SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis.

DNA Melting Analysis (DMA) with a TaqMan probe covering the mutation "hot spot" is a simple, sensitive, and "closed tube" method of mutation detection. However, DMA requires asymmetric PCR to produce single-stranded amplicons capable of interacting with TaqMan probes. This makes quantitative analysis impossible owing to low amplification efficiency. Moreover, bi-strand mutation detection necessitates two independent PCRs. The SLAM-MS (Stem-Loop AMplicon Mutation Scanning) assay, in which symmetric PCR is performed using primers with 5'-universal primer sequence (UPS), has been developed to detect KRAS mutations. Some of the resulting amplicons, sense and antisense, adopt single-stranded stem-loop conformation and become unable to renature, but able to hybridize with TaqMan probes. Hybrids of stem-loops and complementary TaqMan probes are suitable for melting analysis and simultaneous bi-strand mutation scanning. In addition, the areas under the melting peaks are determined by the PeakFit software, a non-linear iterative curve fitting program, to evaluate the wild-type/mutant allele ratio. Thus, the SLAM-MS assay permits quantification of both the number of copies of the target sequence and the percentage of mutant alleles. For mutant enrichment, the SLAM-MS assay uses TaqMan probes as PCR blocking agents allowing an ~10 times higher mutation detection sensitivity than High Resolution Melting (HRM) assay.

[Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes].

Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and   PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.

[Transcripts of satellite DNA in blood plasma: probable markers of tumor growth].

A recent study of human normal and tumor tissues revealed a high transcriptional activity of pericentromeric satellite DNA repeats (they produce half of all transcripts in tumor cells that is many times higher than in normal ones). It was found also that the two subtypes of satellite DNA (HSATII and GSATII) are transcribed reciprocally, i.e. there is a sharp prevalence of HSATII transcription in tumors, while GSATII transcription prevails in the corresponding normal tissues. As different RNAs are present in blood plasma, and some of them serve as effectivetumor markers, we attempted for the first time to evaluate satellite HSATII and GSATII RNAs in the blood plasma of healthy donors and cancer patients. The RT-PCR protocol designed for this purpose allowed us to detect transcripts of both HSATII and GSATII repeats. As it has been shown, HSATII transcripts are more abundant than GSATII ones in plasma of healthy donors and vice versa in plasma of cancer patients; these ratios being diametrically opposed to those that exist within the cells. Some suggestions concerning origins of circulating satellite RNAs and their probable role as tumor markers are discussed.

[Mutation scanning of short amplicons: optimization of DNA melting conditions].

High resolution melting analysis (HRMA) using special "saturating" fluorescent dyes is a new and very effective approach to genotyping and mutation scanning. HRMA, which is carried out usually just after PCR without any intermediate manipulations (the "closed tube" format), is simple and high-throughput method excluding sample cross-contaminations. The "closed tube" format makes, however, HRMA dependent on PCR mixes and, as such, limits its capability. The "open tube" format (post-PCR amplicon shortening and optimization of the ionic medium) proposed by us earlier, although somewhat more laborious, significantly increases sensitivity of the method and makes it possible to scan mutations in the short amplicons using conventional SYBR Green I dye and a standard (not adapted specifically for HRMA) real-time PCR instrument. Detection of mutant K-RAS in DNA of clinical specimens (tumor tissues, formalin-fixed paraffin-embedded samples) reveals equal, at least, sensitivity of this method as compared with the HRMA and much higher as compared with Sanger sequencing. The problem of false-negative results in mutation scanning of K-RAS, which is highly important in some forms of cancer, is discussed.

Isotachophoresis of nucleic acids in agarose gel rods.

A new method of electrophoresis (isotachophoresis in agarose gel rods) in which nucleic acid molecules are not separated but, oppositely, are brought together into one band, was elaborated. Heterogeneous in size DNA and RNA polymers present in a few milliliters of a solution at so low concentration that their isolation by other methods is hardly attainable and fraught with losses are brought together into one visible narrow band when put in a discontinuous electric field. Polynucleotides migrate in dilute (0.1%) semifluid agarose gel that permits easy quantitative isolation of the band of interest. Resulting DNA can be used directly in PCR. The suggested method for isolation of micro amounts of nucleic acids from dilute solutions can be applied to forensic and clinical research and cancer gene diagnostics by the analysis of fragmented circulating DNA from bodily fluids.

[Detection of genetic mutations in clinical specimens from cancer patients: comparison of NIRCA and SSCP scanning methods].

Specimens of tumor with K-RAS mutations were used to compare SSCP and NIRCA efficiencies in screening long target regions for dispersed point mutations. K-RAS mutations were detected in 5 out of 10 tumor tissue samples from colorectal cancer patients (in codon 12-4 and codon 13-1). Mutant alleles occurred most frequently in adenocarcinoma of the ascending colon and rectum. Both methods proved equally efficient. In certain situations, they may be combined or used as complementary. NIRCA is suitable for screening relatively long sequences (up to 1kb) while SSCP is less sophisticated, robust and allows for mutant bands to be extracted from polyacrylamide gel when required.

Detection of mutant k-ras sequences in the urine of cancer patients.

DNA fragments from apoptotic cells crossing the renal barrier retain their matrix functions, which allows PCR identification of mutant sequences in excreted DNA. We investigated the possibility of detecting k-ras mutations in urinary DNA of tumor patients (colon cancer). In some patients with k-ras codon 12 mutations in tumor cell DNA the same changes were detected in the urinary DNA. The possibility of using this approach for early diagnosis and monitoring of tumors is discussed.

[Analysis of DNA excreted from the body as an approach to diagnosis and monitoring tumor growth]. / Analiz ékskretiruemoi iz organizma DNK kak podkhod k vyiavleniiu rastushchei v organizme opukholi.

Proceeding from their early data showing that some portion of DNA originating from apoptotic cells can enter the blood stream and pass through the renal barrier by preserving its template capabilities, the authors analyzed urine DNA from 29 patients with colorectal cancer. PCR was used to compare DNA samples from the normal mucosa surrounding the tumor and from the urine collected just prior to surgery. Six microsatellite loci were studied with oligonucleotide primers. The following results were obtained: i) 3 cases showed differences in one of the studied loci in normal and tissue DNA; ii) some patients displayed changes in urine DNA microsatellite loci, namely: disappearance of some alleles (loss of heterozygocity) and appearance of new ones; iii) there were no differences in microsatellite patterns of lymphocytic DNA (taken as a control) and urine DNA in healthy donors. The findings are discussed in view of current concepts of tumor clonal heterogeneity and interpreted as a promising approach to diagnosing and monitoring tumor growth.

[Metabolic and structural properties of extrachromosomal DNA in mammalian cells]. / Metabolicheskie i strukurnye svoistva vnekhromosomnykh DNK v kletkakh mlekopitaiushchikh.

Discrete bands of free DNAs of approximately 25 kbp were detected in human cell cultures. According to electrophoretic shifts induced by single and double strand breaks, they from topological isoforms (supercoiled, open, and linear). Long-term labeling (24 h) of growing and quiescent cultured cells by [3H]thymidine indicates differences of free versus chromosomal DNAs including (i) significantly lower specific radioactivity in growing cells, (ii) higher specific radioactivity in quiescent cells, and (iii) resistance to fluorodeoxyuridine labeling. During apoptosis of cultured Namalwa cells, heterogeneous fragments are formed which differ from free DNAs. Crosslinking of nascent RNA with DNA template by 8-methoxypsoralen indicated slight transcriptional activity of free DNAs.

[The modifying effect of long-term administration of ascorbic acid with drinking water on asbestos-induced pleural carcinogenesis in Wistar rats]. / Modifikatsiia plevral'nogo asbestovogo kantserogeneza u krys Vistar dlitel'nym vvedeniem s pit'evoi vodoi askorbinovoi kisloty.

Ascorbic acid administered with drinking water, in a concentration of 2.5%, together with sucrose (1%) was found to significantly inhibit the development of mesothelial and pleural tumors induced in Wistar rats by asbestos treatment. Said agents, however, failed to influence spontaneous carcinogenesis.

Factors within the body determining the glycogen reserves in the tissues of rats with transplantable tumours.

Attempts have been made to determine the reason for the depletion of glycogen reserves in tumour-bearing rats. The possible roles of anorexia, competition for glucose by the tumour, and lack of hormonal control of glycogen biosynthesis have been investigated. The glycogen content of the liver, skeletal muscle, and brain, and the levels of glucose and the hormones corticosterone, insulin, and glucagon were determined in healthy rats which had been starved for various periods and in tumour-bearing rats carrying the fast-growing Zajdela ascites hepatoma or the slow-growing solid hepatoma 27. It was found that towards the terminal stages of tumour development there was an increase in the content of corticosterone and glucagon in the blood serum and also an increase in the glycogen reserves in skeletal muscle and brain despite the presence of hypoglycaemia and hypo-insulinaemia. There was at this time a sharp fall in the level of liver glycogen. It is shown that neither anorexia nor excessive competition for glucose by the tumour were the main reasons for liver glycogen depletion and hypoglycaemia. A strong correlation was observed, however, between the occurrence of anaemia and the loss of liver glycogen, which suggests that the former may be an important factor in the changes in host tissue observed in response to tumour growth.

Intracellular factors determining the deposition of glycogen. The role of gluconeogenesis.

The interrelationship between glycogen biosynthesis and do novo glucose formation in rats was studied by determining the oxaloacetic acid, ATP, and glycogen levels in the tissues of healthy starved rats and during the growth of transplantable hepatomas with different rates of growth. From the results obtained it was proposed that glycogen biosynthesis and de novo glucose formation are mutually enhanced and coupled processes. The validity of this conclusion was confirmed by in vitro model experiments involving the use of isolated-liver perfusion. Results of a multiple regression analysis indicated that the efficiency of gluconeogenesis and glycogen deposition is determined by the supply of oxygen to the liver, by the oxaloacetic acid content of this organ, and also by the degree of reduction of the glucose precursor. It was also shown that the ability of glucocorticoids and glucagon to enhance gluconeogenesis is dependent on the supply of oxygen to the liver. From the results of this in vivo and in vitro comparative study it is concluded that deposition of glycogen in the liver and possibly also in the brain is determined by the efficiency of gluconeogenesis.

Effect of putrescine on carbohydrate and lipid metabolism in rats.

Administration of putrescine (tetramethylenediamine) to healthy rats induced rapid and significant changes in carbohydrate and lipid metabolism. On the basis of changes in the stores of glycogen in the liver and muscles, and the concentration of glucose, nonesterified fatty acids, and total lipids in the blood, it appears that the sites of action of putrescine are the liver, muscle, and probably adipose tissue. A low dose of putrescine [30 mumol (100 g)-1 body weight] produced effects similar to those induced by glucagon and adrenaline. The increase in glycogen reserves and the sharp reduction in the levels of nonesterified fatty acids and total lipids seen after administration of high doses of putrescine [60-120 mumol (100 g)-1 body weight] are similar to the combined effects of insulin and glucocorticoids. The induction of hypoglycaemia, the reduction in the levels of nonesterified fatty acids and total lipids, and the increased reserves of glycogen in muscle that were seen with the higher doses occurred alongside a fall in blood insulin level. It is suggested that increased levels of polyamines in the blood might produce disturbances in the link between carbohydrate and lipid metabolism in cancer patients and tumour-bearing animals.