Method for predicting oncological diseases

FIELD: medicine, diagnostics.

SUBSTANCE: the present innovation deals with genetic trials, with diagnostic field of oncological diseases due to analyzing DNA by altered status of gene methylation that take part in intracellular regulation of division, differentiating, apoptosis and detoxication processes. One should measure the status of methylation in three genes: p16, E-cadherine and GSTP1 in any human biological samples taken out of blood plasma, urine, lymph nodes, tumor tissue, inter-tissue liquid, ascitic liquid, blood cells and buccal epithelium and other; one should analyze DNA in which modified genes of tumor origin or their components are present that contain defective genes, moreover, analysis should be performed due to extracting and purifying DNA out of biological samples followed by bisulfite treatment of this DNA for modifying unprotected cytosine foundations at keeping 5-methyl cytosine being a protected cytosine foundation followed by PCR assay of bisulfite-treated and bisulfite-untreated genes under investigation and at detecting alterations obtained according to electrophoretic result of PCR amplificates, due to detecting the difference in the number and electrophoretic mobility of corresponding fractions at comparing with control methylated and unmethylated samples containing normal and hypermethylated forms of genes one should diagnose oncological diseases. The method provides higher reliability in detecting tumors, detection of remained tumor cells after operation.

EFFECT: higher efficiency of therapy.

1 cl, 3 dwg, 4 ex

 

The invention relates to medicine, in particular genetic research to the field of cancer diagnostics by analysis of deoxyribonucleic acid by changing the methylation status of genes involved in the intracellular regulation division, differentiation, apoptosis and detoxification processes.

A known method for diagnosing and monitoring cancer development, including analysis of amplified DNA from blood plasma (U.S. patent No. 5952170, 1999). The disadvantage of this method is that the DNA analysis of tumor origin from a single source reduces the sensitivity of the diagnosis of cancer.

It is now known that impaired methylation genes involved in the intracellular regulation division, differentiation, apoptosis and detoxification processes leads to disruption of the genome in the tumor and some other cells and disruption of the functioning of the respective tissues (patent US 6492144, Dec. 10, 2002, US patent 5726019, Mar. 10, 1998).

Closest to the proposed diagnostic method is a method for the detection of neoplastic cells, including the analysis of the methylation status of the gene P16 (US patent 5856094, Jan.5, 1999).

The disadvantages of this method of diagnostics of oncological diseases is the following:

- different persons with the same type of tumors, hypermethylation of the promotor is Chertkov DNA may differ, i.e. hypermethylation profile for individual tumors;

analysis of a single gene, such as gene P1-6, shows the presence of a tumor in only half the cases when the diagnosis of cancer in the other half of the cases the tumor material gene is not hypermethylation and hypersecretory other genes cell cycle regulation, such as gene E-cadherine.

In addition, unlike DNA isolated from the tumor itself in many biological samples containing DNA of tumor origin, such as DNA in the blood plasma of cancer patients, urine, lymph nodes, percentage hypermethioninemia forms of a gene P16 tumor origin low relative to regimentations the form of normal cells (US patent 6492144, Dec. 10, 2002).

The objective of the proposed method for the diagnosis of cancer is to increase the reliability of detection of tumors, determination of remaining in the body of tumor cells after surgical removal of the tumor or other types of treatment, monitoring of cancer and early assessment of the effectiveness of the treatment, and prognosis of the effectiveness of chemotherapy and other treatments.

The use of the proposed method for the diagnosis of cancer increases the survival rate of cancer patients due to p is increasing the accuracy of diagnosis, timely diagnosis and improve treatment effectiveness, analysis of methylation status of several genes increases the probability of detection of the tumor, and the additional reaction of the nested PCR (nested PCR) allows you to define hypermethioninemia form P16 gene with a low ratio of DNA tumor of origin in relation to DNA from non-neoplastic cells, as well as the opportunity to assess the dynamics of the disease, i.e. the presence of tumor cells after surgical or other treatment, early to predict the Breasts or the spread (metastasis) of disease and to make an early assessment of treatment effectiveness, for the possibility of transition and more suitable in the specific case of chemotherapy or other treatments.

The result is achieved that the proposed method for the diagnosis of cancer by assessing the methylation of genes involved in the regulation of cellular metabolism, simultaneously measure the status of methylation of three genes P16, E-cadherin and GSTP1 in any biological samples of human, taken from blood plasma, urine, lymph nodes, tumor tissue, interstitial fluid, ascitic fluid, blood cells, and buccal epithelium and others analyze DNA that has modified the genes of tumor origin in any of the samples, which can pop the AMB tumor cells or their components, containing the defective genes, and the analysis is carried out by extraction and purification of DNA from biological samples with subsequent bisulfite treatment of DNA for modification of unprotected casinovip grounds while maintaining the 5-methylcytosine, which are protected casinowe basis, subsequent PCR analysis of treated and untreated bisulfite studied genes, and when a change is detected, obtained according to the result of electrophoresis of PCR amplification, by identifying differences in the number and electrophoretic mobility of the corresponding fractions when compared to the control methylated and neetilirovannyj samples containing normal and hypermethioninemia forms of genes diagnosed with cancer.

For control methylated and neetilirovannyj samples used DNA from cultures of human tissue containing normal or hypermethioninemia forms of genes P16, E-cadherin and GSTP1.

Biological samples may be collected from persons surveyed to identify cancer or with established cancer before treatment, during treatment before and after surgery, chemotherapy, radiation therapy, biotherapy, and others.

The DNA extraction is carried out on biological samples, conducting the cleanup, bisulfite modification and further amplification of this DNA to determine the methylation status of genes DNA by comparative analysis of the samples of persons affected by the diagnosis control samples, negative control, DNA from healthy individuals and is known cultures of human tissues with normal methylation status of the studied genes, and positive control DNA cultures of human tissues, where there is a breach in the methylation status. Various impurities in the extraction and purification of DNA interfere with subsequent methylation status, as screen neetilirovannye bases cytosine and the reaction of bisulfite modification they're known as 5-methylcytosine (methylated bases cytosine), which leads to false polozhitelnim diagnoses (Ehrlich M. DNA methylation: normal development, inherited diseases, and cancer. J Clin Ligand Assay 2000; 23: 144-8).

To extract DNA in the present invention uses modified for certain types of biological samples, methods of extraction, which consists in changing the concentration of proteinase K, pH buffer system and the number of repeated phenol extraction and use for deposition of DNA in samples with a low concentration of DNA (e.g., blood plasma) linear polymer of polyacrylamide.

The method is as follows.

9 ml of blood is taken from the ulnar vein one is radovim syringe and transferred into a polypropylene tube with a volume of 15 ml (firm Finbio or similar tubes), containing 1 ml of 1%EDTA-anticoagulant isotonic. Second option: the blood samples are also carried out using a single vacuum system with EDTA - Monovette company Sarstedt, Germany (permission to use the Ministry of health of the Russian Federation No. 2000/403 from 14.08.2000 year).

The first centrifugation is carried out in mild conditions when accelerating 315g for maximum preservation of the integrity of nucleated cells 15 min at 4°C. Use the centrifuge 5804R with bactrocera company Eppendorf (Germany). For this centrifuge such acceleration is provided at 1500 rpm, or can be used another centrifuge with similar conditions of centrifugation. Transfer the plasma to a suspension of nucleated cells into a new tube, being careful not to grab the settled red blood cells, centrifuged at 1260g (3000 rpm, 15 min, 4°C, centrifuge 5804R with bactrocera company Eppendorf, Germany) to precipitate nucleated cells. Then transfer the cell-free plasma in a new tube, leaving about 150-200 ál of sediment. The precipitate nucleated blood cells resuspended in the remaining amount of plasma is transferred to microprobing type Eppendorf 1,5 ml Centrifuged tube with plasma at 3500g (5000 rpm, 15 min, 4° (C) for the complete precipitation of the remaining unit cells. The plasma is transferred into a new tube and used for further work or save the Ute at -80° C.

The isolation of DNA from biological samples

The isolation of DNA from blood cells by the phenol method with pre-treatment with proteinase K. (the same method used for isolation of DNA from sediment cells, urine, cell cultures).

The precipitate nucleated cells transferred to microprobing type Eppendorf 1.5 ml precipitated in Minicentrifuge Microspine company Eppendorf (12000 rpm (8000g), 1 min) and preserved until DNA extraction at -80°C. the precipitated cells resuspended 200 ál lyse buffer (10 mm Tris (pH 8.0), 25 mm EDTA (pH 8.0)and 0.5% SDS). Resuspending and short precipitated by centrifugation. Add 20 ál of a solution of proteinase K (100 µg/ml), mixed and incubated overnight at 56°C. the extraction is carried out proteins from the lysate phenol, balanced buffer (50 mm Tris, pH 8.0, 10 mm EDTA). Centrifuged 5 min at maximum speed (Eppendorf Minispin), the upper (aqueous) phase was transferred into a new tube. This is followed by extraction of the aqueous phase (5) a mixture of phenol - chloroform” ratio of phenol to the chloroform 10:1.

Centrifuged 5 min at maximum speed, transfer the upper (aqueous) phase to a new tube and add an equal volume of chloroform. Shaken and centrifuged 5 min at maximum speed. The upper phase transferred to a new tube. Add 1/10 volume of a solution of 3 M sodium acetate, pH 5,3, and 2 volume okhlazhdennogo to -20°hol is underwater 96%-ethanol. Shaken, incubated 2 hours at -20°C. Centrifuged at +4°15 min at maximum speed. Carefully remove the supernatant, avoiding labilization precipitate DNA. Wall of the tube is washed with 70%ethanol, optionally centrifuged (if labilization sediment), the alcohol is removed, the residue is dried in air. To store the selected DNA is dissolved in water or TE buffer, and stored at -80°C.

DNA isolation from tissues, fixed in paraffin

Prepare 8-15 µm sections from biopsies of prisoners in paraffin blocks. Next, the tissue sections, fixed in paraffin, placed in microprobing type Eppendorf 1.5 ml, add 1 ml of xylene and gently shaken (turning the tube up and down several times). Then centrifuged 5 min at maximum speed, room temperature. Remove the supernatant with a pipette. If necessary, the dewaxing with xylene repeat. Add 1 ml of 96%ethanol to remove residual xylene and mix carefully (turning the tube up and down several times). The alcohol is removed and repeat the washing with alcohol again. Add to the draught of THE buffer, immediately, in order not managed from cuts to buyouts DNA fragments, and centrifuged to remove the supernatant.

Resuspending sediment in lyse buffer (100 mm NaCl, 10 mm Tris (pH 8.0), 25 mm EDTA pH 8.0), 1,5% SDS), 3 slice of 300 ál. Add proteinase K, 50 µg in 100 µl of buffer (usually proteinase is stored in a stock solution of 20 µg/µl). Incubated at 55°With in 24-48 hours until complete lysis of tissues with periodic stirring. If after 24-hour incubation of the tissue is not fully visualized, then add the same amount of a solution of proteinase.

Proteins extracted with phenol. This phenolic fraction remains in the lower phase at the bottom of the tube. Then centrifuged 5 min at maximum speed. The aqueous phase is transferred into a new tube and the phenol phase (bottom) is disposed. The extraction is carried out with a mixture of the phenol-chloroform” ratio of phenol to the chloroform 10:1. Centrifuged 5 min at maximum speed, the aqueous phase is transferred into a new tube, and add to it an equal volume of chloroform. Stirred, centrifuged 5 min at maximum speed. The aqueous phase is transferred into a new tube, the lower phase is disposed. To the aqueous phase add 1/10 volume of a solution of 3 M sodium acetate, pH of 5.2, and 2 volumes of cold 96%ethanol (2 volumes, given the amount of salt solution). Mix, incubated 2 hours at - 20°C.

Centrifuged at +4°30 min at maximum speed (centrifuge Eppendorf Minispin, 13400 rpm (10000g).

Carefully remove the supernatant, avoiding labilization precipitate DNA. Wall of the tube prom which provide 70%ethanol, if necessary, centrifuged (if labilization sediment), the alcohol is removed, the residue is dried in air. Use. To store dissolved in water or TE buffer, and stored in a refrigerator at - 80°C.

DNA isolation from blood plasma.

DNA from plasma was isolated by the phenol method, after pre-treatment with proteinase K. the Previously added to the plasma one part of the two-lyse buffer (1% sodium dodecyl sulphate, 500 mm Tris-Cl, pH 8.0, 20 mm EDTA, pH 8.0, 10 mm NaCl) and proteinase K To a concentration of 500 μg/ml, and then stood overnight at 55°C. For the extraction of proteins was added an equal volume of phenol equilibrated with buffer (50 mm Tris-Cl, pH 8.0, 10 mm EDTA, pH 8.0). Shook 5-10 min and centrifuged 10 min at 3500g at +4°C. Collected the top phase, containing the DNA, and the next stage of deproteinization spent an equal volume mixture of phenol-chloroform, shaking the mixture for 5-10 minutes, and was centrifugally 10 min in the same conditions. Third deproteinization was similar to the second, but used pure chloroform. To precipitate the DNA to the aqueous phase was added 10 M solution of ammonium acetate at a ratio of 1:5, and shoesadidas used linear polyacrylamide, cleaned twice presidenial in ethanol at a final concentration of 25 μg/ml, and had alcohol precipitation of DNA by adding 2 volumes of 96%of etano the a and maintaining the mixture at -20° C for 2 hours. The precipitated DNA was collected by centrifugation for 30 minutes

Next, the precipitate was washed with 1 ml 70%ethanol and after centrifugation was dried in a vacuum concentrator company Eppendorf (model 5301). Next, the precipitate was dissolved in 50 µl TE buffer (50 mm Tris-Cl, pH 8.0, 10 mm EDTA, pH 8.0) and stored at - 80°C.

Bisulfite modification of DNA from biological samples for subsequent methylsilyne PCR genes P16, E-cadherin, GSTP1.

The principle of the method methylsilyne polymerase reaction.

The method is based on MS-PCR lies bisulfite treatment of the analyzed DNA, leading to the conversion of unprotected bases cytosine into uracil and preservation of the protected methyl group of 5-methylcytosine with subsequent PCR with specific to a modified sequence primers. (Singal, R., and Ginder, Ginder, G.D. DNA methylation. Blood, 93: 4059-4070, 1999). Given that proteins can be screened bases cytosine and prevent bisulfite conversion of cytosine to uracil, only used drugs DNA extracted using proteinase K to eliminate false-positive results.

Bisulfite treatment and purification of modified DNA.

The reaction bioliteracy runs only on single-stranded DNA fragments for DNA denatured in an alkaline environment. The DNA sample, dissolved in 18 μl of deionized water, den who was toniroval by adding 2 ál of 3 M NaOH. Incubated at 37°C, 30 minutes (the final concentration of NaOH - 0.3 M).

Bisulfite conversion was performed by adding 208 μl of 2 M sodium metabisulfite (Panso3), pH 5.0, and 12 μl of 10 mm hydroquinone. The mixture is incubated for 16 h at 50°C. Further purification of the modified DNA was performed by sorption on porous glass powder company Sigma. For this, we first added 2 volume lyse buffer 6 M guanidinosuccinic, 0.05 M Tris Hcl pH 5.0, carefully shaking the test tube on a vortex. Added 10 μl of a 10% suspension of sorbent (before adding the sorbent carefully resuspending). Leave the tube at room temperature for 10 min, regularly inverting the tube during this time this time every 2 min to prevent settling of the sorbent. Precipitated sorbent short (30 sec) by centrifugation at 100 rpm, as carefully as possible take away the supernatant. Add 200 ál of 70%ethanol, shaken for mixing or peterout until a homogeneous suspension. Precipitated sorbent short (30 sec) by centrifugation at 1000 rpm, as carefully as possible take away the supernatant.

Add to the precipitate with 200 ál 70%ethanol for washing and suspended sediment to a homogeneous state. Precipitated sorbent short (30 sec) by centrifugation at 1000 rpm, as carefully as possible away super tent. After the last rinsing, dry the precipitate in air for 10 minutes Add to the precipitate in 50 ál TE buffer, mixed and incubated for 10 min at 56°C. Precipitated sorbent long (4 min) by centrifugation at 10000 Rev/min Transfer the supernatant containing the DNA preparation, in another test tube.

Next are exempt from the sulfonates in the DNA sample. To do this, add 3 M NaOH, incubated 15 min at 37°C. Add 20 ál of a solution of 10 M solution of ammonium acetate with pH 7.0, 1 μl of a solution of 0.2%glycogen and 200 ál of 96%ethanol. Place the test tube for 1-2 hours at -20°C. Then centrifuged 15 min at maximum speed. Select the supernatant. Wash the precipitate with 200 ál 70%ethanol, dried at room temperature and dissolved in 50 µl TE buffer.

Samples are immediately used for PCR or store at -80°until analysis. Conduct simultaneously estimate the hypermethylation of 3 genes from one sample DNA after bisulfite modification. For analysis of each gene using 3 µl of DNA in a final reaction mixture volume of 25 µl.

Methylsilyne polymerase chain reaction (MS-PCR).

MS-PCR was used for amplification of the analyzed sequences of the genes P16, E-cadherin and GSTP1. PCR was performed in the apparatus Eppendorf Mastercycler no following General scheme: 3 µl of DNA was added 22 μl of buffer for PCR, with the holding and 1 μl of 200 μm of each deoxyribonucleotide, 3 μl of each solution of the oligonucleotide-primer, 2 μl of 20 mM MgCl2, 10 units of thermophilic DNA polymerase 2 ál, 5 ál of 5x, buffer, 6 μl of deionized water (Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D., and Baylin, S.B. Methylation - specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad.Sci USA, 93: 9821-9826, 1996).

Further modes PCR and the primers used for methylated and demetilirovanny shape of each analyzed gene are individual.

Analysis of the methylation status of the gene P16 using MS-PCR.

In reaction PCR is used for the analysis of bisulfite modified DNA in the presence of methylated and demetilirovanny alleles of the gene p16INK4a. Sequences of primers for analysis of hypermethylation of gene R 16

In the analysis of gene P16 sample analyzed DNA take two test tubes with a volume of 0.5 ml for PCR and each with 3 µl of a solution and the DNA analyzed. In one test tube add 22 ál of the reaction mixture as described above for amplification of methylated sequences containing 15 PM primer RMA 3 ál, 15 PM of primer 16b reverse primer, 3 µl, in another 18 µl reaction mixture for amplification demetilirovanny sequence (3 μl primer 16Ua, 3 ál - 15 PM primer 16Ub reverse primer).

Shake the test tube on the vortex and precipitated a short centrifuger the cation, add one drop of mineral oil and placed in thermal cycler for PCR under the following programs:

For the methylated sequence:

1. 94°C 5 min

3. 65°3 min

4. 72°5 min

For demetilirovanny sequence:

1. 94°5 min

3. 57°3 min

4. 72°5 min

Mix the samples after amplification with the dye for electrophoresis and applied to 10% polyacrylamide gel, conduct electrophoresis to achieve a leading dye the bottom edge of the gel. Paint bromide gel with ethidium within 15 min and evaluate the result by browsing the gel on a UV-transilluminator. The size of amplificata methylated gene is 150 base pairs (BP), and the size of amplificata demetilirovannogo gene 151 BP

For testing a biological sample for PCR analysis was performed to bisulfite conversion of DNA with primers p16Wa and p16Wb, which confirms the suitability of the sample for PCR analysis in the same conditions. PCR amplificate size 140 BP

In the case of identification evaluation hypermethioninemia forms of P16 gene in a DNA sample from blood plasma, where it can be presented in small amounts in the early stages of cancer using nested-PCR (nested PCR) with DNA after bisulfite to the nversio with primers p16Nb, 273 P.N., and then 3 μl of amplificata made as usual for the second PCR with primers RMA and 16b, as stated above (150 BP).

Analysis of the methylation status of the gene E-cadherin (E-cadherine)

In reaction PCR is used for the analysis of bisulfite modified DNA in the presence of methylated and demetilirovanny alleles of E-cadherine. Sequences of primers for analysis of hypermethylation of the gene E-cadherine

E-MA ggT gAA TTT TTA gTT AAT TAg Cgg TAC

E-Mb FFC CAT TAA CCg AAA ACg CCg

Product - 204 bp

E-Ua ggT Agg TgA ATT TTT AgT TAA TTA gTg gTA

E-Ub ACC CAT AAC TAA CCA AAA ACA CCA

Product - 211 bp

In the analysis of gene E-cadherine sample analyzed DNA take two test tubes with a volume of 0.5 ml for PCR and each with 3 µl of a solution and the DNA analyzed. In one test tube add 22 ál of the reaction mixture as described above for amplification of methylated sequences containing 15 PM primer E-MA 15 PM primer E-Mb reverse primer, another 18 µl reaction mixture for amplification demetilirovanny sequence (15 PM primer E-Ua, 15 PM of primer S Ub - reverse primer).

Shake the test tube on the vortex and short precipitated by centrifugation, add one drop of mineral oil and placed in thermal cycler for PCR under the following programs:

Annealing of primers; 57°With 40 seconds

The elongation - 72°With 40 seconds

Denaturate is - 95°C - 50 seconds

35 cycles

Mix the samples after amplification with the dye for electrophoresis and applied to 10% polyacrylamide gel, conduct electrophoresis to achieve a leading dye the bottom edge of the gel. Paint bromide gel with ethidium within 15 min and evaluate the result by browsing the gel on a UV-transilluminator. The size of amplificata methylated gene is 204 base pairs (BP), and the size of amplificata demetilirovannogo gene 211 BP

Analysis of hypermethylation of the gene GSTP1 (glutathione S-transferase P1)

Analysis of hypermethylation is carried out using primers suggested Haslam and others (Goessi et al., 2001).

In the proposed method, determine the optimal conditions for MS-PCR and exclude the possibility of false-positive results, considering the promising studies of blood plasma and cellular urinary sediment of patients for differential diagnosis of adenoma and adenocarcinoma of the prostate, and in some cases also in the evaluation of oncological processes, hepatoma, breast cancer, and others.

As a positive control, the source DNA hypermethioninemia form of the gene GSTP1 used a line of human cells Ln, where the gene is hypermethioninemia form, and as a negative control cell line human Du 14, where there is a normal methylation status (Hidefumi Kinoshita, Yan Shi, Carol Sandefur, Lorraine F.Meisner, Chawnshang Chang Methylation of the Androgen Receptor Minimal Promoter Silences reduced in Human Prostate Cancer (Cancer Res 60, 3623-3630, July 1, 2000). In addition, as the positive control used DNA samples of patients with adenocarcinoma of the prostate with the identified presence hypermethioninemia form of the gene GSTP1 provided by Professor Muller (Free University of Berlin).

The sequence of time and temperature for the GSTP1 gene.

1. Activation of the polymerase 94° (3 min)

2. Annealing of primers

(specific hybridization sequence

DNA matrix). 59°C (30 sec)

2.1. Synthesis of circuits. The elongation 72°C (30 sec)

2.2. The melting. 94°C (30 sec)

3. Excessive annealing 59°C (30 sec)

3.1 Excessive synthesis. 72°C (30 sec)

Primers to the sequence of wild-type unmodified DNA.

GWS 5’ - GACGCCCGGGGTGCAGCGGCCGCC 3’ - SENSE

GWA 5’ - CCGCCCCAGTGCTGAGTCACGGCG 3’ - ANTISENSE

Primers to demetilirovanny sequence of the modified DNA.

GUS 5’ - GATGTTTGGGGTGTAGTGGTTGTT 3’ - SENSE

GUA 5’ - CCACCCCAATACTAAATCACAACA 3’ - ANTISENSE

The primers for the methylated sequence of the modified DNA.

GMS 5’ - TTCGGGGTGTAGCGGTCGTC 3’ - SENSE

GMA 5’ - GCCCCAATACTAAATCACGACG 3’ - ANTISENSE

Electrophoresis of amplification products of the gene GSTP1

Electrophoresis of amplification products was performed is 10% polyacrylamide gel, in a vertical apparatus, under the conditions as described above. Was used marker length of DNA fragments (MB - 50-Base Pair Ladder 1 mg/ml (increments of 50 pairs of nucleotides) in the hole.

Electrophoresis was performed at a constant current of 20 mA. For visualization of the DNA fragments in the gel were able to gel in an aqueous solution of ethidium bromide (Sigma, Molecular Biology Grade) (10 ál ethidium bromide (10 mg/ml) in 100 ml of distilled water). Then watched the gel on a UV-transilluminator and transferred the data to the computer using video GeneScan (DNA-Technology).

Diagnosis is as follows.

If the DNA of the biological sample contains hypermethylation form one of the three genes, two of the three genes, or all three genes, the biological sample probably has a tumor origin.

If DNA from the blood cells do not contain hypermethioninemia forms one of the three genes, two of the three genes, or all three genes, and DNA from blood plasma contains hypermethylation form one of the three genes, two of the three genes, or all three genes, it indicates the presence of tumor in the body.

If DNA from blood plasma contains hypermethylation form one of the three genes, two of the three genes, or all three genes, it indicates the presence of tumor in the body.

If DNA from the tumor contains hypermethioninemia forms one of the sin gene is in, two of the three genes, or all three genes, and DNA from plasma and contains no hypermethylation form one of the three genes, two of the three genes, or all three genes, after surgical removal of the tumor or other treatment, it means the absence or presence of a very small number of remaining tumor cells in the body.

If DNA from the tumor contains hypermethioninemia forms one of the three genes, two of the three genes, or all three genes, and DNA from blood plasma contains hypermethylation form of the same genes, after surgical removal of the tumor or other treatment, it means the presence of remaining tumor cells in the body.

If DNA from the tumor contains hypermethioninemia forms one of the three genes, two of the three genes, or all three genes, it means the functional inferiority of the corresponding gene, which is taken into account when prescribing remedies, the effectiveness of which depends on the state of the listed genes.

If DNA from the blood cells contains hypermethioninemia forms one of the three genes, two of the three genes, or all three genes, and DNA from blood plasma contains hypermethylation form one of the three genes, two of the three genes, or all three genes, it refers to the ability of oncohematological diseases.

Figure 1 presents electrophoregram cont the KTA MC-PCR of viewing P16 gene. Use nested-PCR for amplification of MS-PCR hypermethioninemia forms of P16 gene in DNA biological samples. (DNA blood plasma from a patient with stage 1 breast tumor - T1N0M0).

1 - amplificat DNA from human cell culture RS (hypermethioninemia one allele of the gene P16), using primers for hypermethioninemia genes;

2 - amplificat DNA from human cell culture RS (hypermethioninemia one allele of the gene P16), using primers for the gene P16 with normal methylation status;

3 - amplificat DNA in the blood plasma of the patient with a tumor of the breast the hypermethylation not detected using primers for hypermethioninemia genes;

1A - amplificat DNA from human cell culture RS (hypermethioninemia one allele of the gene P16), using primers for hypermethioninemia genes after pre-enrichment nested PCR;

2A - amplificat DNA from human cell culture RS (hypermethioninemia one allele of the gene P16), using primers for the gene P16 with normal methylation status after pre-enrichment nested PCR;

3A - amplificat DNA in the blood plasma of the patient with a tumor of the breast is detected hypermethylation using primers for hypermethioninemia genes pic the e pre-enrichment nested PCR;

Lad - markers DNA size.

Figure 2 presents electrophoregram product of amplification of the gene E-cadherine.

The products of amplification of the gene E-cadherine after MS-PCR.

1 - markers of DNA, b-pairs.

2, 3 - DNA samples from blood cells of a patient with lung cancer, with primers for methylated (2) and demetilirovanny (3) DNA sequences, respectively.

4, 5 samples with DNA from cell culture RS with a normal allele with primers for methylated (4) and demetilirovanny (5) the DNA sequence, respectively.

6, 7 samples with DNA from cell culture MSF7 with a normal allele with primers for methylated (6) and demetilirovanny (7) the DNA sequence, respectively.

Identified hypermethioninemia (lane 4) and normal (lane 3) form of the gene E-cadherine.

8-9 - DNA samples of blood plasma of a patient with lung cancer, with primers for methylated (8) and demetilirovanny (9) the DNA sequence, respectively.

Detected hypermethylation.

Figure 3 presents electrophoregram product amplification of GSTP1 gene.

1, 2 - samples with DNA cell line LnCap (hypermethylation GSTP1 gene in both alleles), with primers to demetilirovanny and methylated DNA sequences, respectively.

3, 4 - samples with the DNA of the cell sediment of the urine of a patient with adenocarcinoma of the prostate VC is SHL, with primers for methylated and demetilirovanny DNA sequences, respectively. Identified hypermethioninemia (lane 4) and normal (lane 3) form of the gene GSTP1.

5 - markers of DNA size.

Specific examples of the method for the diagnosis of cancer.

Example 1.

Patient A. cystoscope-fibrous mastopathy turned to diagnose possible tumor analysis hypermethioninemia genes of tumor origin. When performing analysis of the methylation status of the gene P16 was found that in the pre-enrichment nested PCR DNA sample from blood plasma reveals hypermethioninemia form of the gene P16 (figure 1). Further clinical examination, biopsy and subsequent operation morphologically revealed adenocarcinoma of the breast in the 1st stage, which confirms the possibility of diagnostics.

Example 2.

Patient Doctor with complaints over a long period of cough and mild fever in the evenings, as well as signs of hepatitis in plasma detected hypermethioninemia gene form E-cadherine, and blood cells are not detected gene E-cadherine (figure 2). Upon further examination in the blood plasma found a hundred-fold excess concentration of carcinoembryonic antigen, and clinically by CT scan of the chest a tumor is egcogi. Further, the presence of distant metastases in the liver. It turned out that the patient has already fourth stage cancer with distant metastases.

Example 3.

Patient Century with prostate cancer were analyzed for the presence of hypermethioninemia form GSTP1 gene in DNA from a cell of urine sediment. Detected the presence of hypermethioninemia and normal form of the gene GSTP1 in this sample. Subsequent biopsy at several points allowed morphologically to detect the focus of adenocarcinoma of the prostate in adenoma (figure 3).

Example 4.

Patient F. with fibrocystic mastopathy was analyzed for the presence of hypermethioninemia forms of these three genes. Revealed the presence of hypermethioninemia forms of a gene P16 and GSTP1 DNA in blood plasma. Not detected hypermethioninemia form of the gene E-cadherine. Blood cell hypermethioninemia forms of these three genes was not found. In the subsequent presence of breast cancers clinically confirmed conducted by biopsy. In remote tumor tissue also revealed hypermethioninemia forms of a gene R 16 and GSTP1, but not the E-cadherine.

The proposed method was investigated about 200 samples, more than 95% of cases the diagnoses were confirmed by the well-known solution in the case of positive analysis.

Our results indicate in which of the possibility of using the proposed method for the diagnosis of cancer in diagnostic laboratories to improve the accuracy and reduce the cost of diagnosis using domestic reagents for conducting modification of DNA and promising DNA studies of biological specimens for the assessment of the status of methylation of three genes listed.

A method for the diagnosis of cancer, characterized in that simultaneously measure the status of methylation of three genes P16, E-cadherine and GSTP1 in any biological samples of human, taken from blood plasma, urine, lymph nodes, tumor tissue, interstitial fluid, ascitic fluid, blood cells, and buccal epithelium, analyze DNA that has modified the genes of tumor origin in any of the samples, which can enter tumor cells or their components, containing the defective genes, and the analysis is carried out by extraction and purification of DNA from biological samples with subsequent bisulfite treatment of DNA for modification of unprotected casinovip grounds while maintaining the 5-methylcytosine, which are protected casinowe basis, subsequent PCR analysis of treated and untreated bisulfite studied genes and the detection of changes obtained by the electrophoresis of PCR amplification, by identifying differences in the number and electrophoretic mobility of the corresponding fractions when compared to the control methylated and neetilirovannyj samples contain asimi normal and hypermethioninemia forms of genes, diagnosed with cancer.



 

Same patents:

FIELD: clinical medicine, pulmonology.

SUBSTANCE: one should carry out complex estimation of interleukin-1β) concentration in blood, saliva, bronchoalveolar liquid. Moreover, one should detect distribution coefficient (DC) for IL-1β as the ratio of IL-1β blood content to IL-1β salivary content. At increased IL-1β blood content by 10 times and more, by 2 times in saliva, unchanged level of bronchoalveolar IL-1β, at DC for IL-1β being above 1.0 one should predict bronchial obstruction. The method enables to conduct diagnostics of the above-mentioned disease at its earlier stages.

EFFECT: higher efficiency of prediction.

2 tbl

FIELD: medicine, juvenile clinical nephrology.

SUBSTANCE: disease duration in case of obstructive pyelonephritis should be detected by two ways: either by detecting the value of NADPH-diaphorase activity, as the marker of nitroxide synthase activity in different renal department and comparing it to established norm, or by detecting clinico-laboratory values, such as: hemoglobin, leukocytes, eosinophils, urea, beta-lipoproteides, lymphocytes, neutrophils, the level of glomerular filtration, that of canalicular reabsorption, urinary specific weight, daily excretion of oxalates, arterial pressure, and estimating their deviation against average statistical values by taking into account a child's age.

EFFECT: higher efficiency of detection.

7 dwg, 1 ex, 6 tbl

FIELD: medicine, clinical toxicology.

SUBSTANCE: at patient's hospitalization one should gather the data of clinical and laboratory values: on the type of chemical substance, patient's age, data of clinical survey and laboratory values: body temperature, the presence or absence of dysphonia, oliguria being below 30 ml/h, hemoglobinuria, erythrocytic hemolysis, exotoxic shock, glucose level in blood, fibrinogen and creatinine concentration in blood serum, general bilirubin, prothrombin index (PTI), Ph-plasma, the state of blood clotting system. The state of every sign should be evaluated in points to be then summed up and at exceeding the sum of points being above "+20" one should predict unfavorable result. At the sum of "-13" prediction should be stated upon as favorable and at "-13" up to "+20" - prediction is considered to be doubtful.

EFFECT: higher accuracy of prediction.

2 ex, 3 tbl

FIELD: medicine, infectology, hepatology.

SUBSTANCE: in hepatic bioptate one should detect products of lipid peroxidation (LP), such as: dienic conjugates (DC), activity of antioxidant enzymes, such as: catalase (CAT)and superoxide dismutase (SOD). One should calculate by the following formula: C = DC/(SOD x CAT)x100, where DC - the content of dienic conjugates, SOD - activity of superoxide dismutase, CAT - activity of catalase. At coefficient (C) values being above 65 one should predict high possibility for appearance of cirrhosis, at 46-645 - moderate possibility and at 14-45 -low possibility for appearance of cirrhosis.

EFFECT: higher accuracy of prediction.

3 ex

FIELD: medicine, hepatology.

SUBSTANCE: one should detect the level of hepato-specific enzymes (HSE) in blood plasma, such as: urokinase (UK), histidase (HIS), fructose-1-phosphataldolase (F-1-P), serine dehydratase (L-SD), threonine dehydratase (L-TD) and products of lipid peroxidation (LP), such as: dienic conjugates (DC), malonic dialdehyde (MDA). Moreover, one should detect the state of inspecific immunity parameters, such as: immunoregulatory index (IRI) as the ratio of T-helpers and T-suppressors, circulating immune complexes (CIC). Additionally, one should evaluate the state of regional circulation by applying rheohepatography (RHG), the system of microhemocirculation with the help of conjunctival biomicroscopy (CB) to detect intravascular index (II). In case of increased UK, HIS levels up to 0.5 mcM/ml/h, F-1-P, L-SD, L-Td, LP products, CIC by 1.5 times, higher IRI up to 2 at the norm being 1.0-1.5, altered values of regional circulation, increased II up to 2 points at the norm being 1 point, not more one should diagnose light degree of process flow. At increased level of UK, HIS up to 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 1.5-2 times, increased IRI up to 2.5, altered values of regional circulation, increased II up to 3-4 points one should diagnose average degree of process flow. At increased level of UK, HIS being above 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 2 and more times, increased IRI being above 2.5, altered values of regional circulation, increased II up to 5 points and more one should diagnose severe degree of process flow.

EFFECT: higher accuracy of diagnostics.

3 ex

The invention relates to the field of medicine
The invention relates to the field of medicine

The invention relates to the field of analytical chemistry and relates to the quantitative determination of antibiotic groups, macrolide azithromycin dihydrate (azithromycin)

The invention relates to medicine, in particular for gastroenterology, Hepatology and General medicine, and can be used for selection of pharmaceuticals for the treatment of hepatic encephalopathy

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

FIELD: biotechnology, medicine, proteins.

SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.

EFFECT: valuable medicinal properties of polypeptide.

19 cl, 33 dwg, 1 ex

The invention relates to the field of biotechnology and can be used in medicine

The invention relates to the field of biotechnology and can be used in veterinary medicine and medicine

The invention relates to medicine and biotechnology and concerns of soluble mutant CTLA4 molecules that bind to the antigen CD86 with higher avidity than wild type CTLA4

FIELD: biotechnology, medicine, proteins.

SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.

EFFECT: valuable medicinal properties of polypeptide.

19 cl, 33 dwg, 1 ex

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