WO2013054666A1 - Amorce pour l'amplification d'une séquence télomérique - Google Patents

Amorce pour l'amplification d'une séquence télomérique Download PDF

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WO2013054666A1
WO2013054666A1 PCT/JP2012/075242 JP2012075242W WO2013054666A1 WO 2013054666 A1 WO2013054666 A1 WO 2013054666A1 JP 2012075242 W JP2012075242 W JP 2012075242W WO 2013054666 A1 WO2013054666 A1 WO 2013054666A1
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primer
sequence
telomere
linker
gtaggg
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PCT/JP2012/075242
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English (en)
Japanese (ja)
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山田　修
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学校法人 東京女子医科大学
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Priority to JP2012549174A priority Critical patent/JP5288387B1/ja
Publication of WO2013054666A1 publication Critical patent/WO2013054666A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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.
  • telomeres are gradually shortened at each cell division, and are known to shorten with age.
  • it is necessary to amplify the repeated sequences and detect them.
  • a polymerase chain reaction (PCR) using an appropriate primer is known.
  • PCR polymerase chain reaction
  • telomere measurement by quantitative PCR Nucleic Acids Research, Vol. 30, No. 10, e47, 2002 .
  • 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.
  • the amount of telomeres was quantified by a relative quantification method.
  • telomere which is a repetitive sequence
  • 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.
  • the present invention provides a telomere sequence amplification primer represented by the following formula (1) or (2).
  • Formula (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.)
  • Formula (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.
  • n 2 represents the number of R 2 as a repeating unit.
  • L 2 represents a linker composed of 4 to 8 bases
  • L 2 represents a linker composed of 8 bases.
  • the present invention also provides a telomere sequence amplification comprising 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 primer set is provided.
  • 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.
  • 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.
  • 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 (PHY marker) used in the leftmost lane.
  • Amplified human Placenta telomeric DNA is observed in a smear state of about 2000 bp or less.
  • FIG. 4 is a graph obtained by analyzing the fluorescence intensity observed in FIG. 3 with NIH ImageNI1.58 software and digitizing the amplification efficiency.
  • FIG. 4 is a graph obtained by analyzing the fluorescence intensity observed in FIG. 3 with NIH ImageNI1.58 software and digitizing the amplification efficiency.
  • H-Yama1 + B-Yama1 means a combination of Yama1F and Yama1R.
  • H-Caw + B-Yama1 is a primer obtained by binding the linker sequence of the forward primer (CawthonF shown in Table 1 below) used by Cawthon 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 composed of the linker sequence of the forward primer used by Carlsson (KarlssonF shown in Table 1 below) and 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 a Yama1F linker sequence to a repeat sequence of KarlssonF and a primer formed by binding a Yama1R linker sequence to a repeat 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 ⁇ x174.
  • 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.
  • ⁇ HindIII 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.
  • ⁇ X174 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.
  • quantification was performed by an absolute quantification method using a primer (Yama1F (3) to (6)) in which 3 to 6 GTAGGG sequences were 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. 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
  • 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).
  • Formula (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.)
  • 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.
  • n 2 represents the number of R 2 as a repeating unit.
  • L 2 represents a linker composed of 4 to 8 bases
  • L 2 represents a linker composed of 8 bases.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a linker consisting of 4 to 8 bases A, G, C or T
  • GTAGGG is 6, 8 bases (A, G, A linker consisting of C or T)
  • 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.
  • 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.
  • L 2 is selected from the group consisting of GTTTATTA, GTTTGTTG and GGGGAGGA, but is not limited thereto.
  • 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.
  • X is TAGGG, GAGGG, GTGGG, GTAGG, AGGG, TGGG, GGGG, GTGG, GAGG, TAGG or GTAG.
  • 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.
  • the first G of the base sequence represented by GTAGGG can be replaced with A, T or C
  • 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
  • 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
  • the sixth G of the base sequence represented by GTAGGG can be replaced with A, T or C.
  • 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.
  • 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.
  • 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.
  • primer synthesis may be outsourced to a synthesis manufacturer such as Sigma-Aldrich Japan.
  • 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.
  • 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.
  • 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 of SEQ ID NOs: 2 to 4. It is a primer set for telomere sequence amplification.
  • the telomere sequence amplification primer consisting of the base sequences represented by SEQ ID NOs: 2 to 4 has the following base sequences.
  • 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 5 GCCTAA sequences are repeated.
  • a base sequence consisting of 9 bases (GGGGCCTAA) is added to the 5 ′ side as a part.
  • the portion that hybridizes to the telomere sequence is a portion of the repetitive sequence. Accordingly, 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 the above SEQ ID NOs: 2 to 4 hybridizes with the telomeric sequence that is a repetitive sequence under appropriate hybridization conditions based on the general knowledge of those skilled in the art as of the filing date of the present application. Can do.
  • 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
  • a reaction can be performed.
  • a 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
  • the primer set can also be made into a kit.
  • the kit includes DNA extraction reagents, PCR buffers such as PCR buffers and DNA polymerase, staining agents, electrophoresis gels, etc.
  • 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.
  • 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.
  • 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. Also, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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; SEQ ID NO: 2 : 73.0 ° C; SEQ ID NO: 7: 73.2 ° C; SEQ ID NO: 4: 78.5 ° C). Therefore, it has the above advantages.
  • 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.
  • 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.
  • telomere sequence refers to a repetitive sequence of the base sequence TTAGGG, and mainly refers to vertebrate telomere sequences, particularly human telomere sequences.
  • telomere sequence 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.
  • 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 telomere 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.
  • 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.
  • 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.
  • 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.
  • a heat-resistant DNA polymerase By repeating the synthesis (for example, 72 ° C.) cycle, for example, 25 to 35 times, only the target gene region can be amplified.
  • 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.
  • 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.
  • the amplification product by PCR can be detected or analyzed as described in WO2003 / 066461 pamphlet.
  • 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.
  • amplification products by PCR can be detected and quantified during amplification reactions 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.
  • the amplification product can be detected and quantified during an amplification reaction by real-time PCR as described in WO2003 / 064615.
  • real-time PCR can be performed using Step One One Plus manufactured by Applied Biosystems.
  • 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.
  • 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.
  • oligomer of single copy gene means an oligonucleotide containing a base sequence specific to the single copy gene.
  • oligomer of 36B4 gene described below means an oligonucleotide containing a base sequence specific to the 36B4 gene.
  • 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.
  • the “telomere oligomer” in the above step (a) has a base sequence composed of TTAGGG repeatedly, for example, 10 to 100 TTAGGGs repeatedly.
  • the number of TTAGGG repeats is preferably 10 to 50, and particularly preferably 14 to 28.
  • “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).
  • 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).
  • 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.
  • albumin, beta globin, etc. can be used.
  • telomere oligomers of both telomeres and 36B4 are used.
  • the number of molecules is calculated from the molecular weight and the synthesized weight.
  • 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.
  • 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 ).
  • 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.
  • 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).
  • telomeres 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Primer design Primers having the nucleotide sequences represented by SEQ ID NOs: 1, 2 and 5 to 16 below were synthesized (Table 1).
  • the primers represented by SEQ ID NOs: 1, 2, 5 to 9, and 12 to 15 are mutated at only one position 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.
  • 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.
  • Example 2 Examination of designed primer ABI 10xPCR buffer II, Taq polymerase, 10 ng of placenta (Placenta) DNA, dNTP, MgCl 2 and primers are used to make a final volume of 20 ⁇ L with purified water for PCR. Using the adjusted master mix, PCR was performed under the following conditions using a PTC-100 Thermal Cycler (manufactured by MJR). 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.
  • PCR cycle conditions are: 95 ° C for 10 minutes 95 cycles at 95 ° C for 15 seconds, 60 ° C for 1 minute, Met.
  • telomeric DNA 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 was used.
  • a master mix adjusted with purified water for PCR to a final volume of 20 ⁇ L of final concentration, dNTP 0.2 mM, MgCl 2 3 mM, SYBR Green X80000, ROX dye 0.3 ⁇ M, 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.
  • 10 ng of Placenta DNA or K562 cell-derived DNA was used as 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 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).
  • 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 Yama1F was obtained by mutating the first T of a 6-mer repetitive sequence 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.
  • 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.
  • 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.
  • 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 ⁇ M, each primer (Yama1F / R) 100 nM and 1 U
  • 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.
  • telomere length 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 ⁇ g of proteinase K per 100,000 cells was added and stirred. 50 ⁇ L 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.
  • the DNA was dissolved in 100 ⁇ L of TE (PH7.5 Tris buffer 10 mM, EDTA 1 mM). 1 ⁇ L of 1 mg / ml RNase was added to the resulting solution and reacted at room temperature for 30 minutes. To this solution, 1 ⁇ L of 10% SDS and 5 ⁇ g 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.
  • TE PH7.5 Tris buffer 10 mM, EDTA 1 mM
  • DNA was dissolved in 100 ⁇ L 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.
  • telomeres by absolute quantification
  • telomere amount in the telomere oligomer was prepared.
  • a calibration curve for the 36B4 gene was prepared based on the report of Nathan et al. (FIG. 9).
  • the vertical axis represents the diploid copy number (cell number) calculated by dividing the copy number of 36B4 gene by 2.
  • 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.
  • 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.
  • 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.
  • Yama1F (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 comprising 4 repeats of GTAGGG sequence on the 3 ′ end side following the linker portion of Yama1F (hereinafter referred to as Yama1F (4)), 3 ′ following the linker portion of Yama1F Primer consisting of 6 repeats of GTAGGG sequence (hereinafter referred to as Yama1F (6)) on the terminal side and primer consisting of 7 repeats of GTAGGG sequence on the 3 ′ end side following the linker part of Yama1F (hereinafter referred to as Yama1F ( 7)) was created.
  • Yama1F (3) a primer comprising 4 repeats of GTAGGG sequence on the 3
  • the amount of telomere was quantified by an absolute quantification method.
  • the prepared primer was used as a forward primer
  • Yama1R was used as a reverse primer
  • 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.
  • 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.
  • the same linker sequence as Yama1F was arranged on the 5 ′ end side.
  • 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.
  • the same linker sequence as Yama1F was arranged on the 5 ′ end side.
  • the first GTAGGG G is T
  • the second T is A
  • 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.
  • first from the linker means between the linker portion and the repeating unit GTAGGG at 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.
  • 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.
  • the amount of telomeres was quantified by an absolute quantification method.
  • the primer prepared as described in (1) or (2) above was used as a forward primer
  • Yama1R was used as a reverse primer
  • 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.
  • 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.
  • the amount of telomeres was quantified by an absolute quantification method.
  • the primer prepared as described in (3) above was used as a forward primer
  • Yama1R was used as a reverse primer
  • 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.
  • a primer (AG D1-2) was prepared.
  • a primer (AG M1-5) was prepared.
  • a primer (AG N6D1-1) was prepared.
  • 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 primer (TG M1-5) was prepared.
  • a primer (TG N6D1-1) was prepared.
  • a primer (TG N6M2-1) was prepared.
  • the amount of telomere was quantified by an absolute quantification method.
  • the primer prepared as described in (4) above was used as a forward primer
  • Yama1R was used as a reverse primer
  • 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.
  • 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.
  • 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.
  • the linker should be composed of 4 to 8 bases. It was confirmed that good telomere sequence amplification efficiency and quantification can be obtained.
  • 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
  • Yama1F was used as a forward primer
  • Yama1R was used as a reverse primer
  • Placenta DNA and K562 cell-derived DNA were used at 10 ng
  • the amount of telomeres was quantified by an absolute quantification method.
  • 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.
  • 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.
  • the TRF method is a gel electrophoresis in which the DNA portion of the telomere is left and the other DNA portion is cut finely by restriction enzyme treatment.
  • telomere DNA remaining in the gel is detected with a complementary telomere probe, and the telomere length that has been conventionally used can be measured (Yamada O et al J. Clin. Invest 95: 1117-1123, 1995).
  • 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.
  • 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.
  • 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.
  • Table 4 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.
  • 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.
  • Qiagen kit QIAamp DNA MiniKit
  • Yama1F and Yama1R a primer of Yama1F and Yama1R
  • sample amount of 10 ng The telomere length was measured in the same manner as described in Example 6.
  • 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.
  • 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.
  • 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, but there was a difference in telomere length for both men and women at the age of 20 years (in Figures 17-19, * indicates p ⁇ 0.05).
  • 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, prognosis management of diseases, confirmation of the effectiveness of therapeutic agents, and the like.
  • 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.

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Abstract

L'objectif de la présente invention est de fournir une amorce pour amplifier une séquence télomérique. La présente invention concerne une amorce représentée par la formule (1) ou la formule (2), et l'utilisation dans l'amplification d'une séquence télomérique. (1) L1-(R1)n1. Dans la formule : les unités répétées R1 représentent une séquence de base représentée par GTAGGG ; n1 représente 5 ou 6 ; lorsque n1 représente 5, L1 est un lieur comprenant 16 bases ou moins ; et lorsque n1 représente 6, L1 est un lieur comprenant 8 bases ou moins. (2) L2-(R2)n2(X). Dans la formule : les unités répétées R2 représentent une séquence de base représentée par GTAGGG ; X représente une séquence obtenue par l'élimination ou la substitution d'une ou deux bases dans la séquence de base représentée par GTAGGG ; n2 représente 5 ou 6 ; lorsque n2 représente 5, L2 est un lieur comprenant 4-8 bases ; et lorsque n2 représente 6, L2 est un lieur comprenant 8 bases.
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JP2018501799A (ja) * 2014-12-30 2018-01-25 テロメア ダイアグノスティクス インコーポレイテッド マルチプレックス定量的ｐｃｒ

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