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1.
Sci Rep ; 10(1): 5476, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32214156

RESUMO

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.


Assuntos
Análise Mutacional de DNA/métodos , Sondas de DNA , Mutação/genética , Reação em Cadeia da Polimerase/métodos , Neoplasias do Colo/genética , Humanos , Desnaturação de Ácido Nucleico , Proteínas Proto-Oncogênicas p21(ras)/genética
2.
Mol Biol (Mosk) ; 51(1): 50-58, 2017.
Artigo em Russo | MEDLINE | ID: mdl-28251966

RESUMO

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.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Análise Mutacional de DNA , GTP Fosfo-Hidrolases/genética , Proteínas de Membrana/genética , Desnaturação de Ácido Nucleico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Colorretais/genética , Humanos , Neoplasias Pulmonares/genética , Melanoma/genética , Mutação , Reação em Cadeia da Polimerase
3.
J Med Life ; 9(3): 249-254, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27974928

RESUMO

The term diabetic foot is usually used to indicate advanced foot pathology (complex clinical situations correlating diabetic foot ulcers, diabetic foot infections, Charcot foot, and critical limb ischemia). The early recognition of the etiology of these foot lesions is essential for the therapeutic decision in order to achieve a good functional result. Several surgical procedures involving the foot have been developed in order to promote healing and avoid complications. Traditionally, surgery has been performed in an open way. The literature regarding the performance and efficacy of classical osteotomies and arthrodesis is inconsistent. This can be attributed to several variables, such as differences in patient clinical aspects and the panel of surgical techniques utilized. As with other surgical specialties, fluoroscopic imaging and minimally invasive tools are now being incorporated in these procedures. The use of high speed burrs associated with specialized osteosynthesis implants, offers several advantages over classical techniques. The ability to associate these gestures to complex protocols is beginning to be currently developed. The respect for the soft tissues is considered one of the first advantages. Despite the limited time since they were introduced in clinical practice, functional results seemed to be consistent, supporting the use of this technology.


Assuntos
Pé Diabético/cirurgia , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Artrodese , Humanos , Osteotomia
4.
J Med Life ; 8 Spec Issue: 87-93, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26361518

RESUMO

Percutaneous techniques are currently more and more used in many surgical procedures on the soft tissues and bones of the foot. Practical advantages include lower complication rates and faster recovery times. Potential disadvantages are related to the need for specific equipment and extensive learning curve. One of the most frequent techniques involves a combination of chevron osteotomy of the first metatarsal with osteotomy of the first phalanx, both internally fixated. Lateral metatarsal misalignment and toe deformities can also be addressed by percutaneous treatment, with lower morbidity rates than open techniques. The most commonly performed percutaneous procedures are described, with their current indications, outcomes, and recent developments.


Assuntos
Ossos do Metatarso/cirurgia , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Fluoroscopia , Humanos , Procedimentos Cirúrgicos Minimamente Invasivos/instrumentação , Osteotomia , Tração
5.
Mol Biol (Mosk) ; 48(6): 999-1007, 2014.
Artigo em Russo | MEDLINE | ID: mdl-25845241

RESUMO

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.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , DNA Satélite/genética , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/genética , RNA Neoplásico/genética , Biomarcadores Tumorais/sangue , Neoplasias do Colo/sangue , Neoplasias do Colo/patologia , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , DNA Satélite/sangue , Humanos , Células MCF-7 , RNA Mensageiro/sangue , RNA Neoplásico/sangue , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Microambiente Tumoral/genética
6.
Mol Biol (Mosk) ; 46(3): 461-8, 2012.
Artigo em Russo | MEDLINE | ID: mdl-22888635

RESUMO

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.


Assuntos
Análise Mutacional de DNA/métodos , DNA/genética , Mutação , Benzotiazóis , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Impressões Digitais de DNA/métodos , Diaminas , Reações Falso-Negativas , Feminino , Corantes Fluorescentes , Formaldeído , Humanos , Desnaturação de Ácido Nucleico , Compostos Orgânicos , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Inclusão em Parafina , Polimorfismo de Fragmento de Restrição , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinolinas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Fixação de Tecidos , Temperatura de Transição
7.
Biochemistry (Mosc) ; 74(11): 1285-8, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19916946

RESUMO

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.


Assuntos
DNA/química , Eletroforese em Gel de Ágar/métodos , RNA/química , Análise Química do Sangue , DNA/genética , Humanos , RNA/genética
8.
Vopr Onkol ; 53(5): 549-53, 2007.
Artigo em Russo | MEDLINE | ID: mdl-18154119

RESUMO

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.


Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , Genes ras , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Adulto , Idoso , Feminino , Genes ras/genética , Humanos , Masculino , Pessoa de Meia-Idade
10.
Bull Exp Biol Med ; 131(3): 283-4, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11427923

RESUMO

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.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/urina , Genes ras , Mutação , Códon , Neoplasias do Colo/diagnóstico , Humanos , Reação em Cadeia da Polimerase
11.
Vestn Ross Akad Med Nauk ; (7): 24-7, 2000.
Artigo em Russo | MEDLINE | ID: mdl-10961144

RESUMO

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.


Assuntos
Adenocarcinoma/urina , Neoplasias Colorretais/urina , DNA de Neoplasias/urina , Mucosa Intestinal/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Neoplasias Colorretais/patologia , Primers do DNA/química , Progressão da Doença , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Urinálise
12.
Clin Chem ; 46(8 Pt 1): 1078-84, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10926886

RESUMO

BACKGROUND: Cell-free DNA from dying cells recently has been discovered in human blood plasma. In experiments performed on animals and humans, we examined whether this cell-free DNA can cross the kidney barrier and be used as a diagnostic tool. METHODS: Mice received subcutaneous injections of either human Raji cells or purified (32)P-labeled DNA. DNA was isolated from urine and analyzed by measurement of radioactivity, agarose gel electrophoresis, and PCR. In humans, the permeability of the kidney barrier to polymeric DNA was assessed by detection in urine of sequences that were different from an organism bulk nuclear DNA. RESULTS: In the experiments on laboratory animals, we found that approximately 0.06% of injected DNA was excreted into urine within 3 days in a polymeric form and that human-specific ALU: sequences that passed through the kidneys could be amplified by PCR. In humans, male-specific sequences could be detected in the urine of females who had been transfused with male blood as well as in DNA isolated from urine of women pregnant with male fetuses. K-ras mutations were detected in the urine of patients with colon adenocarcinomas and pancreatic carcinomas. CONCLUSIONS: The data suggest that the kidney barrier in rodents and humans is permeable to DNA molecules large enough to be analyzed by standard genetic methodologies.


Assuntos
DNA/urina , Animais , Transfusão de Sangue , Morte Celular , Neoplasias Colorretais/química , Neoplasias Colorretais/patologia , Neoplasias Colorretais/urina , DNA/genética , Eletroforese em Gel de Ágar , Feminino , Feto/química , Genes ras , Genoma , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Mutação , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/urina , Reação em Cadeia da Polimerase , Gravidez , Células Tumorais Cultivadas , Cromossomo Y
14.
FEBS Lett ; 432(3): 158-62, 1998 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-9720916

RESUMO

Distinct DNA fractions (fr-DNAs), moving separately from bulk DNA, were revealed by field inversion gel electrophoresis of DNA from intact cells lysed and deproteinized in agarose plugs. These fr-DNAs (approximately 2% of the total DNA) were ubiquitously present in nuclei of all mammalian cells studied, including human normal and tumor tissues, and showed a typical electrophoretic pattern (three bands with constant mobilities termed a-, b-, and c-DNA). Characteristic mobility shifts induced by gamma irradiation of a- and b-DNAs suggest their non-linear conformation. In fact, electron microscopy of a- and b-DNAs from Namalwa cells revealed rosette-like structures stabilized by a central protease-resistant knob. Comparative PCR analysis revealed qualitative differences between genomic fingerprints of a- and b-DNAs on the one hand and chromosomal DNA on the other. The results obtained suggest that fr-DNAs originate either from some specific chromatin regions due to non-random cleavages or from an autonomous intranuclear structure, not identified as yet.


Assuntos
DNA de Neoplasias/química , Apoptose/genética , Fracionamento Químico , Fragmentação do DNA/genética , DNA de Neoplasias/genética , DNA de Neoplasias/ultraestrutura , Eletroforese em Gel de Campo Pulsado , Humanos , Microscopia Eletrônica , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Sefarose , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/citologia
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