RESUMEN
To decrease dependence of effectiveness of isolation of nucleic acids of composition and amount of applied sample a kit was developed for hybridization extraction of DNA HBV RNA HCV and RNA HIV from blood serum in two formats--using up to 250 mkl and up to 1 ml of sample. This kit, in complex with kits for detection using polymerase chain reaction technique in real-time, forms a test characterized by high analytical sensitivity i.e. HBV50 copies per ml, HCV37.5 copies per ml, HIV 13 copies per ml. The developed kit for extraction of target nucleic acids permits to get rid of total DNA and inhibited effect of heparin. It can be adapted for application wit factors B and automated stations of sample preparation.
Asunto(s)
ADN Viral , Infecciones por VIH/sangre , Hepatitis B/sangre , Hepatitis C/sangre , Juego de Reactivos para Diagnóstico , ADN Viral/sangre , ADN Viral/aislamiento & purificación , VIH-1 , Hepacivirus , Virus de la Hepatitis B , Humanos , ARN Viral/sangre , ARN Viral/aislamiento & purificaciónRESUMEN
Polymerase chain reaction (PCR) is widely used to detect specific DNA sequences for purposes of microbial identification, clinical diagnosis, and basic research. The most pernicious problem plaguing this technique is contamination of PCR reagents with previously amplified material. We propose a useful tool for PCR reagent purification from contaminating nucleic acid using DEAE-cellulose and present the analysis of this technique for both decontamination efficiency and an effect on the reagent activity. We also show the suitability of the proposed approach for decontamination of the Taq polymerase, monoclonal antibodies to Taq polymerase, and Moloney murine leukemia virus (M-MLV) reverse transcriptase.
Asunto(s)
DEAE-Celulosa/química , Reacción en Cadena de la Polimerasa/métodos , Descontaminación , Indicadores y Reactivos/aislamiento & purificación , Polimerasa Taq/metabolismoRESUMEN
A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5'-3'-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared. Primer elongation (polymerase reaction) is detected by changes in SYBR Green I fluorescence upon binding to dsDNA during reaction; nuclease activities are detected by changes in fluorescence due to cleavage of the probe, containing the reporter fluorophore and fluorescence quencher, and hybridized in advance to the template single-stranded region. It was also shown that the method can be used for determination of relative activities of DNA polymerase preparations, estimation of temperature-time dissociation parameters of polymerase complexes with specific antibodies to its active center, and analysis of effects of inhibitors and activators of different nature on reaction rates of dsDNA polymerization and 5'-3'-exonuclease cleavage by polymerase. The method can be also used for estimation of endonuclease activities of DNA polymerases.
Asunto(s)
Bioquímica/métodos , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Exonucleasas/aislamiento & purificación , Cartilla de ADN/química , Cartilla de ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Endonucleasas/química , Endonucleasas/aislamiento & purificación , Endonucleasas/metabolismo , Exonucleasas/química , Exonucleasas/metabolismo , Fluorescencia , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Polimerasa Taq , Temperatura , Moldes Genéticos , Factores de TiempoRESUMEN
N.I. Vavilov's theory of the centres of origin of cultivated plants is still as a methodological base for studying processes of domestication. This theory, along with archaeological, botanical, and molecular genetic data, helped to determine the most probable regions where the principal cultivated species were introduced into culture. The search for the mechanisms of origins of morphological changes in the course of domestication is one of evolutionary biology's oldest problems. Employing novd molecular biological data for solving this problem provides a key to the understanding of these mechanisms and helps to reconstruct a possible scenario of how the traits that distinguish domesticated plants from their wild relatives got involved into selection. It is hypothesized that distinct physiological and morphological differences important to ancient agrarians originated from quantitative and/or qualitative changes in the loci regulating the programmes of plant ontogeny. This hypothesis helps to reveal the mechanisms of origin of certain traits in the course of domestication, to study the connection between the direction of selection and how such traits are genetically controlled, and to outline particular ways for further detailed studies of the evolutionary aspect of domestication processes.