RESUMEN
BACKGROUND: Complete following existing guidelines for management of acute coronary syndrome (ACS) is known to be associated with better outcomes. Partly this is explained by lesser adherence to recommendations in high risk patients. Aim of our study was to assess relationship between degree of following current guidelines and in hospital outcomes independently from initial assessment of risk. METHODS: Each key recommendation from guidelines issued between 2008 and 2011 (13 for STE ACS, 12 for NSTE ACS) was given weight of 1. Sum of these units constituted index of guideline adherence (IGA). IGA was retrospectively calculated for 1656 patients included in Russian independent ACS registry RECORD-2 (7 hospitals, duration 04.2009 to 04.2011). The patients were divided into 2 groups according to quartiles of IGA distribution: 1) low adherence group (quartiles I-II); 2) high adherence group (quartiles III-IV). RESULTS: In low adherence compared with high adherence group there were significantly more patients more or equal 65 years (=0.0007), with chronic heart failure [CHF] (<0.0001), previous stroke (<0.0001), atrial fibrillation [AF] (=0.0002), Killip class more or equal II (=0.0065), high risk of death by GRACE score (=0.035). Inhospital mortality was 9.3 and 2.4% in low and high adherence group, respectively (p<0.0001). The following independent predictors of inhospital death were identified: IGA quartiles I-II (odds ratio [OR] 4.0; 95% confidence interval [CI] 2.3-7.1; <0.0001), high GRACE score (OR 3.3; 95% CI 1.8-6.0; <0.0001), admission systolic BP less or equal 100 mm Hg (OR 3.1; 95% CI 1.8-5.4; <0.0001), admission serum glucose more or equal 8 mmol/l (OR 2.9; 95% CI 1.8-4.7; <0.0001), age more or equal 65 years (OR 2.3; 95% CI 1.3-4.0; =0.005), ST elevation more or equal 1 mm on first ECG (OR 1.7; 95% CI 1.1-2.5; =0.013). From groups with low and high adherence to guidelines we selected pairs of patients (n=588) with similar (or close) age, type of ACS, GRACE score, Killip class, presence of other important risk factors (CHF, AF, previous stroke), and formed 2 equal subgroups without significant differences in important demographic, anamnestic, clinical and laboratory data. Hospital mortality was 7.8 and 2.7% in low and high adherence subgroup, respectively (p<0.0001). CONCLUSIONS: In RECORD-2 ACS registry low adherence to guidelines was more frequent among high risk patients and was independent predictor of inhospital death. Association between degree of guidelines adherence and outcomes persisted after equalizing groups by some factors of risk of mortality.
Asunto(s)
Síndrome Coronario Agudo , Técnicas de Diagnóstico Cardiovascular , Adhesión a Directriz , Revascularización Miocárdica/métodos , Evaluación de Procesos y Resultados en Atención de Salud , Síndrome Coronario Agudo/diagnóstico , Síndrome Coronario Agudo/mortalidad , Síndrome Coronario Agudo/terapia , Anciano , Manejo de la Enfermedad , Femenino , Adhesión a Directriz/normas , Adhesión a Directriz/estadística & datos numéricos , Mortalidad Hospitalaria , Hospitalización/estadística & datos numéricos , Humanos , Masculino , Guías de Práctica Clínica como Asunto , Sistema de Registros/estadística & datos numéricos , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Federación de Rusia/epidemiología , Índice de Severidad de la EnfermedadRESUMEN
AIM: Genotype characteristic and determination of serological properties of Vibrio cholerae nonO1/nonO139 strains that caused diseases in population of Rostov region from 2000 to 2009. MATERIALS AND METHODS: 15 clinical strains of V. cholerae nonO1/nonO139 were studied. Serotyping was performed by using a kit of monospecific typing sera against serogroup 02-084 cholera vibrios obtained from Rostov Research Institute for Plague Control, PCR and VNTR-genotyping--by using specific primers described in scientific publications and constructed by us. RESULTS: Serologic features of strains are very diverse and strains contain various combination of pathogenicity factor genes that seem to be interchangeable. Similar pattern was observed for VNTR-genotyping. Distribution of the examined strains by VNTR-genotyping did not correlate with either PCR-genotyping data or serotyping, or place and time of isolation. CONCLUSION: The data obtained indicates a lack of general source of human infection even in the same location and time period. On the other hand, serological and genotypic features of V. cholerae nonO1/nonO139 may undergo changes in the process of staying in the macro organism or environment due to high plasticity of their genome.
Asunto(s)
Cólera/epidemiología , Cólera/microbiología , Vibrio cholerae O139/clasificación , Vibrio cholerae no O1/clasificación , Toxina del Cólera/genética , Brotes de Enfermedades , Genes Bacterianos , Genotipo , Humanos , Repeticiones de Minisatélite/genética , Federación de Rusia/epidemiología , Serotipificación , Vibrio cholerae O139/genética , Vibrio cholerae O139/aislamiento & purificación , Vibrio cholerae no O1/genética , Vibrio cholerae no O1/aislamiento & purificación , Factores de Virulencia/genéticaRESUMEN
We have developed a simple method for fast analysis of single nucleotide polymorphisms and identification of target clones from cloned complex PCR products. The method utilizes Kamchatka crab duplex-specific nuclease and universal fluorescent probe and is alternative to laborious screening procedures using radioactive probes, restriction analysis followed by gel electrophoresis or expensive sequencing. The method efficacy was demonstrated in several model experiments.
Asunto(s)
Braquiuros/enzimología , Análisis Mutacional de ADN/métodos , ADN/genética , Desoxirribonucleasas/química , Polimorfismo de Nucleótido Simple/genética , Animales , Proteínas de Ciclo Celular/análisis , Clonación Molecular , ADN/química , Humanos , Proteínas Nucleares/análisis , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/análisis , Receptores de Antígenos de Linfocitos T/químicaRESUMEN
High contents of non-coding RNA in total bacteria RNA complicates considerably transcriptome analysis using standard approaches like high-throughput sequencing, gene expression profiles, subtractive hybridization. We suggest a procedure of preparation of bacterial cDNA for transcriptomics that includes rRNA and tRNA depletion with preservation of relative abundance of coding sequences. The method is based on the second order hybridization kinetics and unique properties of Kanchatka crab duplex-specific nuclease. The method efficacy was demonstrated on a model experiments.
Asunto(s)
ADN Complementario/aislamiento & purificación , Perfilación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Genoma Bacteriano , Células Procariotas/química , ARN no Traducido/químicaRESUMEN
Until recently, the production of reactive oxygen species by NADPH oxidase has been considered only in the context of the oxidative damage to pathogens inside the phagosome. However, homologues of phagocytic NADPH oxidase have been found in almost all cell types, where they produce hydrogen peroxide and thereby regulate the initial intracellular stages of MAP kinase cascades. In the present work, the activation of two MAP kinase cascades, p38 and Erk1/2, during phagocytosis has been studied. It was found that phagocytosis activates both cascades. The activation of Erkl/2 is dependent, and the activation of p38 is not dependent, on the activity of NADPH oxidase. Thus, it can be stated that the activation of MAP kinases in phagocytes during phagocytosis occurs by a mechanism similar to that operating in nonphagocytizing cells, indicating the universality of the function of NADPH oxidases in different cell types.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , NADPH Oxidasas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Activación Enzimática , Sistema de Señalización de MAP Quinasas , Ratones , Fagocitos/metabolismo , FagocitosisRESUMEN
Case histories of 147 patients with new-onset destructive pulmonary tuberculosis (PT) (infiltrative, disseminated, fibrocavernous) with the pathogen of the disease showing multidrug resistance (MDR) were analyzed, by evaluating the efficiency of treatment involving sputum abacillation after termination of the inpatient stage of treatment. A control group included 220 patients with PT of the similar lesion and clinical forms, who preserved drug resistance (DR) to antibacterial agents. Most patients with MDR disseminated destructive PT are young people aged 36.6 +/- 1.6 years, this disease-associated disabled individuals (65.3%), alcoholics (48.3%), opium addicts (11.5%), ex-prisoners (26.5%), single, homeless; more frequently suffer from gastrointestinal (40%) and chronic nonspecific lung (24.5%) diseases. The course of MDR PT is significantly more commonly complicated by the development of respiratory failure (48.9%) and hemoptysis (6.1%) (in DR PT 19.1 and 2.3%, respectively). In terms of negative smear tests and the results of sputum cultures, the abacillation rates were 70.1 and 67.3%, respectively (in DR PT, these were 88.18 and 86.34%, p = 0.01). A negative reaction of sputum occurred in 42.1% of patients at 2-3 months of treatment while its culture did in 46.2% at 4-5 months. In the control group wherein drug sensitivity of M. tuberculosis was preserved, sputum abacillation occurred in the larger proportion of the patients within the first 2 months (in 68.6 and 56.3% of cases with sputum smear and culture, respectively). The efficiency of inpatient therapy is greatly affected by short-term treatment caused by voluntary withdrawal and irregular uses of antituberculous drugs, mainly due to alcoholization.
Asunto(s)
Atención Ambulatoria/métodos , Antituberculosos/uso terapéutico , Cooperación del Paciente/estadística & datos numéricos , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Pulmonar/tratamiento farmacológico , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores Socioeconómicos , Resultado del Tratamiento , Adulto JovenRESUMEN
The Val-Val-Tyr-Pro-Asp bone marrow peptide (MP-5) and an analogue (MP-5-Lys) were synthesized. Fluorescent derivatives, Ftc-MP-5 and MP-5-Lys(Ftc), were prepared. The biological activity of MP-5 and MP-5-Lys was studied in vitro and in vivo. The MP-5 peptide caused 60-84% inhibition of growth of the following mouse cancers: lymphatic leukemia P-388, melanoma B-16, and cervical carcinoma CUC-5. These peptides also restored functional activity of T lymphocytes that was inhibited by metabolic products of the HL-60 leukemic cell line. MP-5-Lys(Ftc) was shown to preserve the functional properties of MP-5 toward T lymphocytes, but Ftc-MP-5 was practically inactive.
Asunto(s)
Antineoplásicos/síntesis química , Colorantes Fluorescentes/síntesis química , Factores Inmunológicos/síntesis química , Oligopéptidos/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Ratones , Oligopéptidos/química , Oligopéptidos/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Myelopeptide-4 (MP-4) (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), inducing the terminal differentiation of HL-60 leukemia cells, was labeled with fluorescein isothiocyanate. The specific binding of this modified peptide to the surface of HL-60 cells and its ability to penetrate into the cells were studied. It was shown by cytometry and confocal microscopy to be bound on the HL-60 cell surface, to penetrate into their cytoplasm, and finally to concentrate around the cell nucleus. These phenomena are probably necessary for the exhibition of MP-4 differentiating activity.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Citoplasma/metabolismo , Oligopéptidos/farmacología , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacología , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/farmacología , Células HL-60 , Humanos , Microscopía Confocal , Microscopía Fluorescente , Oligopéptidos/química , Oligopéptidos/metabolismo , Transporte de Proteínas/efectos de los fármacosRESUMEN
The bone marrow myelopeptide MP-2 (Leu-Val-Val-Tyr-Pro-Trp), exhibiting antitumor activity, and its retro-analogue (Trp-Pro-Tyr-Val-Val-Leu) were synthesized, and their properties were studied. The in vitro and in vivo activities of retro-MP-2 were comparable with those of MP-2. Both peptides equally restored the functional activity of T-lymphocytes inhibited by toxins released by HL-60 cells and inhibited by 70-82% the growth of various types of transplantable solid tumors: Ca-755 adenocarcinoma of the mammary gland, Lewis adenocarcinoma of the lung, and S180 sarcoma. The positions and intensities of the Cotton effects in CD spectra of the MP-2 peptide and its retro-analogue in various solvents are almost indistinguishable. The positions of extrema and integral intensities of the amide I and amide A bands in IR spectra of both peptides were practically identical. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Adyuvantes Inmunológicos/química , Animales , Células HL-60 , Humanos , Ratones , Oligopéptidos/químicaRESUMEN
We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.
Asunto(s)
ADN Complementario/química , ADN de Cadena Simple/química , Biblioteca de Genes , Animales , Braquiuros , Endonucleasas/química , Humanos , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
The cDNAs encoding the genes of new proteins homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria were cloned. Two green fluorescent proteins from one un-identified anthojellyfish, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthojellyfish were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Hidrozoos/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas Fluorescentes Verdes/genética , Hidrozoos/genética , Datos de Secuencia Molecular , Espectrometría de FluorescenciaRESUMEN
A family of genes of the agamic race of planarian Girardia tigrina were described that encode proteins that belong to the superfamily of C-type lectins and were demonstrated to have a unique domain organization. The genes are differentially expressed in the planarian body. The protein products of at least two genes (scarf2 and gtlec1) are expressed in specifically differentiated gland cells of the planarian and secreted into the environment through long cell necks. A comparison of the results obtained by electron microscopy and immunohistochemistry with literature data allows the assignment of these cells to the group of adhesion glands. The observation of the regeneration of the cell necks in normal and artificial two-headed planaria indicated that the dorsoventral contact at the edge of the head part of the planarian body directs and maintains the growth of the gtLec1-producing cell necks during regeneration. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.
Asunto(s)
Proteínas del Helminto/genética , Lectinas Tipo C/genética , Planarias/genética , Regeneración/fisiología , Secuencia de Aminoácidos , Animales , Proteínas del Helminto/metabolismo , Inmunohistoquímica , Intrones , Lectinas Tipo C/metabolismo , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Planarias/metabolismo , Planarias/ultraestructura , Regeneración/genéticaRESUMEN
The previously described gene sip1 belongs to transcription factors of the zinc finger family. It has been ascertained recently that this gene is involved in TGF signaling cascade. Mutations in human gene sip1 cause Hirschprung syndrome. The expression of gene sip1 during embryonic mouse development was studied by in situ hybridization and immunostaining. Starting at E12.5, sip1 transcripts are present in a number of tissues: in the cortical plate, ventricular zone of the basal ganglion, thalamus, pons and midbrain, in specific nuclei of the brain stem and in the dorsal part of the spinal cord. In the developing cerebral cortex, sip1 expression is region-specific. In the brain of adult mice, sip1 expression is mostly detected in hippocampus, dentate gyrus, and white matter of the neocortex. Sip1 protein expression in the cerebral cortex is mostly confined to glutamatergic neurons.
Asunto(s)
Sistema Nervioso Central/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Animales , Secuencia de Bases , Sistema Nervioso Central/embriología , Clonación Molecular , Cartilla de ADN , ADN Complementario , Hibridación in Situ , RatonesRESUMEN
The gene of a new red fluorescent protein zoan2RFP from a coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the Aequorea victoria jellyfish, was cloned. At early maturation stages, zoan2RFP exhibits a green fluorescence, which then turns into the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.
Asunto(s)
Antozoos/genética , Colorantes Fluorescentes/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Escherichia coli/genética , Fluorescencia , Proteínas Fluorescentes Verdes , Cinética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Mutación , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/química , Homología de Secuencia de Aminoácido , Espectrometría de FluorescenciaRESUMEN
A combination of suppression subtraction hybridization (SSH) and a new technique of mirror orientation selection (MOS) was used to compare the total DNA for two, sexual (SR) and asexual (AR), races of freshwater planarian Giradia tigrina. Several race-specific DNA fragments were found. A new element termed planarian extrachromosomal virus-like element (PEVE) was revealed in AR. The PEVE genome contains two unique regions, Ul and Us, which are flanked by inverted repeats. Two variants observed for the PEVE genome differ in combination of single- and double-stranded regions corresponding to Ul and Us. The PEVE genome codes for two helicases, one homologous to the circovirus replication initiation protein (Rep) and one corresponding to the helicase domain of papillomavirus E1. PEVE is nonuniformly distributed though the planarian body and is possibly replicated only in certain parenchymal cells.
Asunto(s)
Elementos Transponibles de ADN , Proteínas de Unión al ADN , Herencia Extracromosómica/genética , Planarias/genética , Secuencia de Aminoácidos , Animales , Cromosomas/genética , Circovirus/genética , Clonación Molecular , ADN Helicasas/genética , ADN de Cadena Simple , Agua Dulce , Orden Génico , Variación Genética , Hibridación in Situ/métodos , Datos de Secuencia Molecular , Papillomaviridae/genética , Planarias/fisiología , Secuencias Repetitivas de Ácidos Nucleicos , Reproducción/genética , Homología de Secuencia de Aminoácido , Transactivadores/genética , Virus/genéticaRESUMEN
Peptide Leu-Val-Cys-Tyr-Pro-Gln, identical to the bone marrow peptide MP-3, and its Val3 and Ser3 analogs, lacking SH-group, were synthesized by conventional methods of peptide chemistry in solution and, along with the MP-3 S-S-dimerization product, were studied with respect to their effect on the macrophage phagocytic activity. It was shown that the activity was only enhanced by peptide MP-3, which demonstrated the essential role of the SH-group in this function. The dimer analog of MP-3, unlike dimer analogs of other monocysteine-containing peptides, glutathione and HP5b, did not exhibit the inhibitory effect.
Asunto(s)
Activación de Macrófagos/efectos de los fármacos , Oligopéptidos/farmacología , Compuestos de Sulfhidrilo/farmacología , Secuencia de Aminoácidos , Médula Ósea/química , Cromatografía Líquida de Alta Presión , Oligopéptidos/síntesis química , Oligopéptidos/química , Compuestos de Sulfhidrilo/síntesis química , Compuestos de Sulfhidrilo/químicaRESUMEN
The results of the study on the role of some wild mammals and birds as feeding sources of for ticks Hyalomma marginatum, the main vectors of Crimean haemorrhagic fever virus in the Stavropol Territory, in the preimago phases of their development are presented. These phases (larvae and nymphs) were found on rooks, hooded crows, partridges, European brown hares and eared hedgehogs. The examination of domestic fowl resulted in finding larvae and nymphs in small amounts on turkeys. According to the data of the epizootological survey carried out in summer and autumn of 2000 in the Stavropol Territory, rooks and, to a lesser extent, hares were found to be the main feeding sources for ticks in the preimago phases. Rodents seemed to be of minimal importance as feeding sources under the conditions of the Stavropol Territory. Of all animals, rooks must be the main object in the epizootological survey of the territory.
Asunto(s)
Vectores Arácnidos , Aves/parasitología , Liebres/parasitología , Virus de la Fiebre Hemorrágica de Crimea-Congo , Fiebre Hemorrágica de Crimea/transmisión , Ixodes , Animales , Erizos , Larva , Ninfa , Vigilancia de la Población , Federación de Rusia , Estaciones del Año , Pájaros Cantores , PavosRESUMEN
The primary structure of the crusta gene encoding alpha-latrocrustoxin (alpha-LCT), a high molecular mass neurotoxin specific to crustaceans, was determined in the black widow spider Latrodectus mactans tredicimguttatus genome. The total length of the sequenced DNA was 4693 bp. The structural part of the black widow spider chromosome gene encoding alpha-LCT does not contain introns. The sequenced DNA contains a single extended open reading frame (4185 bp) and encodes a protein precursor of alpha-LCT, comprising 1395 aa. We assume the Met residue at position -10 relative to the N-terminal residue of Glu1 of the mature toxin to be the first one in the protein precursor. The calculated molecular mass of the precursor (156147 Da) exceeds that of the mature toxin by approximately 30 kDa. These data are in agreement with the notion that over the course of maturation the protein precursor undergoes double processing--cleavage of a decapeptide from the N-terminal part and of a approximately 200-aa fragment from the C-terminal part. alpha-LCT displayed a number of imperfect ankyrin-like repeats and areas of structural homology with earlier studied latrotoxins; the highest homology degree (62%) was revealed with alpha-latroinsectotoxin (alpha-LIT).
Asunto(s)
Araña Viuda Negra/genética , Venenos de Araña/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Genes de Insecto , Datos de Secuencia Molecular , Neurotoxinas/genética , Alineación de Secuencia , Análisis de SecuenciaAsunto(s)
Enfermedad de Alzheimer/genética , Peptidil-Dipeptidasa A/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Cartilla de ADN/genética , Femenino , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , Medición de Riesgo , Factores de RiesgoRESUMEN
The selective suppression of the polymerase chain reaction and methods based upon it (construction of cDNA libraries from low amounts of biological material, subtractive hybridization and differential display of mRNA, fast cloning of full-size cDNA, chromosome walking, cloning in vitro, and others) are reviewed. These methods display a high effectiveness and, taken together, enable intricate DNA analyses to be performed--from the search for nontrivial sequences to the total sequencing of the corresponding genes.