RESUMEN
A systematic characterization of the effects of important physical parameters on the sensitivity and specificity of methods in searching for unknown base changes (mutations or single nucleotide polymorphisms) over a relatively long DNA segment has not been previously reported. To this end, we have constructed a set of molecules of varying G+C content (40, 50, and 60% GC) having all possible base changes at a particular location - the "DNA toolbox". Exhaustive confirmatory sequencing demonstrated that there were no other base changes in any of the clones. Using this set of clones as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base mutated to all other bases were generated. The behavior of these constructs in manual and automated heteroduplex analysis was analyzed as a function of the size and overall base content of the fragment, the nature and location of the base change. Our results show that in heteroduplex analysis, the nature of the mismatched base pair is the overriding determinant for the ability to detect the mutation, regardless of fragment length, GC content, or the location of the mutation.
Asunto(s)
ADN Viral/análisis , Mutación , Ácidos Nucleicos Heterodúplex/análisis , Estudios de Evaluación como AsuntoRESUMEN
GelStar nucleic acid gel stain can be used for sensitive fluorescent detection of both double-stranded (ds) and single-stranded (ss) DNAs, oligonucleotides and RNA in gels. The stain can be added to agarose gels at casting for immediate imaging after electrophoresis or can be used after electrophoresis with both agarose and acrylamide gels. GelStar stain is highly fluorescent only when bound to nucleic acids thus giving superior signal-to-noise ratios and obviating the need to destain the gel. The detection limits of GelStar strain are 20 pg for dsDNA, 25 pg for ssDNA and 10 ng for native or glyoxal-treated RNA.
Asunto(s)
Colorantes , Ácidos Nucleicos/análisis , Biotecnología , Análisis Mutacional de ADN , Electroforesis en Gel de Agar , Etidio , Estudios de Evaluación como Asunto , Colorantes Fluorescentes , Geles , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Sensibilidad y Especificidad , Secuencias Repetidas en TándemRESUMEN
Accurate resolution of PCR products in the range of 15-40 kb may be obtained in agarose gels without pulsed field electrophoresis. A gel of 0.3% SeaKem Gold agarose cast on GelBond support film provides good resolution and sufficient get strength to reliably allow staining and photography. This paper describes a test system for Long PCR and demonstrates analysis of the PCR products on a gel run under standard low-voltage electrophoresis conditions.
Asunto(s)
ADN Viral/análisis , Electroforesis en Gel de Agar/métodos , Reacción en Cadena de la Polimerasa , Bacteriófago lambda/genética , Electroforesis en Gel de Agar/instrumentación , Magnesio/administración & dosificación , Fotograbar , Coloración y Etiquetado , Moldes GenéticosRESUMEN
Electrophoresis in polyacrylamide gels is one of the most powerful tools used for the analysis of proteins. However, this technique is not widely used for protein purification for a variety of reasons such as the following: less than quantitative recoveries; involved, time-consuming methodologies; and impurities in the protein preparations from gel-polymerization by-products that can modify the proteins and interfere with subsequent experiments. As an alternative, we have developed a simple and quantitative recovery procedure for proteins separated by electrophoresis in the all-agarose ProSieve gel system. Using this procedure, greater than 90% of each protein examined was recovered, and these proteins were unaffected by the recovery procedure.