Method of quantitative evaluation of terminal nucleotides of g-chain of telomeric human rna by means of polymerase chain reaction and duplex-specific analysis, sets of synthetic oligonucleotide primers and probes for implementation of above said method. RU patent 2508407.

FIELD: biotechnologies.

SUBSTANCE: invention proposes a method involving stages of preliminary extraction of genomic RNA, extraction of a specific fraction of single-stranded G-overhanges of telomeric RNA and further amplification of their negative chain with duplex-specific analysis; besides, amplification stages include modification of 3'-ends of telomeric overhangs by means of terminal deoxynucleotidyl transferase and are implemented using a set of primers SEQ ID NO: 1-5. The duplex-specific analysis is based on hybridisation of minus chains of overhangs with a set of specific fluorescent marked probes SEQ ID NO: 6-11, which correspond to six possible versions of nucleotide endings of G-chain of telomeric human RNA. A buildup degree of probes in presence of a ferment of duplex-specific nuclease serves as a criterion of presence of the corresponding version of the nucleotide ending and a measure of its quantitative content. Obtained profiles of terminal nucleotides of G-chain of telomeric RNA serve as proliferative markers pointing at an activation degree of cell fission and determining time duration of a cellular cycle.

EFFECT: invention is a new kind of RNA diagnostics, intended for detection of changes of nucleotide endings of RNA of telomeric areas of chromosomes, which occur in the cellular cycle process, and can be used in medicine for diagnostics and treatment of cancer, in immunology, transplantology, cosmetology, dermatology, gerontology, cellular biotechnology and other areas involved in evaluation of a proliferative state of a cell and methods of its directed control.

15 cl, 9 dwg, 8 tbl, 5 ex

The technical field

The invention relates to the field of genetics and molecular biology of the cell, in particular to molecular diagnosis of processes occurring in such areas, the telomeres of chromosomes during cell cycle. The claimed method is used to build a profile of terminal nucleotides terminal domains of G-chain of telomere DNA of a man, reflecting their quantitative percentage. Snapshots are abnormal proliferative markers indicating the activation or inhibition of cell division and controlling the duration of the cell cycle.

The invention is a new form of DNA-diagnostics of proliferative status of cells, tissues and the organism as a whole. The method can be used in diagnostics and treatment of oncological diseases determining and monitoring of the immune status of an organism of the person, in gerontology, cosmetology, dermatology, Transplantology, cell biotechnology and other areas where the required control and directional influence on cell division.

The level of technology

The research object of this invention are leaf plots G-chain of telomere DNA, called «» (from the English. overhang «makeweight»), as they cause the greatest interest in the issues related to cell division. To date there are two ways of identification of terminal nucleotides G- telomere DNA based on the use of the principle of ligation. These methods have significant methodological limitations associated with the impossibility of direct ligation to free 3'-end of the .

In 2001 for the study of this question from the simplest organism Tetrahymena thermophila proposed a method Ligation-mediated primer extension protocol, which allowed to measure the length and nucleotide the end of the G- containing replays TTGGGG. Basic information given in the article: Naduparambil K. Jacob, Rose Skopp and Carolyn M. Price. G-overhang dynamics at Tetrahymena telomeres. The EMBO Journal, Vol.20, No.15, 2001, pp. 4299-4308. Due to the impossibility of direct ligation to 3'-end of the this method is based on ligation with universal sequence of Unique oligo, which is pre- with a complementary area Guide oligo sequence. The authors of the articles were developed six options Guide oligos sequences labelled using a 32 P and contain in addition to the complementary Unique oligo region, plot corresponding to the possible version of the end of the G-.

According to the research Protocol first mixed Unique oligo and Guide oligo sequence in equimolar concentrations. Then the obtained duplexes, bearing singlestranded plot of 5 nucleotides one of six options repeat, added to the analyzed sample and ligation performed using T4 DNA ligase. Ligation was carried out at 16 degrees C during the night. After removal duplexes proceeded to completion of the 3'-end Guide oligo sequence the entire length of the matrix using T4 DNA polymerase. The criterion of the presence of one option or the other end of the was the presence of the labeled DNA that could be synthesized only if ligation and full compliance with the 5'-end Unique oligo sequence and the 3'-end . Presence of received fragments was assessed by the presence of tags in a polyacrylamide gel.

The critical moment for the achievement of efficiency ligation, and therefore the success of the considered analysis, is the formation of nick-like (nick-like) structure between 5'-end Unique oligo sequence and 3'-end , which is sewed by ligase. For education Nika need to plot Guide oligo sequence complementary repeat , , at the very end. Despite the short sizes G- Tetrahymena from 14 to 27 N.D. not exclude the possibility of an annealing to the left of the terminal site . The last factor, given the spatial configuration duplex, educated Unique oligo and Guide oligo sequences, can hinder the further annealing plot located to the right of the terminal site matrix . The dependence of the efficiency of the method on the features of the annealing and ligation is the main factor limiting this method.

The only method used to identify the terminal nucleotides human chromosomes containing replays TTAGGG, is the Single Telomere length analysis (STELA)proposed Agnello J. Sfeir et al. (2005, 2006). Basic information is given in the work: Agnello J. Sfeir, Weihang Chai, Jerry W. Shay and Woodring E. Wright. Telomere-End Processing: the Terminal Nucleotides of Human Chromosomes. Molecular Cell, Vol.18, 2005, pp. 131-138.

The authors propose methods to investigate the terminal nucleotides ends of the chromosomes of both strands of DNA. For the study of terminal nucleotides C-rich chain method was used Primer-Ligation Assay, as well as modification of the method of Single Telomere length analysis (strand C STELA), which are based on the principle of ligation to free the 5'-end C-chain. The great difficulty is the G-rich chain of telomere DNA as a direct ligation to 3'-end of the impossible. In this regard, the article suggests a corresponding modification of the STELA method, intended for research purposes G-rich chain of telomere DNA (G strand STELA). This method is the closest analogue of the claimed invention.

In this method, due to the impossibility of immediate ligation to 3'-end of the same as the previous method, a procedure is proposed annealing artificial sequence Guide platform [3'-AATCCC-5']10 on the matrix complementary G-rich chain. But in contrast to the proposed Jacob et al., annealing platform length of 60 nucleotides on the matrix of 3'- took place at the first stage to hybridization with other complementary sequences. According to the authors of this ensured the formation of artificial 5'-, which allowed to conduct further ligation synthetic 5'-phosphorylated cassettes (G telorettes) directly to the 3'-end of the .

Each cassette included a site in length of 7 N.D. corresponding to one of the six options telomeric endings, and the sequence complementary to the universal reverse G teltail . Ligation of each of the six variants of the tapes was carried out only in full compliance with its 5'-end and 3'-end of the investigated G-rich chain. For further amplification used reverse G teltail primer and direct universal subtelomeric primer-specific plot the field of chromosomes.

Amplification samples led to the formation of products of different lengths that were analyzed in the gel using southern blot and served as a measure of the presence and quantitative measurement of the defined options endings G-rich chain of telomere DNA. For this split in agarose gel DNA fragments moved nylon filter and hybridized with 32 P-labeled breakdown, formed in the course of PCR using forward and reverse primers.

A significant limitation of the method as the method proposed for the study of Tetrahymena, is the dependence of its efficiency of annealing conditions sequence complementary G-. The need for terminal annealing Guide platform sequence at the end of presupposes the formation of artificial 5'-, the formation of which depends on conditions of hybridization, the length of the platform and sizes of the matrix .

The dimensions of G- telomere DNA of a person vary from 12 to 624 N.D. Under other equal conditions, hybridization 3'- and platform for success adequate analysis of heterogeneous length population is difficult.

Efficiency analysis is determined by the condition in which at least one unit of the platform shall form an artificial 5'- the optimal length of not less than 6-7 N.D., as defined by the dimensions of telomere sequence in each cartridge. When reducing the size of the produced 5'- to 1-3 N.D. efficiency of subsequent ligation with declared temperature 35 C decreases up to absence. If we assume that, subject to pre-emptive annealing full-length sequences Guide platform with declared temperature 70 C now far repeat -5' platform falls on the end of a 3'-, then there are the following possible variants of critically short 5'- (underlined):

1. GGGTTAGGGTTAGGG-3' CCC -5'

will not be formed 5'-;

2. GGGTTAGGGTTAGG-3' -5'

will produce 5'- length of 1 N.D.;

3. GGGTTAGGGTTAG-3' -5'

will produce 5'- length of 2 N.D.;

4. GGGTTAGGGTTA-3' MOP-5'

will produce 5'- length 3 N.D.;

5. GGGTTAGGGTT-3' -5'

will produce 5'- length of 4 N.D.;

6. GGGTTAGGGT-3' -5'

will form the 5'- length of 5 N.D.

In this regard advantage when get the tapes, which parts are annealed with the formation of 5'- length of 5 N.D. and more. Under such conditions, hybridization of platform in the most adverse conditions are ending in triplets GGG, AGG and TAG signal ligation which may be understated.

Difficult point in this method is the analysis of the shortest less than 30 nucleotides due to the formation of unstable duplex at hybridization with a high temperature of 70 deg C. This restriction partially cleared when the temperature falls, the initial hybridization to room temperature, which was implemented by the authors of much later, in the work of 2009. However, in this study, the method G strand STELA was only used to determine the length of telomeres without identification of the terminal nucleotides (Yong Zhao, Agnello J.Sfeir et al. Telomere extension occurs at most chromosome ends and is designated as uncoupled from the fill-in in human cancer cells. Cell. Vol.138, No.3, 2009, pp. 463-475).

We consider the possible cases in which the annealing platform may lead to retention of the 3'-. A possibility of its formation is increased at the expense of primary annealing full-length sequences of the platform during a declared high temperature hybridization in 70 degrees C. While this will always be present options such hybridization, in which the site far repeat -5' platform falls on complementary him the latest iteration of TTAGGG (underlined). There are the following possible combinations of hybridization depending on the finishing :

1. GGGTTAGGGTTAGG-3' MOP-5'

will produce a residual 3'- long 5 N.D.;

2. GGGJTAGGGTTAG-3' MOP-5'

will produce a residual 3'- long 4 N.O;

3. GGGTTAGGGTTA-3' MOP-5'

will produce a residual 3'- long 3 N.O.;

4. GGGTTAGGGTT-3' MOP-5'

will produce a residual 3'- long 2 N.O.;

5. GGGTTAGGGT-3' MOP-5'

will produce a residual 3'- long 1 N.O.;

6. GGGTTAGGG-3' MOP-5'

will not be formed residual 3'-.

G-, especially ending in triplets GGG, the result of a blunt 3'-end, General risk to remain not , and therefore can completely fall out of the analysis. Perhaps, this is associated with low frequency of occurrence of this option, the end gained by the authors as a result of artifacts ligation. In the same harsh conditions are ending in GGT, GTT and TTA triplets, so how can form too small residual 3'-phase, insufficient for annealing another unit platform and subsequent ligation.

A significant part of the population of telomeric G- presents fragments of length greater than one hundred and even several hundred nucleotides. At length over 60 N.D. likely annealing of several units of the platform along the entire length of . It is always not excluded the possibility of hybridization:

a) with the formation of the blunt end, especially if the study ends with triplet GGG-3', that is determined by the characteristics of the end of the platform on the triplet 5'-SSS;

b) with the formation of residual 3'- with minimal length and with a melting point of much less than the selected authors for hybridization and ligation;

a) with the formation of very small 5'-, length of 1-3 N.D. insufficient for subsequent annealing G telorettes and ligation.

These factors can lead to the formation of a very short, is insufficient for the further analysis, 5'- or its complete absence. Thus, further study for part becomes impossible and signal ligation is significantly underestimated.

Thus, the main drawback of the analyzed method lies in the fact that different in length matrix with different efficiency. Under other equal conditions, hybridization of some matrix benefit, and ligation of the others can be difficult.

In addition, the success of the analyzed method depends on the degree of degradation of telomere DNA. The ultimate amplification may be feasible only upon the condition that the area between the direct and reverse primer G teltail primer contains of two-chain breaks. These areas, reaching lengths of several thousands of nucleotides can be broken down in the process of extraction of DNA. It is also possible to destruction of integrity the field of chromosomes during processing of genomic DNA by EcoRI at the initial stage of analysis.

Evidence that ligation and amplification during the G strand STELA method each time takes place in different ways, are obtained by the authors screenshots of which had different sets of bands in the repetition of the analysis from 2 to 8 times on each version of the end of the G-.

The above disadvantages of the method described associated with the occurrence of artifacts during ligation and amplification may substantially affect the final result of the analysis and lead to erroneous determination of the frequencies of occurrence of options nucleotide endings G-. Eventually this can lead to the construction of a distorted profile terminal nucleotides G-chain of telomere DNA, which plays a key role in the assessment of proliferative status of the cells.

Disclosure of the invention

The main object of the invention is development of a new effective method for the study of terminal nucleotides G-rich chain of telomere DNA to eliminate the shortcomings of the existing methods.

The technical result of the invention is to increase the specificity and sensitivity of the method, which are expressed in the possibility of a successful study of the entire faction of the G- of different length and produce an accurate profile of terminal nucleotides telomere DNA.

The problem is solved using a new approach to the study of terminal nucleotides of DNA, consisting in combination of amplification of telomere DNA with subsequent duplex-specific analysis.

Summary of the invention consists in the creation of synthetic fluorescently-labeled probes, which correspond to the six possible options telomeric endings. The presence or absence of certain variants of terminal nucleotides reveal in the course of hybridization probes with the study of telomere DNA and splitting them with the help of an enzyme duplex-specific nucleases. Probe cleavage occurs only under condition of full complement of compliance with the sequence. This ensures a high specificity analysis and leads to specific fluorescent signal in test-tubes, which is registered . The degree of outbreaks of probes, corresponding to the possible options for the endings of telomere DNA, not only serves as a criterion of their presence, but also a measure of their quantitative ratio.

The claimed process includes the possible ways of registration and interpretation of results of the analysis. Result of evaluation of the level of the signals are invited to submit a bar chart that reflects the percentage of certain variants of nucleotide endings G- and referred to as «the profile to the terminal nucleotides G-chain of telomere DNA.

The main characteristic of the declared method is determined by the specifics of the implementation of the duplex-specific analysis in relation to the object of research of telomere DNA. Duplex-specific analysis or DSNP-analysis (from the English. Duplex-specific nuclease preference-based enzyme duplex-specific nucleases (LTOs, DSN) of Kamchatka crab, which has a specific feature to split DNA and remain inactive in relation to single-stranded DNA. DSNP-method was developed by Russian scientists for the analysis of single nucleotide polymorphisms of genes and used successfully for the study of point mutations of genes (Shagin D.A. et al. A Novel Method for SNP Detection Using a New Duplex-Specific Nuclease From Crab Hepatopancreas. Genome Research, 2002, Vol.12, pp. 1935-1942).

It was shown that efficiency of the LTO-hydrolysis committed DNA duplexes significantly higher than imperfect, containing at least one base. This feature of the duplex-specific nucleases formed the basis of the developed method of the study of polymorphism of terminal nucleotides G-chain of telomere DNA of the human DSNP-analysis that formed the basis of the declared method.

The General principle DSNP analysis is about using a short length of 10-12 nucleotides, synthetic probes (of signal samples), bearing the donor fluorescence at the 5'-end and a fluorescence quencher on the opposite side of the 3'-end. When fully complementary interaction between the probe and studied DNA target activity LTOs leads to program fluorescent signal. If this shows a high sensitivity of the method in connection with the ability of the duplex-specific nucleases distinguish between variations of a single nucleotide (Shagin D.A. et al., 2002). In the study of single nucleotide polymorphisms in case of point mutations of genes analyzed DNA using the polymerase chain reaction, and then aliquot of PCR product is mixed with the signal samples and incubated in the presence of the enzyme LTOs.

The essential difference of the proposed method from the classic duplex-specific analysis of nucleotide polymorphisms is that he used to study not DNA sequence of the genes and end portions of tandem-of repetitive DNA telomeres. The last property of the object of the study identified a fundamental modification of the stages of amplification investigated telomere DNA by polymerase chain reaction using a special set of primers, presented in table 1 (SEQ ID NO: 1-5).

Extreme 3'-end location of the G- prevents the use of natural reverse primer for its amplification. The last fact caused the necessity of the use of artificial increasing the 3'-end using the terminal and mononucleotides that affected the structure of signal samples. A set of six SRES samples are presented in table 2 (SEQ ID NO: 6-11), in which the position of discrimination is located at a distance of 4-5 nucleotides from the 3'-end in the sphere of the natural end of the G- and start poly(dC) tail, completed transferase.

List used as primers for PCR

Title primer

Sequence in the direction 5'-3'

Oligo dT 25 Adapter Primer

CTCACTGACACACTATGGTTTTTTTTTTTTTTTTTTTTTTTTT

Oligo dA 25 Adapter Primer

CTCACTGACACACTATGGAAAAAAAAAAAAAAAAAAAAAAAAA

Adapter Primer

CTCACTGACACACTATGG

Oligo dT 30 Primer

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

Oligo dA 30 Primer

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

List used as a fluorescently-labeled probes (of signal samples) if the duplex-specific analysis of

Name probe

Sequence in the direction 5'-3'

Probe TAG

F∗-GGTTAGCCCC-Q

Probe AGG

F∗-GTTAGGCCCC-Q

Probe GGG

F∗-TTAGGGCCCC-Q

Probe GGT

F∗-TAGGGTCCCC-Q

Probe GTT

F∗-AGGGTTCCCC-Q

Probe TTA

F∗-GGGTTACCCC-Q

Note. F∗ means a fluorescent label at the 5'end, Q denotes the molecule of a damper fluorescence (Quencher) at the 3'end.

The choice of dCTP as a building material was due to the fact that this is the only nucleotide, in the natural repeat TTAGGG rights, and providing unambiguous discrimination of all six possible options nucleotide endings G-chain of telomere DNA. In the examples of realization of the invention proposed to use a combination of dye (FAM) at the 5'end and its effective absorber of RTQ1 the 3'end, that allow a quantitative analysis of all possible variants of the end of the G- growth rate of green fluorescence in time. When designing probes possible to use any dyes and dampers in the corresponding fields of fluorescence, including multiband, which is reflected in the formula of the invention in item 6 and 15.

The effectiveness of using one or another variant of a labelling will be defined by the spectral characteristics fluorophore-related fluorescence of DNA at the current time of the study. During the development of the proposed method it was established that the nucleotide sequence of DNA have a specific fluorescence in visible wavelength range of light associated with the establishment of the General quantum-associated spectrum with the molecule of the fluorophore (dye). According to the data received by DNA is not only known fluorescence in the UV range, but has integrated range of fluorophore-related fluorescence in visible wavelength range of light. The specificity of this fluorescence in each of the colors of the visible spectrum depends on the nucleotide sequence of the DNA. In this regard, the nucleotide sequence of the probe labeled with a fluorescent dye, determines the specifics of its fluorescence in the corresponding spectrum of the visible range, and speed of its flare UPS when conducting DSNP analysis.

Studies have shown that the spectral code fluorescence of DNA dynamic in time and the relationship between the levels of fluorescence of the primary color channels subject to fluctuation (fluctuations). These changes are to be monitored, as they directly affect the levels of background fluorescence probes and the specifics of their flare UPS when conducting DSNP analysis. Adequacy of use concentrations probes and probe-specific coefficients when performing the analysis must be verified control analysis of equimolar mixture of synthetic analogues endings , as in example 5.

Schematic diagram of identification of terminal nucleotides G-rich chain of telomere DNA by PCR and DSNP analysis.

At the first stage of the study of genomic DNA sample obtained in any manner that preserves its , separate faction of single-chain telomere DNA. For this purpose, using the enzyme duplex-specific nuclease, the effectiveness of which has been shown in the work of research length of telomeric (Yong Zhao et al. Quantitative telomeric overhang determination using a double-strand specific nuclease. Nucleic Acids Research, 2008, Vol.36, No. 3). Research the authors have shown that the use of LTOs in a concentration of 0.2 per every 5 mcg genomic DNA allows effectively split DNA fragments length of not more than 10 base pairs and preserve native faction of single-chain .

For concentration and purification from the remnants of genomic DNA in the claimed method proposed modification of the method with the use of covered paramagnetic particles, described in the article Woodring E. Wright et al. (Normal human chromosomes have long G-rich telomeric overhangs at one end. Genes&Development. 1997, Vol.11, No. 21, pp. 2801-2809). According to the authors of 40 mcg genomic DNA contains about 1 (fmole) telomeric ends. In accordance with this for precipitation even the longest is not more than 1 (pmole) biotinylated probe () 4 and 3 El covered magnetic particles in a concentration of 10 mg/ml

In the examples of the implementation of the proposed use of the invention of the magnetic particles to streptavidin at a concentration of 1 mg/ml production firm «» (Russia) with the efficiency binding on average 8 pmol thrice biotinylated product on 10ul particles. In connection with the fact that precipitate faction different length is slower than linking short oligonucleotides with complexes of particle probe is proposed to use 10ul particles on every 20 mcg original genomic DNA. This amount is sufficient, even with some loss of magnetic particles at .

For pre-splitting genomic DNA, instead of restrictase Hinfl use the duplex-specific and binding native with probes precedes the incubation last magnetic particles covered with . The use of cleaning in buffer ensures effective removal is not associated with magnetic particles probes at the first stage, and the remnants of genomic DNA double-strand in the next step. This sequence of steps provides reduced time precipitation up to 1.5 hours, compared with the initial methodology Wright et al., where the final stage of incubation suspension DNA and magnetic particles was carried out during the night. A temperature increase of final elution to 70-75°allows more effective to concentrate faction studied in volume, suitable for a further stage of completing the poly(dC) tail. The use of triple Biotin for modifying the probe provides a more solid connection to streptavidin on magnetic particles when performing high-temperature elution. To receive purified fraction of single-chain any other type of group hybridization or sorption, including the use of various membranes, columns, particles or other surfaces to immobilization and the subsequent removal from the surface of the investigated molecules that appear in claim 2.

On the second stage of the research, water eluent, containing a single-stranded G- use as a substrate for the enzyme terminal , which completes the 3'-end of the chain. At the stage of completion sequence () as a construction material used dCTP . The area of event DSNP-analysis is the area of contact of the natural end of the 3'- and the beginning of poly(dC) sequence. Duration of synthesis is chosen empirically and done within a time sufficient for completing the sequence length 100-200 nucleotides that it is necessary to ensure the effectiveness of subsequent deposition short at their cleaning.

At the third stage, after the standard procedure and cleaning is not included into the chain dCTP mononucleotides sample used for a similar completing poly(dA)/poly(dT) tail using each dATP/dTTP mononucleotides and enzyme terminal . The combination of the successive stages of completing the and () sequences allows you to separate the studied border contact the end of and poly(dC) tail from customers at the subsequent synthesis of copies of sub-zero circuit. Save this border is the main condition for obtaining adequate results of the analysis.

At the fourth stage is realized by synthesis negative chain using DNA polymerase and oligo dT 25 Adapter primer (SEQ ID NO: 1) in the case of limit at the previous stage. In the other variant of the method of synthesis of negative chain are using oligo dA 25 Adapter primer (SEQ ID nos: 2)if the third stage was the accession of sequence (end ). Both options primers contain at its 5'-end adapter sequence SEQ ID NO: 3). As the adapter can be used in any sequence, not complementary repetition and the corresponding optimal temperature annealing in conducting the synthesis of negative chain, which is reflected in the formula of the invention in item 4. The number of cycles synthesis negative chain lead up to 100-200 cycles, essential to produce a sufficient level of fluorescence signal during the final DSNP-analysis. The latter condition is especially important when small initial number of analyzed DNA.

At the fifth stage after cleaning and deposition fraction negative chain carry out the completion of its 3'-end using terminal transferase and each dATP mononucleotides if the third stage was used end- chain , or dTTP mononucleotides, if applied its .

At the sixth stage cleansed and faction minus circuits constructed with poly(dA) tail subjected to final asymmetric amplification using the adapter sequence SEQ ID NO: 3), or its equivalent, as a direct primer and oligo dT 30 primer (SEQ ID NO: 4) as the reverse. In the second variant of the method of freezing chain with poly(dT) tail using the same adapter sequence as a direct primer, but in combination with oligo dA 30 a primer (SEQ ID NO: 5) as a reverse primer. The number of cycles is determined experimentally, no more than 18-20, so as to ensure the preservation of the initial ratio of variants of the end of the G-.

The use of asymmetric PCR with a ratio of direct and reverse primers 10:1 ensures priority synthesis negative chain and its subsequent hybridization with signal samples. The dominance adapter primer lead to the displacement of oligo dT 30 (oligo dA 30 ) primer, which can partially as direct on poly(dA/dT) sequence, which is located directly behind the adapter.

At the seventh stage of conduct DSNP-analysis. Purified PCR product is divided into six equal parts and mix with signal samples in the presence of the enzyme duplex-specific nucleases.

Design of signal samples is presented in table 2 (SEQ ID NO: 6-11) and selected taking into account the specifics of the study area, namely the border between the end of the 3'- and the beginning of poly(dC) sequence. Signal sample is a synthetic oligonucleotide probe length 10 pairs of nucleotides, presented the positive chain G- and corresponding to one of the six options expiration.

Structure probe intended for duplex-specific analysis assumes the presence of a molecule donor fluorescence 5'/3'-end of the corresponding absorber of fluorescence on the opposite 3'/5'end. In the examples of realization of the invention used a combination of dye (FAM) and suppressor RTQ1. Possible to use other dyes in combination with damper and corresponding channel fluorescence, including multiband, which is reflected in the formula of the invention in item 6 and 15.

After making the signal samples to the appropriate tube containing the sample, before the addition of the enzyme LTOs, carry out the measurement of the background signal, which is then deducted when rationing values increase fluorescence at splitting the signal samples.

Splitting the signal samples is performed in the presence of the duplex-specific nucleases only when it is fully complementary accordance site amplified the negative chain G-. Thus there is a decomposition of the sample primarily between the fourth and fifth or the fifth and sixth nucleotides (Anisimova V.E. et al. Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab. BMC Biochemistry, 2008, Vol.9, no. 14). Spatial separation of dye and suppressor leads to fluorescence corresponding to a particular channel detection. For the dye FAM is the wave length of 520 nm in the green range of visible light.

A flare-up of a signal in a test tube with a known signal breakdown indicates the presence of a corresponding end of the G-. This may not only qualitative determination of presence of an end, but semi-quantitative measurement of their content. It is very important to track changes in the correlation of different variants of the end of the G- during the cell cycle.

Measurement of the fluorescence signal is carried out in test tubes by any known method with the help of specially designed for this purpose devices (, photometers-, fluorescent scanners and detectors, fluorescent stereomicroscopes etc), as indicated in paragraph 7 of the claims.

Interpretation of results splitting probes includes the regulation signals, which consists in subtracting the original background set to analysis, and dissemination of the obtained numerical values on the probe-specific ratio, empirically calculated for each fluorescent probe. The coefficients calculated when building the growth curves of fluorescence in time for a signal samples during a follow-up study of equimolar mixture . For this purpose use synthetic analogues G-, having passed all stages of analysis, ranging from the synthesis of poly(dC) of the tail, and ending with the final amplifying the negative chain, as in example 5.

Factors specific to each signal samples separately and for each combination «dye-quencher». They reflect the rate of splitting of the probe, which is constant for a given period of time analysis and match the current spectral code fluorophore-related fluorescence of DNA in the visible range. As previously noted, the rates reflect the specifics of the energy potential of telomeric repeats included in probes. Basically it is determined by the spectral code fluorescence guanine at the time of research. In the following examples, the implementation of the invention shows the coefficients of regulation for six versions of signal samples labelled by a combination of dye FAM and suppressor RTQ1, after one and two hours of analysis (table 3).

Changes of spectral code fluorescence of DNA, other concentration ratio probes as other variants of marking, require a new calculation of the coefficients of regulation of signal values for each probe. So, periodic study of the control sample equimolar mixture of synthetic analogues endings purpose of verification of the coefficients of rationing signals is required. The special significance it has in the conditions of unstable spectral properties of DNA.

The resulting value of the signal for each probe is calculated using the coefficients of regulation for one and two hours in parallel, and then averaged. The result is given in the form of a bar chart that shows the average percentage of received signals of each probe of the total level of signal received from all probes. Values are used to build the chart percentages of cases nucleotide endings G- telomere DNA, called a profile of terminal nucleotides G-chain of telomere DNA.

Table 3.

Coefficients of rationing signals fluorescence probes FAM-RTQ1 during DSNP-analysis

Option probe and terminal triplets (stressed)

A final concentration in the reaction

1 hour analysis

2 hours analysis

1. GGTTAGCCCC

1.1 microns

2. GTTAGGCCCC

0.8 microns

3. TTAGGGCCCC

0,4 micron

4. TAGGGTCCCC

0.8 microns

5. AGGGTTCCCC

1.5 microns

6. GGGTTACCCC

1.5 microns

Thus, the first aspect of the invention is a set of specific primers, presented in table 1 and are used to stage amplification negative chain of telomere DNA prior to DSNP-analysis.

The second aspect of the invention is a set of specific oligonucleotide probes listed in table 2 are used as signal samples when conducting duplex-specific analysis of terminal nucleotides telomere DNA.

The third aspect of the invention is directly method of identification of terminal nucleotides G-rich chain of telomere DNA, including amplification of the negative chain G- telomere DNA and its subsequent duplex-specific analysis. The claimed method differs from the previously known fact that: a) the final amplification subjected () at the 3'-end of the copies of the negative chain containing adapter sequence used as a direct primer taken in excess with respect to the reverse poly(dT)/poly(dA) ; b) the results of analyzed by hybridization with fluorescently-labeled developing probes in the presence of the enzyme duplex-specific nucleases; C) the results are registered according to the extent of splitting probes and level outbreaks of fluorescence using any of the hardware-software complex, allowing to display the fluorescence of tubes with probes on the computer screen and quantify the level of the signal in each of them.

Comparative analysis of the prototype allows to make a conclusion that the claimed method of the study of terminal nucleotides G-chain of telomere DNA is fundamentally different from the above-described method Single Telomere length analysis (G strand STELA)proposed Agnello J. Sfeir et al., and is based on a completely different approach with the use of enzyme duplex-specific nucleases without using the procedure ligation. The claimed method includes the ability to effectively analyze the entire faction of the G- of any length and different endings. This fact is the main technical result of the claimed invention, which is expressed in increasing the specificity and sensitivity analysis compared with the prototype. In this regard, the claimed method allows to determine the true ratio options endings telomeric . Based on that adequate profile terminal nucleotides G-chain of telomere DNA is a kind of marker processes associated with cell division. Thus, the claimed method may become a new tool for fundamental research in the field of cell biology and solution of applied tasks in the field of medicine and biotechnology.

Brief description of drawings

The claimed invention is illustrated by the following figures.

Figure 1. The result DSNP-analysis of equimolar mixture of synthetic analogues endings telomeric in the form of a graphic file (example 5). Is a series of consecutive shots to capture tubes in the green spectrum obtained using the fluorescence detector FDG-001. Picture tubes in the form of round branches correspond to the zones of measurement of optical density in the program LabWorks (UVP, England) and are located line by line for each variant of the probe. The first line corresponds to the TAG option the end of the G-chain of telomere DNA, and the last TTA option. In each row of the first zone corresponds to the starting value of the background, second and third zones - signal levels after one and two hours of carrying out DSNP analysis respectively.

Figure 2. Result of the final data processing DSNP-analysis sample 1 in the form of building a profile of terminal nucleotides G-chain of telomere DNA. In the further description of the drawings under the profile understand percentage of different variants of nucleotide endings (triplets) of the study population G- telomere DNA. Sample 1 shows the genomic DNA extracted from the culture of human peripheral blood lymphocytes, not stimulated to division with PHA (example 1).

Figure 3. Result of the final data processing DSNP-analysis sample 2 in the form of building a profile of terminal nucleotides G-chain of telomere DNA. Sample 2, submitted genomic DNA extracted from the culture of human peripheral blood lymphocytes, stimulated to divide the processing of PHA within 2 hours (example 2).

Figure 4. Result of the final data processing DSNP-analysis sample 3 in the form of building a profile of terminal nucleotides G-chain of telomere DNA. Sample 3 shows a genomic DNA extracted from the culture of human peripheral blood lymphocytes, stimulated to divide the processing of APF for 4 hours (example 3).

Figure 5. Result of the final data processing DSNP-analysis sample 4 in the form of building a profile of terminal nucleotides G-chain of telomere DNA. Sample 4, presented genomic DNA extracted from the culture of human peripheral blood lymphocytes, stimulated to divide the processing of PHA within 11 hours (example 4).

Figure 6. Result of the final data processing DSNP analysis of a sample of 5 in the form of building a profile of terminal nucleotides G-chain of telomere DNA. Sample 5, is presented by a mixture of synthetic analogues of endings telomeric in equimolar ratio (example 5).

Figure 7. The combined chart of dynamics of changes in the profile of terminal nucleotides G-chain of telomere DNA blood lymphocytes. Chart based on the time scale of the impact on lymphocyte culture stimulating division factor (APF) during the first eleven o'clock.

Figure 8. Profile of terminal nucleotides G-chain of telomere DNA leukemia culture Jurkat, synchronized in the mitotic phase.

Figure 9. Profile of terminal nucleotides G-chain of telomere DNA leukemia culture K-562, synchronized in the mitotic phase.

Realization of the invention

The method is illustrated by the following examples.

Example 1. Analysis of terminal nucleotides G-chain telomere human DNA extracted from the culture of peripheral blood lymphocytes, not stimulated to division with PHA (PHA).

To highlight the faction of single-strand 3'- G-chain of telomere DNA sample used genomic DNA, selected by a set of Diatom™ DNA Prep100 («Biocom», Russia). Extraction was carried out in accordance with manufacturer's recommendations to some modifications. Incubation of the sample in buffer at the first stage the extraction and the final stage of DNA elution was performed at a temperature of 55 C, instead of 65, in order to avoid denaturation of DNA. Next 20 mcg genomic DNA introduced into the reaction mixture consisting of the following components:

4 MKL of the buffer for the duplex-specific nucleases (Eurogen, Russia);

0.8 u duplex-specific nucleases (Eurogen, Russia).

The volume of the reaction mixture is brought up to 40 cells and delicately mixed on vortexing.

The reaction of splitting of the double-stranded DNA was carried out at 37 C for up to 2.5 hours. Then enzyme LTOs were removed from the reaction mixture 3 times the processing solution sorbent BlueSorb («», St. Petersburg). For this to sample each time added on 6 MKL of sorbent, mix carefully shook the vortexing and besieged the sorbent brief centrifugation followed by separation of the liquid phase, containing DNA.

After the removal of nucleases sample was incubated with magnetic particles carrying on its surface oligonucleotide probes, complementary repetition G-chain of telomere DNA. For these purposes, used particles coated with at a concentration of 1 mg/ml production firm «» (Russia) and synthetic oligonucleotide probes, consisting of a sequence of containing at its 5'-end of three molecules of Biotin («Synthol», Russia). Experiments have shown that an average of 100 MKL of magnetic particles were 80 3xBiotin probe. Accordingly 10ul magnetic particles tied around 8 probe. This amount is more than enough for precipitation of single-stranded . The redundancy of the number of particles and the probe in relation to the number of telomeric is necessary, first, to ensure the effectiveness of linking magnetic particles and extended telomeric sequences, and, secondly, for the convenience of work with the volume of magnetic particles in order to avoid their loss .

All the manipulation of magnetic particles produced according to the manufacturer's recommendations, using original solutions and magnetic tripod. magnetic particles in the amount of 10 MKL were washed three times 0,5x solution for 200 cells, suspended 100 MKL of the same solution and incubated with 10 3xBiotin probe. Conjugation of streptavidin and Biotin were carried out at room temperature for 30 minutes. The sample gently suspended with the help of a pipette dosing every 5-7 minutes. Then magnetic particles once were washed 3 times 0,5x solution from probe, suspended in 50 ul 1x solution and added the analyzed DNA sample after the treatment and sorbent.

Linking probes with native faction of single-chain G- carried out at room temperature for 40 minutes, gently stirring the contents of a test tube with the help of a pipette dosing every 5-7 minutes. After that, the magnetic particles with immobilized G- washed from the remnants of the double-stranded genomic DNA using 0,1x solution for 200 cells four times. After careful removal of residues buffer magnetic particles suspended in distilled purified water in a volume of 20 ml and incubated in a thermostat at 70-75 degree for 5 minutes. Temperature were performed in two stages, every time gathering magnetic particles with magnetic tripod and selecting the solution of eluent pipette dosing in new tube. The second time carried out in the amount of 15 ul. Joint eluent used for the reaction of synthesis of poly(dC) tail (end ) in the amount of 40 ml reaction mixture, containing:

water eluent ;

8 ml 5x buffer for («», Russia);

2 MKL of 5 mm mortar dCTP («», Russia);

2.5 u terminal («», Russia).

The volume of the reaction mixture if necessary, brought up to 40 cells using treated distilled water.

The reaction was carried out at a temperature of 37 C for 40 minutes, carrying a gentle stirring vortexing every 10 minutes.

Without special enzyme inactivation carried out DNA sample, which in the reaction mixture was successively added:

1 ml solution of glycogen concentration of 20 mg/ml («», Russia);

25 ml of 7M solution of ammonium acetate;

165 MKL of 96% of chilled ethyl alcohol.

The mixture is thoroughly mixed on vortexing and put in a freezer at a temperature of -20 degrees for at least 12 hours.

Deposition was carried out by centrifugation at 15 thousands of rpm for 60 minutes. After careful washing sludge chilled 70% alcohol in the amount of 300 MKL of carried out re-centrifugation at the same speeds within 30 minutes. Dried open vitro residue was dissolved in 25 ml of purified water.

Later in the test tube, similar to the above method, carried out the reaction , with the only difference that instead of the solution of dCTP added 2 MKL of 5 mm solution of each dATP. The reaction was carried out at a temperature of 37 C for 30 minutes, and carried DNA sample as shown above. After the final centrifugation residue was dissolved in 25 ml of purified water.

Fusion minus chains conducted in the volume of 40 cells on a programmable thermocycler «» («DNA-Technology», Russia). Sample after thorough mixing of the vortexing placed in 0.5 ml PCR tube containing the following components:

4 MKL of 10x buffer Taq DNA-polymerase, containing 25 mM MgCl 2 («», Russia);

3 El 5 mm solution of dNTP («», Russia);

4 MKL of 20 mm mortar primers SEQ ID NO: 1;

2.5 u Taq DNA polymerase («», Russia).

The volume of the reaction mixture is brought up to 40 cells and covered with mineral oil to avoid evaporation of the reaction. Application amplifier with cover avoids the need for oil.

The synthesis was performed in 4 rounds, 50 cycles, each with the following temperature regime: 94 Degrees - 40, 43 Degrees - 30 s, 72 Degrees - 40 C. In the first round of initial denaturation at 95 was 30 seconds, and in subsequent rounds to be increased up to two minutes. Final completion of the chain while 72°increased to 3 minutes in the first three rounds and up to 5 minutes in the last round. Before each subsequent round was added to the new portion of the enzyme Taq DNA polymerase up 2.5 units of the Total number of cycles synthesis negative chain amounted to 200.

After the last round of the sample is carefully selected from under the oil and poured into the volume of 1,5 ml where added with an equal volume of chloroform. After intensive shaking on vortexing conducted centrifugation at 10 thousands of rpm for 3 minutes. To the selected added:

1 ml solution of tRNA with a concentration of 10 mg/ml («», Russia);

25 ml of 7M solution of ammonium acetate;

165 MKL of 96% of chilled ethyl alcohol.

The mixture is thoroughly mixed on vortexing and put in a freezer at a temperature of -20 degrees for at least 12 hours. Use as solution tRNA has allowed to avoid deposition not to become involved in the chain of the «dummy» primers. Use for these purposes of glycogen or linear polyacrylamide not acceptable, as they beset short oligonucleotides.

Deposition was carried out by centrifugation at 15 thousands of rpm for 60 minutes. After a delicate washing sludge chilled 70% alcohol in the amount of 300 MKL of carried out re-centrifugation at the same speeds within 30 minutes. Dried open vitro residue was dissolved in 25 ml of purified water.

Reaction 3'-end of the obtained fractions negative chain was performed in the same where consistently made:

8 ml 5x buffer for («», Russia);

4 MKL of 5 mm solution of each dATP («», Russia);

15 units of terminal («», Russia).

The volume of the reaction mixture is brought up to 40 cells using treated distilled water. The reaction was carried out at a temperature of 37 C, implementing a gentle stirring the mixture on vortexing every 10 minutes. The synthesis was carried out for 30 minutes without temperature enzyme inactivation.

Immediately after the reaction to conduct DNA consistently added:

1 ml solution of glycogen concentration of 20 mg/ml («», Russia);

25 ml of 7M solution of ammonium acetate;

165 MKL of 96% of chilled ethyl alcohol.

The mixture is thoroughly mixed on vortexing and put in a freezer at a temperature of -20 degrees for at least 12 hours.

Deposition was carried out by centrifugation at 15 thousands of rpm for 60 minutes. After a delicate washing sludge 70% alcohol in the amount of 300 MKL of carried out re-centrifugation at the same speeds within 30 minutes. Dried open vitro residue was dissolved in 50 ul of purified water.

The reaction of the final asymmetric amplification negative chain was performed in two a volume of 0.5 ml in a finite volume reaction 40 cells on a programmable thermocycler «» («DNA-Technology», Russia). Sample after thorough mixing of the vortexing divided into two parts and transferred in two PCR test tubes, each of which contained a mixture of the following components:

4 MKL of 10x buffer Taq DNA-polymerase, containing 25 mM MgCl 2 («», Russia);

4 MKL of 5 mm solution of dNTP («», Russia);

4 ml solution containing a mixture adapter primer (SEQ ID NO: 3) and Oligo dT 30 primer (SEQ ID NO: 4) at a concentration of 20 mm and 2 mm, respectively;

5 ed Taq DNA polymerase («», Russia).

The volume of the reaction mixture into each tube brought up to 40 cells and after a delicate blending mixture covered mineral oil in order to avoid evaporation during the PCR. Application amplifier with cover avoids the need for oil.

After polymerase chain reaction samples of carefully selected from under oil and transferred into the General volume of 1,5 ml where added with an equal volume of chloroform. After intensive shaking on vortexing conducted centrifugation at 10 thousands of rpm for 3 minutes. To the selected added:

1,5 ml solution of glycogen concentration of 20 mg/ml («», Russia);

50ul of 7M solution of ammonium acetate;

329 MKL of 96% of chilled ethyl alcohol.

The mixture is thoroughly mixed on vortexing and put in a freezer at a temperature of -20 degrees for 1.5 hours. Incubation for more than this time may lead to undesired excessive deposition of «dummy» primers.

Deposition was carried out by centrifugation at 15 thousands of rpm for 40 minutes. After washing sludge 70% alcohol volume of 400 cells was carried out re-centrifugation at the same speeds within 20 minutes. Dried open vitro residue was dissolved in 90 MKL of purified water.

To conduct the duplex-specific (DSNP) analysis prepared 6 thin PCR test tubes firm «Axygen with a volume of 0.5 ml, in each of which added:

2 MKL of 10x buffer for duplex-specific nucleases (Eurogen, Russia);

3 ml solution of one of the 6 probes presented in sequences SEQ ID NO: 6-11 (table 2) in next version. Oligonucleotides were synthesized by the company «Synthol» (Russia) and contain a molecule (FAM) at the 5'end and suppressor of fluorescence RTQ1 on the opposite side of the 3'-end.

The original stock concentration of solutions for each probe selected empirically. They comply with the following concentrations: 10 microns, SEQ ID NO: 10 and SEQ ID NO: 11; 7,5 m for the SEQ ID NO: 6; 5 microns, SEQ ID NO: 7 and SEQ ID NO: 9; 2,5 microns, SEQ ID NO: 8. It should be noted that the concentration of probes, as well as the conditions and the processing of results DSNP analysis satisfy the conditions which existed at the moment of study of the spectral code fluorophore-related fluorescence of DNA (December 2010 - October 2011).

Significant changes of spectral code require the recalculation of concentration values of the stock of solutions and coefficients of the regulation signals for each probe in table 3. The use of other dyes in a non-green fluorescence spectra also requires a separate establishment of the concentration of the solutions of the probes and the coefficients regulation signals in accordance with the current spectral code fluorescence of DNA. Their determination is made, in the course of the study, the control sample, which is a mixture of different endings synthetic analogues of telomeric (example 5). In connection with the possibilities of spectral instability fluorescent properties of DNA periodic study of control sample is very expedient and necessary.

To conduct DSNP analysis in each tube contains the LTOs buffer and probe were 15 ul thoroughly mixed aliquot of the sample. Immediately after mixing of the sample and the probe was measured background signal (background) each tube using a hardware-software complex consisting of the instrument fluorescence detector FDG-001 (joint development of the author of the patent and the Institute of theoretical and experimental Biophysics, Pushchino, Russia) and data processing program LabWorks 4.6 (UVP Biolmaging Systems, England).

The device FDG-001 is intended for detection of fluorescence signals in different spectra of visible light. Green channel fluorescence detection of the device correspond to spectral characteristics (FAM) and had a range of exciting radiation, equal 450-490 nm, and the detected radiation - 510-560 nm. For fluorescence excitation of optical label tube probes evenly illuminated LEDs blue light of the appropriate wavelength. To get the image fluorescent monoclonal tubes of light passing through the green interference filter, hit the lens monochrome camera WAT-120N (Watec Co Ltd, Japan). Capture card Studio TM iMovieBox (Pinnacle Systems GmbH, Germany) transformed the analogue TV signal into digital form and allowed to display the image tubes on the computer screen and save it as an image file.

After saving the image background signal for each probe each tube was added to 0,125 ed enzyme duplex-specific nucleases (LTOs). The splitting of the probes was carried out for two hours at 37 C, gently stirring the vortexing every 30 minutes. After one and two hours of analysis carried out a video capture of each tube in the form of a circumference of a fixed area similar to the picture 1, obtained for example 5.

Series of consecutive shots to capture tubes in the green spectrum analyzed in the program LabWorks, which allows the level measurement of optical density of the specified area of the image. Obtained with the help of the program magnitude significant optical density (Mean Density) in conventional units served as a measure of the intensity of fluorescent probe signal at the appropriate vitro. For level measurement of fluorescence signals possible to use any devices and hardware-software complexes, as indicated in paragraph 7 of the claims.

1st stage: the subtraction of the background signal and the receipt of the signal level for each probe after 1-St and 2 hours of the analysis;

2nd stage: standardization of the signal level by multiplying by the appropriate each probe factor from table 3 (implemented in parallel for the 1-St and 2 hours of analysis);

3rd stage: determination of the levels of the signals from each probe in percent after the 1st and 2 hours of the analysis.

Steps 1-3 for processing of the obtained data presented in table 4.

4th stage: the averaging of the percentage of the levels of the signals received after one and two hours of analysis. The result of the final processing of the data was creating a column chart that shows the average percentage of cases nucleotide endings G-, and referred to as «the profile to the terminal nucleotides G-chain of telomere DNA» (figure 2).

The variant of the terminal triplet

1 hour DSNP-analysis

2 hours DSNP-analysis

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

Example 2. Analysis of terminal nucleotides G-chain telomere human DNA extracted from the culture of peripheral blood lymphocytes, stimulated to divide in the presence of PHA (APF) for 2 hours. Stimulation division lymphocytes carried addition PHA to the cultural environment RPMI-1640 at a final concentration of 10 mcg/ml.

The study of gene expression in the culture of cells by the method of standard RT-PCR revealed a drastic increase in the number of mRNA D2 against the absence of A2, B1, D3 and E1. The level of mRNA D2 towards mRNA normalization gene GAPDH amounted to 1.32, that was a significant progression of early G1 phase of the cell cycle in large parts of the cells. Lack apparently detected gene expression groups A, b and E testified to the lack of mitosis and preceding synthetic phase. In connection with this culture of lymphocytes to satisfy the requirements of status and significant activation of early preparation for cell division.

All stages of analysis was conducted similarly to the methodology described in example 1.

Successive stages of processing the data received in the course of the duplex-specific analysis, are presented in table 5.

The variant of the terminal triplet

1 hour DSNP-analysis

2 hours DSNP-analysis

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

averaged 11.92

The final result processing is represented by a bar graph of average percentages of fluorescent probes signals, corresponding options nucleotide endings G- in the studied population of lymphocytes. This chart presents 3 and corresponds to the type of terminal nucleotides G-chain of telomere DNA extracted from cultures of human lymphocytes, stimulated to divide in the presence of PHA within 2 hours.

Example 3. Analysis of terminal nucleotides G-chain telomere human DNA extracted from the culture of peripheral blood lymphocytes, stimulated to divide in the presence of PHA (APF) for 4 hours.

The study of gene expression in the culture of cells as well as in the previous example, revealed a high level of mRNA D2 (1,19) on the background of absence of A2, B1, D3 and E1. In connection with this culture of lymphocytes and culture in the previous example, to satisfy the requirements of status and significant activation of early preparation for cell division.

All stages of analysis was conducted similarly to the methodology described in example 1. Successive stages of processing the data received in the course of the duplex-specific analysis are shown in table 6.

The variant of the terminal triplet

1 hour DSNP-analysis

2 hours DSNP-analysis

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

The final result processing is represented by a bar graph of average percentages of fluorescent probes signals, corresponding options nucleotide endings G- in the studied population of lymphocytes. This chart presents figure 4 and corresponds to the type of terminal nucleotides G-chain of telomere DNA extracted from cultures of human lymphocytes, stimulated to divide in the presence of PHA within 4 hours.

Example 4. Analysis of terminal nucleotides G-chain telomere human DNA extracted from the culture of peripheral blood lymphocytes, stimulated to divide in the presence of PHA (PHA) within 11 hours.

The study of gene expression in the culture of cells by the method of standard RT-PCR revealed the presence of all detected . The values of the levels of mRNA towards mRNA normalization gene GAPDH: A2 (0,25), B1 (0,95), D2 (1,47), D3 (0.91) and E1 (0,17). High levels of mRNA D2 and D3 testified about a significant progression G1 phase of the cell cycle in large parts of the cells. At the same time, another part of the cell was on the synthetic phase of preparation for the division ( E1 and A2) and stage of mitosis (cyclin B1). Apparently originally this culture of lymphocytes was a mixed cell population, different stages of the cell cycle. Part of the cells before stimulation RNA was already in G1 and S phases. Further progression of the cell cycle led after 11 hours of PHA to the emergence of synthetic and mitotic phases, respectively.

The examined culture is of particular interest, as it enables to establish the nature of the profile of terminal nucleotides, the existing large part of the cell cycle. It allows you to determine which profile is available during the late G1 phase, synthetic and phases, as well as during the mitosis, except during the first hours of the occurrence of the earlier of the G1 phase of the trigger mechanism of the beginning of activation of cell division.

All stages of analysis was conducted similarly to the methodology described in example 1.

Successive stages of processing the data received in the course of the duplex-specific analysis, presented in table 7.

The variant of the terminal triplet

1 hour DSNP-analysis

2 hours DSNP-analysis

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

27.0%of the

The final result of processing the data presented profile terminal nucleotides G-chain of telomere DNA extracted from cultures of human lymphocytes, stimulated to divide in the presence of PHA within 11 hours (figure 5).

Example 5. Analysis of terminal nucleotides telomere DNA sample mixture of synthetic analogues endings G- in equimolar concentrations. This sample was used as a reference for the establishment of the concentration ratios of the probes and the coefficients of rationing signal their cleavage when conducting DSNP analysis.

In volume of 1,5 ml of the prepared mixture of analyzed DNA, for which added 0.5 each of the six options endings G-. Synthetic analogues were a synthetic length of 25 nucleotides, synthesized standard method in the company Eurogen (Russia) and ending with its 3'-end of triplets: TAG, AGG, GGG, GGT, GTT and TTA.

Mixture used for reactions in a finite volume 40 ml. Next, all subsequent stages of , synthesis negative chain etc. carried out according to the methodology described in example 1.

Figure 1 is a graphic image fluorescence each probe separately after one and two hours of carrying out DSNP analysis. Processing of the images received conducted as described in example 1, the procedure of analysis using the program LabWorks. Successive stages of processing of the obtained values of fluorescence for each probe and the corresponding terminal end are presented in table 8.

The variant of the terminal triplet

1 hour DSNP-analysis

2 hours DSNP-analysis

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

Level of the signal after subtracting the background

Rationing signal multiplied by the coefficient

The percentage of signal

As can be seen from the chart, the resulting profile indicates the presence of all the options nucleotide triplet endings in equal ratio of approximately 16.7%, which fully corresponds to the initial ratio in a mixture of synthetic analogues of endings G-. The maximum deviation of the obtained in this analysis, the values from theoretically expected not exceed 0.5%. Carrying out of similar researches of reference samples with known in advance proportion endings telomeric revealed a margin of error of the proposed method is not more than 1%. This fact allowed to apply the selected concentration of solutions of the probes and the coefficients of rationing signals with a high degree of reliability of the results in the study of native factions telomeric in examples 1-4.

In conclusion, the above examples illustrate the possibility of applying the declared method not only for the qualitative determination of the presence of options nucleotide endings G-chain of telomere DNA, but also to quantify their relationship during the cell cycle. The claimed method is suitable for the study of very short and for longer. Shows the efficiency of its application for the analysis of synthetic analogues of telomeric and natural factions G- presented varied in length .

In the proposed method, due to the complete departure from the procedures ligation, dependence of effectiveness analysis of the effectiveness of its passage, namely the length of the matrix G-, which is observed in the way proposed Agnello J. Sfeir et al. This fact demonstrates the undeniable technical advantage of the declared method over the existing analogues. It has allowed the author of the patent identify previously unknown changes terminal nucleotides telomeric occurring in the first hours after the activation of cell division. It was found the existence and mutual transition of equilibrium and non-equilibrium state of the balance profile of terminal nucleotides G-chain of telomere DNA of the person who determines the duration of the cell cycle.

Activation of cell division is accompanied by the appearance of unbalanced equilibrium profile of terminal nucleotides which characteristic feature is the dramatic increase in the content of TTA variants of the end of the G-chain of telomere DNA. The quantity reaches the level of 60-70%, was observed in the second and third examples of realization of the invention, where studied the culture of lymphocytes after 2 and 4 hours of PHA (figure 3 and 4).

Thus, were obtained marked differences between the profile of terminal nucleotides in the 1st example and profiles in the 2nd and 3rd examples. In the first example, profile match culture, not stimulated to division with PHA, mainly state with weak natural activation at an early stage of preparation for cell division. In this profile, the presence of a sufficiently large number of endings on the triplets TAG and AGG, the number of TTA end was not above 30%. Such a profile by percentages options nucleotide endings refers to partially balanced.

With the help of the declared method were analyzed other culture of lymphocytes, not stimulated with PHA, which were characterized by a delay in G0 phase most of the cells on the background of absence of visible progression G1 phase. The analysis of such crops in full compliance with the requirements of lack of activation of preparation for cell division, revealed the presence of a fully balanced equilibrium profile of terminal nucleotides. It is characterized by the following ratio of the percentage of the terminal triplets:

(TAG=TTA=16-20%)+(GGG=GGT=5-7%)=(AGG=GTT=25-27%).

The profile resembled the shape of a symmetrical crown» like profile, shown in figure 5. A comparison of such symmetric profile with the profile obtained in the 1st example, the fact that in the studied culture of cells already running the activation mechanisms of cell division. This is evidenced by the drop in the content of the AGG variants of the end of telomeric against the background of moderate growth TTA end. The further tendency of change in the profile of terminal nucleotides in activation fission as a result of the impact of the stimulus (APF) for 2 and 4 hours examples 2 and 3.

For the first time with the help of the declared method has been shown that the initiation of cell division under the influence of some of the stimulus, for example PHA, it is always accompanied by rearrangements of terminal nucleotides telomere DNA. The transition of the majority of nucleotide endings G-chain of telomere DNA in the end of TTA is the triggering of the mechanism for resetting the preparation of the cell to divide, namely, the transition G0 phase in early G1 phase.

The use of the claimed process provides a unique opportunity for a dynamic survey of changes in the profile of terminal nucleotides by establishing the exact percentage. The possibility of constructing graphs displaying changes in the quantitative content of options endings telomeric during the cell cycle distinguishes the proposed method from all the existing analogues. Tracking these dynamics of the changes in the profile of terminal nucleotides possible to establish the nature of their emergence.

When considering Fig.7, where a similar dynamics shows that the curves corresponding to certain choices nucleotide endings (triplets) have a wavelike character. Change the amount of a certain variant of completion is associated with changes in the neighboring options. This suggests mutual transition of some variants endings in others as a result of impact on their terminal nucleotides. Such a character of the possible adoption of a variant of the end of in another clearly traced in the first 11 hours of incentive division and was carried out under the direction of: AGG→TAG→TTA→GTT→GGT→GGG→AGG.

In the first two hours of signal TTA end of the rapidly grew by TAG and AGG endings in the serial off AGG→TAG→TTA. In the same time interval slightly varied content triplets GTT and GGT and GGG by consecutive crossings endings TTA→GTT→GGT→GGG. Such transitions neighboring options endings each other displayed throughout graphics Fig.7. If this were strictly observed their quantitative ratio. If decreased level of one terminal of a triplet, the increased level of neighboring triplet with the shift of one-two nucleotides. The existing mechanism offset endings telomeric is a kind of «nucleotide clock», which on the one hand provide the trigger mechanism of activation division, and on the other hand, determine the existence of a balanced equilibrium profile of terminal nucleotides for longer periods of cell cycle.

The existence of a balanced equilibrium profile of terminal nucleotides for the greater part of the cell cycle is a protective molecular mechanism of healthy cells. It prevents premature start of a new cell division and determines the duration of the cell cycle. For healthy peripheral blood lymphocytes, stimulated to division with PHA, it is approximately 48 hours (Morice et al., 1993). The following unbalanced nonequilibrium profile may not occur earlier than the set number of hours after the start of the primary impact of the stimulating agent. Offensive balanced equilibrium state, ranging from 10-12 hours after the start of stimulation of the division, and its maintenance during the greater part of the cell cycle are an important diagnostic feature of healthy cells.

A reduction in the duration of the cell cycle due to an earlier onset of the next state of non-equilibrium profile of terminal nucleotides and its existence for a longer time than the G1 phase of the cell cycle, is a characteristic feature of malignant cells. Thus impair the formation of a balanced equilibrium profile of terminal nucleotides, namely lock cycle transitions endings G-chain of telomere DNA, can lead to long-term dominance of TTA variants of its end. Presumably TTA end of the G-chain of telomere DNA, while at the end of most of chromosomes, is responsible for the activation of them in the expression of genes determine the beginning of the preparation of cells for cell division. Due to the long dominance TTA end of the G-chain of telomere DNA conditions are created permanent activation of cell division and shorten the duration of the cell cycle, which can lead to cancer of rebirth.

With the help of the declared method of study was conducted leukaemic cultures of human lymphocytes Jurkat and K-562 synchronized in the mitotic phase of the cell cycle. Synchronization cells was carried out within 12 hours adding colchicine in the culture medium RPMI-1640 in the final concentration of 0.5 mg/ml The long duration of impact colchicine ensured maximum accumulation of cells in the mitotic phase.

Synchronization of leukaemic cells in metaphase mitotic division revealed the lack of a balanced equilibrium profile of terminal nucleotides. The degree of violation balance profile correlated with the degree of malignancy cells and their rate of reproduction. For culture Jurkat characterized by a factor of two times greater rate of fission compared to other studied culture was characterized by highly unbalanced profile terminal nucleotides (Fig.8). This profile was presented mainly by ending TTA (82,3%) and very low presence of GTT (15,9%), and GGT (1.8%) of the terminal triplets. The absence of other variants of terminal endings, especially AGG and TAG triplets, testified about blocking cycle transitions endings G-chain of telomere DNA. The result of the partial suspension of transitions terminal triplets TTA→GTT→GGT and complete blocking of the navigation GGT→GGG→AGG→TAG has been the lack of education balanced equilibrium profile.

Culture K-562 also meet unbalanced profile terminal nucleotides, but with a lower degree of imbalance (Fig.9). On the background of dominating TTA triplets (57,5%) were the end of the AGG (22,1%), GGT (14.5%) and GTT (5,9%), indicating a decrease progression cycle transitions endings towards formation of a balanced equilibrium profile. A characteristic feature of both cultures was the absence of the TAG option end, that, probably, is connected with its complete shift in TTA triplet.

Thus, the presence or absence of a balanced equilibrium profile of terminal nucleotides, as well as its degree of imbalance in synchronization cells in specific phases of the cell cycle (for example, mitotic), is an important diagnostic criterion in Oncology. Investigation of changes in the terminal nucleotides during the cell cycle of cancer cells, namely, the duration of a non-equilibrium profile and violations associated with the emergence of the equilibrium state, may be a sign of determining the degree of malignant cells.

Application of the claimed method allows to catch minimum changes in the percentage of those or other variations or nucleotide endings of telomere DNA and can serve as a universal tool for the study of the cell cycle of any cells and tissues. Profiles terminal nucleotides G-chain of telomere DNA can be used to determine the proliferative status of various cells and tissues and be a criterion for the assessment of their ability to activate the division. For example, profiling terminal nucleotides telomere DNA immune cells can detect the degree of their activation and readiness to divide and be a criterion for the immune status of the organism. Study of proliferative potential of stem cells, as well as any able to cell division, using the declared method can be used for transplantation as a criterion for the survival of cells in dermatology and gerontology as a factor of regenerative abilities tissues and capacity to the rejuvenation of the organism as a whole.

2. The method according to claim 1, wherein the separation of telomeric G- at the stage of (a) use the method of preserving -stranded DNA from the group hybridization or sorption methods with the use of various membranes, columns, particles or other surfaces to immobilization and the subsequent removal from the surface of the investigated molecules.

3. The method according to claim 1, characterized in that the amplification of the faction of telomeric G- at the stage of (b) after spend by the enzyme terminal , hereinafter referred to consistently implement the amplification of the negative chain with the use of the primer SEQ ID nos: 2, 3'-modification of the received minus copies via the connection sequence using the enzyme terminal and their final amplification using pairs of primers, which consist of given in SEQ ID NO: 3 and SEQ ID NO: 5 sequences, subject to the excess, adapter primer.

4. The method according to claim 1 or 3, wherein the amplification of the faction of the G- at the stage of (b) as an adapter sequence contained in SEQ ID NO: 1-3, use of an appropriate structure and temperature annealing sequence, not complementary repetition G-chain of telomere DNA.

5. The method according to claim 1, characterized in that the conduct of the duplex-specific analysis at the stage of (a) instead of probes presented in SEQ ID NO: 6-11 uses variations of length 10 - 12 nucleotides.

6. The method according to claim 1, characterized in that the conduct of the duplex-specific analysis at the stage of (a) use one form or combination of several kinds of fluorescent labeling probes with a combination of «fluorophore-damper on any of the opposing ends of the probe, consisting of dyes FITC, FAM, R110, TET, R6G, HEX, JOE, VIC, YakimaYellow, TAMRA, Cy3, ROX, SuperROX, Cy 3.5, Cy 5, Cy 5.5, Cy 7, Cy 7.5 and similar and related absorbers of a number of RTQ, BHQ, and Dabcyl like them.

7. The method according to claim 1, characterized in that the registration of the results of the duplex-specific analysis of the stage (g) are species of hardware and software that help to quantify the level of meaningful fluorescent signal for each probe, including multiband a combination of several types of tagging 6.

8. The method according to claim 1, characterized in that the mathematical processing of the signal levels at registration of the results of the duplex-specific analysis of the stage (g) is the standardization of values multiplied by the factors listed in table 3, for each probe from the group FAM-RTQ1 after 1 and 2 hours of the analysis.

9. The method according to claim 1, characterized in that the mathematical processing of the signal levels at registration of the results of the duplex-specific analysis of the stage (g) is the standardization of values by multiplying the coefficients are different from tables 3 and calculated for options probes in various concentration ratios, such as, for groups fluorescent labeling 6 and relevant to the current state of the spectral properties of the fluorophore-related fluorescence of DNA at the time of research.

10. The method according to claim 1, characterized in that obtained in the course of its implementation results are applied for the purposes of medical diagnostics of diseases and aging of the body, the assessment of proliferative properties and tissue specific features of cells and tissues, the definition of the forecast of their development and selection of specific therapy using as a criterion of a certain profile of terminal nucleotides G-chain of telomere DNA.

11. A set of specific presented in SEQ ID NO: 1-5, used as primers for amplification of minus chains telomeric G- during the PCR method according to claim 1.

12. A set of specific according to cl.11 used as primers for amplification of minus chains telomeric G- during PCR, notable as the adapter sequence contained in SEQ ID NO: 1-3, use of an appropriate structure and temperature annealing sequence, not complementary repetition G-chain of telomere DNA.

13. A set of specific presented in SEQ ID NO: 6-11 used as a fluorescently-labeled probes when conducting duplex-specific analysis in the method according to claim 1.

14. A set of specific by p. 13 used as a fluorescently-labeled probes length of 10 - 12 nucleotides.

15. A set of specific on 13 used as a fluorescently-labeled probes when conducting duplex-specific analysis, which uses one kind or a combination of several types of fluorescent labeling with a combination of «fluorophore-damper on any of the opposing ends of the probe, consisting of dyes FITC, FAM, R110, TET, R6G, HEX, JOE, VIC, Yakima Yellow, TAMRA, Cy 3, ROX, SuperROX, Cy 3.5, Cy 5, Cy 5.5, Cy 7, Cy 7.5 and similar and related absorbers of a number of RTQ, BHQ, Dabcyl and similar.