related diseases, the determination of stress, the age of organisms and cells, the anti-aging field and the cosmetics field. is there.

In order to quantify the amount of telomere with higher accuracy, it is preferable to use the following as a primer.
A linker represented by the above formula (1), n ₁ is 5, L ₁ is less than 16, preferably 12 or less bases, such as GTTT, GGTTTT, CGGTTTG, GTTTATTA, GTTTGTTG, GGGGAGGA, A primer representing a linker selected from the group consisting of ATTTATTA and GTTTATTAGTTT (SEQ ID NO: 21).
A primer which is represented by the above formula (2) and does not contain a sequence obtained by deleting two bases from the base sequence represented by GTAGGG at the 3 ′ end.
A linker represented by the above formula (2) and L ₂ consisting of 8 bases, for example, a linker selected from the group consisting of GTTTATTA, GTTTGTTG and GGGGAGGA, is represented by GTAGGG at the 3 ′ end. A primer that does not include a sequence in which two bases are deleted from the base sequence.
In the formula (1) or (2), L ₁ or L ₂ represents a linker represented by GTTTATTA, and 2 bases are deleted from the base sequence represented by GTAGGG at the 3 ′ end. A primer that does not contain the sequence that is formed.

  Hereinafter, the effects of the present invention will be described in detail with reference to examples. However, the scope of the present invention is not limited to these examples.

(Example 1) Primer design Primers having the nucleotide sequences represented by SEQ ID NOs: 1, 2 and 5-16 below were synthesized (Table 1). The primers represented by SEQ ID NOs: 1, 2, 5-9 and 12-15 are mutated at only one place in the TTAGGG or CCCTAA repeat sequence, and the base sequence on the 5 ′ side for the purpose of adjusting the Tm value. 6 forward and reverse primers with close Tm values were selected from those with the.
Regarding these primers, International Publication No. 2003/064615, “Richard M. Cawthon, Telomere measurement by quantitative PCR, Nucleic Acids Research, Vol. 30, No. 10, e47, 2002” Or “Andreas O. Karlsson et al., Estimating human age in forensic sample by analysis of telomere repeats, Forensic Science International: Genetics Supplement Series 1: pp. 569-571, Comparison with the primers disclosed in 2008 (SEQ ID NOs: 10, 11, 13, and 16) (Table 2).

Table 1: Names and base sequences of synthesized primers

Table 2: Comparison of 6-mer repetitive sequence mutation sites

In the primer names in Table 1 above, the symbols R and F represent a forward primer and a reverse primer, respectively.
CawthonF and CawthonR are disclosed in WO2003 / 064615 and “Richard M. Cawthon, Telomere measurement by quantitative PCR, Nucleic Acids Research, Vol. 30, No. 10, e47, 2002”. Are the same as the primers specifically described in.
KarlssonF and KarlssonR are described in WO2003 / 064615 and "Andreas O. Karlsson et al., Estimating human age in forensic sample by analysis of telomere repeats, Forensic Science. International: Genetics Supplement Series 1: pp. 569-571, 2008 ”.
Yama3R, Yama6R and CawthonR have the same sequence.
The Tm values listed in the above table are calculated based on the nearest neighbor method calculation formula.

(Example 2) Examination of designed primer
Using a master mix containing ABI 10xPCR buffer II, Taq polymerase, 10 ng Placenta DNA, dNTP, MgCl ₂ and primers, adjusted to a final volume of 20 μL with purified water for PCR, PCR was performed using 100 Thermal Cycler (manufactured by MJR) under the following conditions.
Placenta DNA was obtained from a healthy human placenta.
The final concentrations in the master mix are dNTP 0.2 mM, MgCl ₂ 3 mM, each primer 100 nM and 1 U Taq polymerase. The same applies to the following embodiments.
For Taq polymerase, Gene Gene of Nippon Gene or Hot-Start Gene Taq was used. Gene Taq is difficult to handle because there is a concern that the polymerase reaction may progress during reagent preparation. As a result of using Hot-Start Gene Taq, the enzyme reaction is activated by heating at 95 ° C for 5 minutes. Became stable. The same applies to the following embodiments.
Primers were used in the combinations of Yama1F-Yama1R, Yama2F-Yama2R, Yama3F-Yama3R, Yama4F-Yama4R, Yama5F-Yama5R, Yama6F-Yama6R, CawthonF-CawthonR and KarlssonF-KarlssonR designed in Example 1. For primer concentrations, see “Andreas O. Karlsson et al., Estimating human age in forensic sample by analysis of telomere repeats, Forensic Science International: Genetics Supplement Series 1: 569 ˜p. 571, 2008 ”, a test using a forward primer of 100 nM and a reverse primer of 900 nM and a test using an equal amount of a forward primer and a reverse primer of 500 nM for comparison.
PCR cycle conditions are:
95 ° C for 10 minutes
95 cycles at 95 ° C for 15 seconds, 60 ° C for 1 minute,
Met.

When the obtained PCR reaction solution was subjected to ethidium bromide-containing agarose gel electrophoresis and the PCR amplification product visualized by fluorescence was observed, smear of telomeric DNA was confirmed (FIG. 3). In particular, it was confirmed that remarkable amplification efficiencies were brought about when the Yama1F-Yama1R primer combination was used.
Therefore, the amplification efficiency was digitized by performing image processing analysis on the fluorescence intensity observed in FIG. 3 using NIH Image software 1.58 (FIG. 4). The amplification efficiency of telomeric DNA when using the Yama1 primer was about 2 to 3 times or more compared with the case where the primer used by Cawthon and Karlsson et al. Was used.

As shown in Table 2, in the combination of Yama1F-Yama1R, the difference in Tm value was 2.7 ° C. (F: 75.7 ° C. and R: 73.0 ° C.). On the other hand, in the combination of Yama3F-Yama3R, the difference in Tm value was 1.3 ° C. (F: 76.4 ° C. and R: 77.7 ° C.), and the Tm value was closer than that of the combination of Yama1F-Yama1R. Further, in the combination of Yama4F-Yama4R, the difference in Tm value was 2.9 ° C. (F: 69.3 ° C. and R: 72.2 ° C.), which was similar to the combination of Yama1F-Yama1R.
However, from the results of Example 2, it was found that particularly remarkable amplification efficiency was brought about when the Yama1F-Yama1R combination was used. From this, it can be seen that the combination of Yama1F-Yama1R has a significant effect that far exceeds the benefits expected to be obtained simply because they both have similar Tm values.

(Example 3) Examination of effective site of designed primer The following primer set was prepared.
(I) A set of a primer formed by binding a CawthonF linker sequence to a Yama1F repeat sequence and a primer formed by binding a CawthonR linker sequence to a Yama1R repeat sequence.
(Ii) A set of a primer formed by binding a linker sequence of KarlssonF to a repeat sequence of Yama1F and a primer formed by binding a linker sequence of KarlssonR to a repeat sequence of Yama1R.
(Iii) A set of a primer obtained by binding a Yama1F linker sequence to a CawthonF repeat sequence and a primer obtained by binding a Yama1R linker sequence to a CawthonR repeat sequence.
(Iv) A set of a primer formed by binding a Yama1F linker sequence to a KarlssonF repeat sequence and a primer formed by binding a Yama1R linker sequence to a KarlssonR repeat sequence.

Next, a master mix adjusted with purified water for PCR to a final volume of 20 μL of final concentration, dNTP 0.2 mM, MgCl ₂ 3 mM, SYBR Green X80000, ROX dye 0.3 μM, each primer 100 nM and 1 U Taq polymerase. The PCR was carried out using the PTC-100 Thermal Cycler (manufactured by MJR) under the following conditions. As a sample, 10 ng of Placenta DNA or K562 cell-derived DNA was used.
Placenta DNA was obtained from a healthy human placenta as in Example 2. K562 cells are a human chronic myeloid leukemia cell line and are known to have short telomeres. The same applies to the following embodiments.
As the primers, the above primer sets (i) to (iv) were used, respectively, and compared with the combination of Yama1F-Yama1R.
PCR cycle conditions are:
95 ° C for 10 minutes
40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute,
Met.
The obtained PCR reaction solution was subjected to ethidium bromide-containing agarose gel electrophoresis, and a PCR amplification product visualized by fluorescence was observed. Only when a primer having a repeat sequence of Yama1F / R was used, telomere extension was performed. Happened (FIG. 5). Therefore, it was found that the essential portion for amplification of the telomere sequence is the Yama1F / R repeat sequence, and the sequence of the linker portion can be modified without affecting the amplification efficiency.
It was also found that short telomeres such as telomeres derived from K562 cells can be sufficiently amplified by PCR using a primer having a Yama1F / R repeat sequence.

(Example 4) Examination of primers
In Yama1F, the first T of the 6-mer repetitive sequence was mutated to G. Therefore, the case of mutating to C (SKF1) or A (SKF4) in addition to G was examined. For each of the three types, there are three possibilities of base substitution in the reverse primer, so a total of nine combinations were examined. Table 3 shows the base sequences of the primers used.

Table 3: Names and base sequences of the primers used

PCR was performed under the same conditions as in Example 3 except that the primers were used in the following combinations.
1. Yama1F-Yama1R
2. Yama1F-SKR1
3. Yama1F-SKR4
4). SKF1-Yama1R
5. SKF1-SKR1
6). SKF1-SKR4
7). SKF4-Yama1R
8). SKF4-SKR1
9. SKF4-SKR4

The obtained PCR reaction solution was subjected to ethidium bromide-containing agarose gel electrophoresis, and the PCR amplification product visualized by fluorescence was observed. As a result, Yama1F in which the first T of the 6-mer repetitive sequence was mutated to G was identified. Only when it was used, telomere elongation occurred regardless of the type of reverse primer used (FIG. 6).
From these results, it was confirmed that the optimal primer combinations were Yama1F-Yama1R, Yama1F-SKR1, and Yama1F-SKR4.

(Example 5) Verification of telomere sequence amplification efficiency of primer of the present invention Final concentration, dNTP 0.2 mM, MgCl ₂ 3 mM, SYBR Green X80000, ROX dye 0.3 μM, each primer (Yama1F / R) 100 nM and 1 U Using a master mix of Taq polymerase adjusted with purified water for PCR to a final volume of 20 μL, PCR was performed under the following conditions using Step One Plus manufactured by Applied Biosystems. As samples, 5 ng, 10 ng, 20 ng, 40 ng or 80 ng of Placenta DNA was used.
PCR cycle conditions are:
95 ° C for 10 minutes
40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute,
Met.

  The obtained PCR reaction solution was subjected to ethidium bromide-containing agarose gel electrophoresis, and when PCR amplification products visualized by fluorescence were observed, smear of telomeric DNA could be confirmed even in the case of each template DNA amount. It was confirmed that the amount of PCR reaction product increased according to the amount (left of FIG. 7). Moreover, from the result of Southern blotting performed according to the standard method, it was confirmed that the primer of the present invention was telomere-specific (FIG. 7 right). From these results, it was found that the primer of the present invention has high specificity, and that the telomere sequence can be effectively amplified even if the amount of template DNA is small if the primer of the present invention is used.

(Example 6) Comparison by absolute quantification method of telomere length between CD34 positive cells including hematopoietic stem cells and granulocytes which are mature and differentiated cells Extraction of genomic DNA from cells 5 μg of proteinase K per 100,000 cells was added and stirred. 50 μL of cell lysis buffer (PH8.0 Tris buffer 50 mM, EDTA 10 mM, SDS 1% at final concentration) was added, and the mixture was shaken at 60 ° C. for 2 hours for reaction. Purified extraction twice with phenol and twice with chloroform. DNA was ethanol precipitated and washed with 70% ethanol. After air drying, the DNA was dissolved in 100 μL of TE (PH7.5 Tris buffer 10 mM, EDTA 1 mM). 1 μL of 1 mg / ml RNase was added to the resulting solution and reacted at room temperature for 30 minutes. To this solution, 1 μL of 10% SDS and 5 μg of 1 mg / ml proteinase K were added and shaken at 60 ° C. for 1 hour. Purified and extracted twice with phenol and twice with chloroform. DNA was ethanol precipitated in the presence of NaCl at a final concentration of 100 mM, and then washed with 70% ethanol. After air drying, the DNA was dissolved in 100 μL of TE (PH7.5 Tris buffer 10 mM, EDTA 1 mM). DNA concentration and purity were measured.
The cells used were CD34-positive cells and granulocytes (mainly neutrophils) obtained from normal persons aged 42, 52 or 60.
For comparison, K562 cells and human placenta (Placenta) cells were similarly tested.

2. Determination of telomeres by absolute quantification Final concentration, dNTP 0.2 mM, MgCl ₂ 3 mM, SYBR Green X80000, ROX dye 0.3 μM, each primer (Yama1F / R) 100 nM and 1 U Taq polymerase, final volume 20 μL Using a master mix adjusted with PCR-purified water as described above, a cycle of 95 cycles at 95 ° C for 10 minutes, 95 ° C for 15 seconds and 60 ° C for 1 minute for 40 cycles using a real-time PCR device (Applied Biosystems Step One Plus) PCR was performed under the conditions, and a calibration curve (FIG. 8) showing the relationship between the telomere amount in the telomere oligomer and the Ct value was prepared. In addition, a calibration curve for the 36B4 gene was prepared based on the report of Nathan et al. (FIG. 9). In the graph shown in FIG. 9, the vertical axis represents the diploid copy number (cell number) calculated by dividing the copy number of 36B4 gene by 2.
As the telomere oligomer, a synthetic oligomer in which TTAGGG was repeated 14 times was used. This oligomer was 84 bp and the weight average molecular weight was 26667.2. The base sequence is shown below.
5'-ttagggttagggttagggttagggttagggttagggttagggttagggttagggttagggttagggttagggttagggttaggg-3 '(SEQ ID NO: 19)
As the oligomer of the 36B4 gene, one having 75 bp and a weight average molecular weight of 22953 was used. The base sequence is shown below.
5'-cccattctatcatcaacgggtacaaacgagtcctggccttgtctgtggagacggattacaccttcccacttgctg-3 '(SEQ ID NO: 20)

Next, the above 1. Real-time PCR was performed on the genomic DNA in the sample obtained in the above under the same conditions as described above. The obtained Ct value is applied to the calibration curve shown in FIG. The amount of telomeres in the sample obtained in (1) was calculated.
Similarly, using the real-time PCR and the calibration curve shown in FIG. The number of each cell used in (the diploid copy number) was calculated.
Above 1. The amount of telomeres in the sample obtained in (1) was divided by the number of cells (diploid copy number) to determine the amount of telomeres per cell. An example of calculating the amount of telomeres for CD34 positive cells including hematopoietic stem cells and granulocytes, which are mature and differentiated cells, by the above technique is shown here, and the results are shown in FIG.

CD34 positive cells, which are markers for hematopoietic stem cells, differentiate into granulocytes. During this time, cells are considered to repeat division several times, and as a result of the disappearance of telomerase activity with cell differentiation, it is thought that telomeres become shorter with differentiation.
As described above, when the amount of telomeres in CD34 positive cells and granulocytes obtained from normal persons of 42 years, 52 years and 60 years was compared by an absolute quantification method, shortening of telomeres could be detected in all cases. That is, by using the primer of the present invention, it can be confirmed that the telomere length of CD34 positive cells is shortened with aging and that the telomere length is further shortened in mature differentiated granulocytes. did it. Therefore, in the method of the present invention, it is considered that the difference in several mitotic lifetimes can be identified. Furthermore, it is considered that the method of the present invention can detect telomere shortening accompanying aging between different individuals.

(Example 7) Examination of mutants based on the sequence of Yama1F Examination of the number of GTAGGG sequence repeats
Yama1F contains a sequence consisting of 5 repeats of GTAGGG sequence. Furthermore, a primer consisting of 3 repeats of GTAGGG sequence on the 3 'end side following the linker portion of Yama1F (hereinafter referred to as Yama1F (3)) ), A primer that repeats four GTAGGG sequences on the 3 'end side following the linker portion of Yama1F (hereinafter referred to as Yama1F (4)), and a GTAGGG sequence on the 3' end side following the linker portion of Yama1F A primer consisting of 6 repeats (hereinafter referred to as Yama1F (6)) and a primer consisting of 7 repeats of the GTAGGG sequence on the 3 'end side following the linker portion of Yama1F (hereinafter referred to as Yama1F (7)) were prepared. .
Next, in the same manner as in the method described in Example 6, the amount of telomere was quantified by an absolute quantification method. However, the prepared primer was used as a forward primer, Yama1R was used as a reverse primer, and Placenta DNA and K562 cell-derived DNA were used as samples. The case where these primers were used was compared with the case where Yama1F and Yama1R were used. The results are shown in FIG.

As shown in FIGS. 11 (a) to 11 (e), telomere amplification using Yama1F (3), Yama1F (4), Yama1F (5) (ie, Yama1F) and Yama1F (6) depended on the telomere amount. Good linearity was observed.
However, as shown in FIG. 11 (f), when quantification is actually performed, it is almost the same when Yama1F (6) is used as compared with the measurement result when Yama1F is used as 100%. Amplification was obtained, but when Yama1F (3) was used, it was 6% for K562 and 15% for Placenta, and when Yama1F (4) was used, it was 27 for K562. % And Placenta were low at 24%.
Therefore, in order to efficiently amplify and quantify telomeres, it was considered that the GTAGGG sequence should be present 5 to 6 times.

2. Study on deletion and substitution of bases in GTAGGG sequence and change in length and sequence of linker base 5 or 6 times repeated sequence of GTAGGG, further 1 or 2 bases deleted or substituted from GTAGGG With respect to the primer added with, it was examined whether the positional relationship between the GTAGGG repeat sequence and the deletion sequence or the mutant sequence (referred to as a sequence with a substituted base) affects telomere amplification.
(1) 5 repetitions of GTAGGG + deletion sequence or mutant sequence In addition to 5 repetitions of GTAGGG, the deletion sequence TAGGG lacking the first G of GTAGGG is counted from the first to the sixth from the linker, respectively. Arranged primers (D1-1, D1-2, D1-3, D1-4, D1-5, and D1-6) were prepared. The same linker sequence as Yama1F was arranged on the 5 ′ end side.
In addition to 5 GTAGGG repeats, a primer (M1-1, M1-2, M1) in which a mutant sequence CTAGGG obtained by mutating the first G of GTAGGG to C is counted from the linker in the first to sixth positions, respectively. -3, M1-4, M1-5, and M1-6). The same linker sequence as Yama1F was arranged on the 5 ′ end side.
In addition to 5 GTAGGG repeats, a primer (D2-1, D2-2, D2-) in which the deletion sequence AGGG lacking the first 2 bases of GTAGGG is arranged from the first to the sixth counted from the linker, respectively. 3, D2-4, D2-5, and D2-6). The same linker sequence as Yama1F was arranged on the 5 ′ end side.
In addition to 5 GTAGGG repeats, the first GTAGGG G is T, the second T is A, and TAAGGG, a sequence mutated by 2 bases, is placed in the first to sixth positions counted from the linker. Primers (M2-1, M2-2, M2-3, M2-4, M2-5, and M2-6) were prepared. The same linker sequence as Yama1F was arranged on the 5 ′ end side.

  Here, “first from the linker” means between the linker moiety and the repeating unit GTAGGG on the 5 ′ end side. The “second from the linker” means between the most repeating unit GTAGGG at the 5 ′ end side and the second repeating unit GTAGGG from the 5 ′ end side. The “third from the linker” means between the second repeating unit GTAGGG from the 5 ′ end side and the third repeating unit GTAGGG from the 5 ′ end side. The “fourth from the linker” means between the third repeating unit GTAGGG from the 5 ′ end side and the fourth repeating unit GTAGGG from the 5 ′ end side. “Fifth from the linker” means between the fourth repeating unit GTAGGG from the 5 ′ end side and the fifth repeating unit GTAGGG from the 5 ′ end side. “6th counted from the linker” means the 3 ′ end side of the 5 times repeated sequence of GTAGGG. The same applies hereinafter.

(2) 5 GTAGGG repeats + linker mutation 5 GTAGGG repeats with zero linker base number (L0-5), only the first 4 bases of Yama1F linker sequence 5 'Linked to the terminal side (L4-5), Linked to the 5' terminal side by adding 4 bases (GTTT) to the 3 'terminal side of the linker sequence of Yama1F to make 12 bases (L12- 5) or two Yama1F linker sequences linked to each other at 16 ′ base (L16-5) were prepared.
A linker having 5 GTAGGG repeats and having all of A in the linker sequence of Yama1F mutated to G bound to the 5 ′ end (Yama1FL (AG)), in the linker sequence of Yama1F A linker in which T is all mutated to G is bound to the 5 ′ end (Yama1FL (TG)), and a linker in which all G in the linker sequence of Yama1F is A is bound to the 5 ′ end ( Yama1FL (GA)), one having a CawthonF linker on the 5 ′ end side (Cawthon + GTAGGG × 5), or one having a KarlssonF linker on the 5 ′ end side (Karlsson + GTAGGG × 5) were prepared.

  Next, in the same manner as in the method described in Example 6, the amount of telomere was quantified by an absolute quantification method. However, the primer prepared as described in (1) or (2) above was used as a forward primer, Yama1R was used as a reverse primer, and Placena DNA and K562 cell-derived DNA were used as samples. The case where these primers were used was compared with the case where Yama1F and Yama1R were used. The results are shown in FIG.

(3) 6 GTAGGG repeats + deletion sequence or mutant sequence From the results shown in FIG. 12, in addition to the 6 GTAGGG repeats, the sequences with extremely high and low amplification efficiency were considered. Primers were designed as follows for those to which sequences or mutant sequences were added.
In addition to 6 GTAGGG repeats, a primer (N6D1-1) was prepared in which a deletion sequence TAGGG lacking the first G of GTAGGG was first arranged from the linker. The same linker sequence as Yama1F was arranged on the 5 ′ end side.
In addition to 6 GTAGGG repetitions, a primer (N6M1-1) was prepared in which a mutant sequence CTAGGG obtained by mutating the first G of GTAGGG to C was counted first from the linker.
In addition to 6 GTAGGG repeats, the deletion sequence AGGG lacking the first 2 bases of GTAGGG is first arranged from the linker (N6D2-1) and the seventh arranged primer (N6D2 -7) was created. The “seventh from the linker” means the 3 ′ end side of the 6 times GTAGGG repeat sequence.
In addition to 6 GTAGGG repeats, a primer (N6M2-) that has TAAGGG, the first GTAGGG sequence with 2 base mutations in T and 2nd T in A, counted from the linker. 1) created.

  Next, in the same manner as in the method described in Example 6, the amount of telomere was quantified by an absolute quantification method. However, the primer prepared as described in (3) above was used as a forward primer, Yama1R was used as a reverse primer, and Placena DNA and K562 cell-derived DNA were used as samples. The case where these primers were used was compared with the case where Yama1F and Yama1R were used. The results are shown in FIG.

  From the results of FIG. 12 and FIG. 13, good results were obtained in both cases. When adding a sequence in which two consecutive bases from GTAGGG have been deleted or substituted, than adding a sequence in which one base has been deleted or replaced, or a sequence in which two consecutive bases have been deleted or replaced, is added from GTAGGG. It is usually considered that the function as a primer is easily affected. However, when a sequence in which two consecutive bases from GTAGGG were deleted or substituted was added, good results were obtained, so when a sequence in which any one or two bases were deleted or substituted from GTAGGG was added. The functions as the primer of the present invention were considered to be good.

(4) 5 or 6 repeats of GTAGGG + deletion sequence or mutant sequence + linker mutation 5 repeats of GTAGGG, and only the first 4 bases of the linker sequence of Yama1F are bound to the 5 ′ end, Furthermore, a primer (L4 D1-2) was prepared in which the deletion sequence TAGGG was arranged second from the linker.
A primer (L4 M1-5, which has 5 GTAGGG repeats), which binds only the first 4 bases of the linker sequence of Yama1F to the 5 ′ end side, and that the mutant sequence CTAGGG is located at the 5th position counted from the linker. )created.
A primer (L4 M2-1) having 5 GTAGGG repeats, binding only the first 4 bases of the linker sequence of Yama1F to the 5 ′ end side, and the mutant sequence TAAGGG counted first from the linker )created.
A primer (L4 N6D1-) that has 6 repeats of GTAGGG, binds only the first 4 bases of the linker sequence of Yama1F to the 5 ′ end, and further positions the deletion sequence TAGGG first from the linker. 1) created.
A primer (L4 N6M2-1) having 6 GTAGGG repeats, binding only the first 4 bases of the linker sequence of Yama1F to the 5 ′ end side, and the mutant sequence TAAGGG counted first from the linker )created.

A linker that has 5 GTAGGG repeats, in which A in the linker sequence of Yama1F is all mutated to G, is linked to the 5 ′ end, and the deletion sequence TAGGG is located second from the linker. A primer (AG D1-2) was prepared.
A linker having 5 GTAGGG repeats, in which all A in the linker sequence of Yama1F was mutated to G, was linked to the 5 ′ end side, and the mutated sequence CTAGGG was placed in the fifth position counted from the linker. A primer (AG M1-5) was prepared.
A linker having 5 GTAGGG repeats, in which all of A in the linker sequence of Yama1F were mutated to G was linked to the 5 ′ end side, and the mutated sequence TAAGGG was arranged first from the linker. A primer (AG M2-1) was prepared.
A linker having 6 GTAGGG repeats, in which A in the linker sequence of Yama1F is all mutated to G, is linked to the 5 ′ end, and the deletion sequence TAGGG is first counted from the linker. A primer (AG N6D1-1) was prepared.
A linker having 6 GTAGGG repeats, in which all of A in the linker sequence of Yama1F were mutated to G, was linked to the 5 ′ end side, and the mutated sequence TAAGGG was first arranged from the linker. A primer (AG N6M2-1) was prepared.

A linker that has 5 GTAGGG repeats and mutated all Ts in the linker sequence of Yama1F to G is linked to the 5 ′ end side, and the deletion sequence TAGGG is arranged second from the linker. Primer (TG D1-2) was prepared.
A linker having 5 GTAGGG repeats, in which all Ts in the linker sequence of Yama1F were mutated to G, was linked to the 5 ′ end side, and the mutant sequence CTAGGG was placed in the fifth position counted from the linker. A primer (TG M1-5) was prepared.
A linker having 5 GTAGGG repeats, in which all Ts in the linker sequence of Yama1F were mutated to G, was linked to the 5 ′ end side, and the mutated sequence TAAGGG was arranged first from the linker. A primer (TG M2-1) was prepared.
A linker having 6 GTAGGG repeats, in which all Ts in the linker sequence of Yama1F have been mutated to G, is linked to the 5 ′ end, and the deletion sequence TAGGG is arranged first from the linker. A primer (TG N6D1-1) was prepared.
A linker having 6 GTAGGG repeats, all of which was mutated to T in the linker sequence of Yama1F, was linked to the 5 ′ end side, and the mutant sequence CTAGGG was first arranged from the linker. A primer (TG N6M1-1) was prepared.
A linker that has 6 GTAGGG repeats and in which all T in the linker sequence of Yama1F has been mutated to G was linked to the 5 ′ end side, and the mutant sequence TAAGGG was first arranged from the linker. A primer (TG N6M2-1) was prepared.

Next, in the same manner as in the method described in Example 6, the amount of telomere was quantified by an absolute quantification method. However, the primer prepared as described in (4) above was used as a forward primer, Yama1R was used as a reverse primer, and Placenta DNA and K562 cell-derived DNA were used as samples. The case where these primers were used was compared with the case where Yama1F and Yama1R were used. The results are shown in FIG.
When using L4 N6D1-1, K562 showed an extremely high value of 232% and Placenta of 265%, but the calibration in the calibration sample was inferior to that using Yama1F. It is considered that it is not suitable for accurate quantification of Yama1F.
In addition, in the primer tested in Example 7, the specificity with respect to telomeres has any primer, and also in terms of calibration, all the primers are good except that L4 N6D1-1 is inferior as described above. Has calibration.
From the above results, when a sequence having 5 GTAGGG repeats and having 1 or 2 bases deleted or substituted from GTAGGG is added, the linker should be composed of 4 to 8 bases. It was confirmed that good telomere sequence amplification efficiency and quantification can be obtained. In addition, when a sequence having 6 GTAGGG repeats and one or two bases deleted or substituted from GTAGGG is added, a good telomere can be used if the linker consists of 8 bases. It was confirmed that sequence amplification efficiency and quantification can be obtained.

(Example 8) Comparative verification of telomere sequence amplification efficiency and quantitativeness of the primer of the present invention
Using Yama1F as a forward primer, Yama1R as a reverse primer, and 10 ng of Placenta DNA and K562 cell-derived DNA, the amount of telomeres was quantified by an absolute quantification method according to the method described in Example 6, respectively. did. Similarly, a comparison experiment with the case of using primers of Corson, Carlson et al.
The amplification efficiency by each primer was compared with the amount of PCR product by Yama1F as 100. The results are shown in FIG. When using the primers used by Corson, 11% for K562, 3% for Placenta, 25% for K562 and 31% for Placenta when using the primers used by Carlson. This result was consistent with the result of gel electrophoresis described above, and it was confirmed that the amplification of Yama1F was the best and resulted in remarkable amplification efficiency.
In addition, as shown in FIG. 12, when D2-6 or L16-5 was used, the amplification efficiency was slightly lower than when Yama1F was used. However, when the amount of PCR product by Yama1F is 100 and D2-6 is used, it is 55% for K562 and 53% for Placenta, and when L16-5 is used, it is 60% for K562 and Placenta. It was 64%. From these results, even when D2-6 or L16-5 is used, it is about 5 to 10 times or more for the primer used by Corson, and about 2 times for the primer used by Carlson. It was observed that the above amplification efficiency was brought about.

Next, a dilution series is prepared by diluting the sample for the calibration curve (telomere oligomer used in Example 6) every 10 times, and the standard dilution oligo is PCR using the primer (Yama1F / R) of the present invention. It was measured whether or not amplification was performed in a capacity-dependent manner. PCR conditions and the like were performed according to the method described in Example 6. The results are shown in FIG.
As shown in FIG. 16, when Yama1F / R was used, volume-dependent amplification in accordance with the sample dilution series was observed. On the other hand, when a Corson or Carlson primer is used, it is considered that accurate quantitativeness is not recognized.

(Example 9) Correlation between absolute quantification method using primer of the present invention and TRF method
The TRF method is a method in which the DNA portion of the telomere remains, the other DNA portion is cut into pieces by restriction enzyme treatment, washed away by gel electrophoresis, and the telomeric DNA remaining on the gel is detected with a complementary telomere probe. There is a conventional method for measuring telomere length (Yamada O et al J. Clin. Invest. 95: 1117-1123, 1995). However, compared to the method of the present invention, the sample amount is required to be about 1000 times, the time is required about 10 times, the procedure is troublesome, enormous costs and special equipment are required, etc. Convenience is not good.

Using DNA extracted from fibroblasts (ASF4-1 to ASF4-4) of 36, 47, 56, 62 years of the same individual, telomere length (TRF) by Southern blotting, and the primer of the present invention Comparative study of absolute quantification method using sapphire (Table 4). The absolute quantification method using the primer of the present invention showed a good correlation with the result of the TRF method. The reason why the telomere length is slightly shorter than that of TRF is considered to be because only the telomere portion is amplified without reflecting the subtelomere portion. In addition, by using human cell DNA collected from the same individual with aging as described above as a standard line for absolute telomere length quantification, it is possible to control the quality between facilities.
The results are shown in Table 4 below.
From this result, it was proved that the quantification by the absolute quantification method using the primer of the present invention reflected the telomere length.

(Example 10) Comparison of telomere length of normal human DNA measured using the primer of the present invention 10 healthy men in their 30s, 40s, 50s and 60s, and health in their 70s 7 males and 10 healthy women in their 30s, 40s, 50s, 60s and 70s, and measured and compared DNA telomere length using the primer of the present invention did.
After separating the leukocyte layer from the peripheral blood of each person and extracting DNA using Qiagen kit (QIAamp DNA MiniKit), using a primer of Yama1F and Yama1R, using a sample amount of 10 ng, The telomere length was measured in the same manner as described in Example 6. However, Hot-Start Gene Taq NT was used for Taq polymerase. The experiment was performed in triplicate, and when one of the three samples showed a value far from the other, it was determined that there was a problem with the procedure or reaction, and the one sample was removed and counted. The results are shown in Table 5 below and FIGS.

As a result, it was proved that the telomere length decreased with aging even in the normal population. What the inventor normally uses as a standard sample of telomeric DNA accompanying the aging of the same male is 698 kb at the age of 36, 603 kb at the age of 47, 519 kb at the age of 56, 623 kb, 62 when converted per cell. It is 404 kb at age. Compared to this value, it was shown that the telomere length was shortened to the same extent in the group.
There was no significant difference in telomere length at the age of 10 years between the ages, but there was a significant difference in telomere length for both men and women at the age of 20 years (in FIGS. 17-19, * indicates p <0.05). ** represents p <0.01). Since telomere length is born and has individual differences, an age difference of about 10 years old is affected by natural differences. However, the effect of individual differences can be ignored by increasing the number of samples sufficiently. From the above, quantification by the absolute quantification method using the primer of the present invention is considered to have reproducibility in quantifying telomere length even in a normal population.

  The present invention is useful in medical diagnosis, disease prognosis management, confirmation of the effectiveness of therapeutic agents, and the like. In addition, the present invention is useful for use in auxiliary diagnosis of cancer from biopsy material, police knowledge field, prediction of lifestyle-related diseases, determination of stress, biological age and cell age, anti-aging field, cosmetics field, etc. It is.

Claims (8)

A primer for telomere sequence amplification represented by the following formula (1) or (2).
(1) L _1- (R ₁ ) n ₁
In the formula (1), R _{1 as} a repeating unit represents a base sequence represented by GTAGGG, n ₁ represents the number of R ₁ as a repeating unit, 5 or 6, and when n ₁ is 5, ₁ represents a linker composed of 16 or less bases, and when n ₁ is 6, L ₁ represents a linker composed of 8 or less bases.
(2) L _2- (R ₂ ) n ₂ (X)
In the formula (2), R ₂ which is a repeating unit represents a base sequence represented by GTAGGG, and X is the 5 ′ end side of the repeating unit, between any two adjacent repeating units, or of the repeating unit. This represents a sequence in which 1 or 2 bases are deleted or substituted from the base sequence represented by GTAGGG, which is bound to any one position on the 3 ′ end side, and n ₂ is a repeat unit of R ₂ The number is 5 or 6, and when n ₂ is 5, L ₂ represents a linker composed of 4 to 8 bases, and when n ₂ is 6, L ₂ represents a linker composed of 8 bases. Represent. Represented by the formula (1), n ₁ is 5, represents a linker L ₁ is composed of 12 or fewer bases, the primer of claim 1. L ₁ is, GTTT, GGTTTT, CGGTTTG, GTTTATTA , GTTTGTTG, GGGGAGGA, is selected from the group consisting of ATTTATTA and GTTTATTAGTTT, primers of Claim 2.   The primer according to claim 1, which is represented by the formula (2) and does not include a sequence in which two bases are deleted from the base sequence represented by GTAGGG at the 3 'end of the primer. L ₂ is, GTTTATTA, is selected from the group consisting of GTTTGTTG and GGGGAGGA, primers of claim 4.   A primer set for telomere sequence amplification, comprising a combination of the primer according to any one of claims 1 to 5 and at least one primer comprising the base sequence represented by any of SEQ ID NOs: 2 to 4.   A method for amplifying a telomere sequence by performing a polymerase chain reaction using the primer set according to claim 6. A method for quantifying telomeres per test diploid cell, comprising:
(A) For a synthetic telomere oligomer that is a repetitive sequence of a base sequence consisting of TTAGGG, using real-time PCR, a step of creating a calibration curve showing the relationship between the telomere amount and the Ct value,
(B) subjecting a cell population sample of test diploid cells to real-time PCR under the same conditions as in step (a) to obtain a Ct value;
(C) applying the Ct value of step (b) to the calibration curve of step (a) to obtain the telomere amount (X) in the cell population sample; and
(D) Applying X in step (c) to the following formula to obtain the amount of telomeres (Y) per test diploid cell:
Y = X / Z
Z: including the number of cells contained in the cell population sample obtained using a single copy gene present in the test diploid cell as an index,
The said method characterized by performing real-time PCR of a process (a) and (b) using the primer set of Claim 6.