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Method for determination of the length of the g-tail sequence and kit for the method

Inactive Publication Date: 2009-12-03
HIROSHIMA UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0118]Hereinafter, the advantageous effects of the present invention will be described. According to the method of measuring the length of a G tail sequence according to the present invention, it is possible to measure a short-length G tail of up to 20 nucleotides specifically and quantitatively at high sensitivity, only in 3 steps without the denaturation of a telomere or any tedious processing.
[0119]In addition, the measuring method according to the present invention allows analysis not only of nondenatured chromosomal DNAs but also of cells directly as samples and thus, acceleration of analysis, and is applicable, for example, to the high-throughput screening for analyzing a great number of samples.
[0120]Furthermore, it is possible to analyze a small amount of cells of 5×105 cells or less directly; the chromosomal DNA is not lost during sample preparation; and thus, the method of the present invention is useful in the cases where the number of the cells obtained is limited, for example, during blood test or analysis of clinical samples such as biopsy and urine for cancer cell analysis.
[0121]With the kit for measuring the length of a G tail sequence according to the present invention, it is possible to measure the length of the G tail sequence in a sample within 40 minutes by using only a single container (e.g., test tube).
[0122]The measuring method according to the present invention can be used clinically for patients with various diseases associated with cancer and aging, which may proceed as a result of G tail loss.
[0123]The measuring method according to the invention is useful in basic research on cancer, aging, and the biological impacts caused by telomere abnormality.EXAMPLES

Problems solved by technology

This is probably because there was no method of measuring the length of a G tail accurately and quantitatively, as the G tail is much shorter than the telomere.
However, as will be described below with reference to Table 1, all these methods demand autoradiography and gel preparation with a radioactive label (such as 32P), which are troublesome in handling.
For these reasons, all these methods are tedious assays demanding at least two days for completion, which are unfavorable for the real-time monitoring of progress of cancer and rapid diagnosis of clinical outcome.
In addition, these methods could not be applied easily to the high-throughput screening for analyzing a great number of samples.
Specifically, because the signal intensity of a G tail obtained by the method is so low at the noise level, it is difficult to determine the G tail quantitatively and accurately and also to identify whether the signal is specific to the G tail.

Method used

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  • Method for determination of the length of the g-tail sequence and kit for the method

Examples

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Effect test

example 1

Test for Confirming Dose-Response of Synthetic Single-Stranded G Tail

[0125]For the confirmation of dose-response relationship by the measuring method according to the invention, the following hybridization buffer diluents containing a 84-base single-stranded synthetic G tail, 5′-(TTAGGG)14-3′ (manufactured by Prorigo) at various concentrations and an AE-labeled G tail HPA probe (5′-CCCTAACCCTAACC*CTAACCCTAACCCTA-3′, SEQ ID No. 1, *: AE-labeling site, 29 bases) having a amount of chemiluminescence of 3×107 relative light units (hereinafter, referred to simply as rlu) were incubated and allowed to hybridize with each other in 100 μL of the following hybridization buffer at 60° C. for 20 minutes.

[0126]The AE-labeled G tail probe was prepared by labeling, with AE, an amino linker-introduced oligonucleotide (SEQ ID No. 1) prepared by using the linker-introducing reagent 3, according to the method described in Japanese Patent No. 3483829.

TABLE 2Composition of hybridization buffer0.1 mol / ...

example 2

Measurement of G tail sequence length, directly by using cell pellet (1)

[0152]The SiHa cancer cell line pellet prepared in the section below for the measurement of the length of the G tail in cell pellet was resuspended in 100 μL of the hybridization buffer above, and the suspended solution was mixed by pipetting and sheared with a 26G syringe before use. The AE-labeled G tail HPA probe in an amount of 3×106 rlu and the cell pellet were incubated and hybridized under a condition similar to that in above. Hydrolysis and chemiluminescence detection of the AE in the unhybridized probe were performed under a condition similar to that in above. The cell pellet (1 / 10 volume) was denatured similarly to above and hybridized with 3×106 rlu of the AE-labeled Alu probe, for normalization of the total genomic DNA amount.

Preparation of Cell Pellet

[0153]A cell pellet of a SiHa cancer cell line was prepared by collecting cells after the centrifugation of the SiHa cancer cell line at 1,000 G ...

example 3

Test for Comparing the Measuring Method According to the Invention with that Described in Japanese Unexamined Patent Publication No. 2001-95586

[0164]The dominant negative allelic gene (TRF2ΔBΔM) was infected to various cells (HeLa cancer cell, SiHa cancer cell, MCF-7 cancer cell, MRC-5-hTERT normal fibroblast, and 90p normal mammary epithelial cell) according to a method similar to that in ; The nondenatured DNA was isolated from each cell (HeLa cancer cell, SiHa cancer cell, MCF-7 cancer cell, MRC-5-hTERT normal fibroblast, and 90p normal mammary epithelial cell) according to a method similar to that in ; The nondenatured DNA (5 μg) was used in the G tail measuring method according to the present invention for measurement of the G tail length; and, as a Comparative Example, the measuring method described in Japanese Unexamined Patent Publication No. 2001-95586 was applied to each denatured DNA (0.5 μg) for measurement of the entire telomere length. ExoI treatment was performed sim...

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Abstract

A method of measuring the length of a G tail sequence, characterized by hybridizing the G tail of an nondenatured chromosomal DNA in a sample with a labeled DNA probe having a sequence complementary to the telomere repeat sequence, measuring chemiluminescence from the hybridized DNA probe, and determining the length of the G tail sequence from the measured value, and a kit used for use in such a method.

Description

TECHNICAL FIELD[0001]The present invention relates to a method of measuring the length of a G tail sequence and a kit for use in the method.BACKGROUND ART[0002]Human chromosomal DNA have double-stranded DNA with repeated sequences of 5′-TTAGGG-3′ at the terminus, which are called telomeres. However, the terminal of the telomere has a structure in which the 3′-terminal is an overhang, and a single-stranded DNA region of 75 to 300 bases (G tail, hereinafter referred to simply as G tail). The G tail is normally in a protected state, as a loop is formed, except during the access of a telomere-elongating enzyme telomerase or during the replication of DNA (see, for example, Nonpatent Document 1).[0003]A telomere double-stranded region occupying the most of the telomere is known to be shortened after each cell division and thus involved in cell aging, but the G tail retains a certain length of 75 to 300 bases even after repeated cell division. Contradictorily, there are reports showing tha...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q1/6841C12Q2545/114C12Q2525/151
Inventor TAHARA, HIDETOSHIIDE, TOSHINORI
Owner HIROSHIMA UNIVERSITY
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