RESUMEN
The PowerPlex(®) ESI 16 Fast, ESI 17 Fast, ESX 16 Fast, and ESX 17 Fast Systems represent faster cycling versions (50min or less) of the PowerPlex(®) ESI and ESX Systems released by Promega in 2009 to accommodate the ENFSI and EDNAP groups' call for new STR multiplexes for Europe. In addition to amplification of purified DNA samples, these new faster cycling systems allow for direct amplification from single-source blood and buccal samples deposited on FTA(®) and nonFTA paper as well as from SwabSolution™ extracts of buccal swabs without the need for purification and quantitation. There are no changes to the autosomal primer pair sequences in the PowerPlex(®) ESI Fast and ESX Fast Systems compared to the original multiplexes, and full concordance at all autosomal loci and amelogenin was observed with data generated previously with the original PowerPlex(®) ESI and ESX Systems. This paper describes the developmental validation study performed on these new fast systems following guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and those of the DNA Advisory Board (DAB). Validation data demonstrate that these systems are sensitive for detecting low levels of DNA while also being capable of generating robust profiles from the high amount of input DNA present in direct-amplification samples. These systems are also tolerant to both high concentrations of PCR inhibitors as well as to slight variations in the final concentration of master mix and primer pair present in the amplification reaction that might be encountered due to pipetting error. The results of this validation study demonstrate that these systems may be used on multiple thermal cyclers and capillary electrophoresis platforms.
Asunto(s)
Dermatoglifia del ADN , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa Multiplex/instrumentación , Animales , Degradación Necrótica del ADN , Electroforesis Capilar , Humanos , Masculino , Reproducibilidad de los Resultados , Especificidad de la Especie , Manejo de Especímenes/métodosRESUMEN
Many complex viruses use an assembly pathway in which their genome is packaged into an empty procapsid which subsequently matures into its final expanded form. We utilized Pseudomonas phage 6, a well-established virus assembly model, to probe the plasticity of the procapsid maturation pathway. The 6 packaging nucleoside triphosphatase (NTPase), which powers sequential translocation of the three viral genomic single-stranded RNA molecules to the procapsid during capsid maturation, is part of the mature 6 virion but may spontaneously be dissociated from the procapsid shell. We demonstrate that the dissociation of NTPase subunits results in premature capsid expansion, which is detected as a change in the sedimentation velocity and as defects in RNA packaging and transcription activity. However, this dead-end conformation of the procapsids was rescued by the addition of purified NTPase hexamers, which efficiently associated on the NTPase-deficient particles and subsequently drove their contraction to the compact naive conformation. The resulting particles regained their biological and enzymatic activities, directing them into a productive maturation pathway. These observations imply that the maturation pathways of complex viruses may contain reversible steps that allow the rescue of the off-pathway conformation in an overall unidirectional virion assembly pathway. Furthermore, we provide direct experimental evidence that particles which have different physical properties (distinct sedimentation velocities and conformations) display different stages of the genome packaging program and show that the transcriptional activity of the 6 procapsids correlates with the number of associated NTPase subunits.
Asunto(s)
Bacteriófago phi 6/fisiología , Pseudomonas syringae/virología , Virión/fisiología , Ensamble de Virus , Bacteriófago phi 6/genética , Bacteriófago phi 6/ultraestructura , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virión/genética , Virión/ultraestructuraRESUMEN
The Y-chromosomal diversity among Finnish males is characterized by low diversity and substantial geographical substructuring. In a 12-locus data set (PowerPlexY), especially the eastern parts of the country showed low levels of variation, and the western, middle, and eastern parts of Finland differed from each other by their Y-short tandem repeat (STR) haplotype frequencies (Palo et al., Forensic Sci Int Genet 1:120-124, 2007). In this paper, we have analyzed geographical patterns of Y-STR diversity using both 12-locus (PowerPlexY) and 17-locus (Yfiler) data sets from the same set of geographically structured samples. In the larger data set, the haplotype diversity is significantly higher, as expected. The geographical distribution of haplotypes is similar in both data sets, but the level of interregional differences is significantly lower in the Yfiler data. The implications of these observations on the forensic casework are discussed.
Asunto(s)
Cromosomas Humanos Y , Dermatoglifia del ADN , Genética de Población , Secuencias Repetidas en Tándem , Finlandia , Variación Genética , Haplotipos , Humanos , MasculinoRESUMEN
Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids). This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis. At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors. In the case of the double-stranded RNA bacteriophage phi 6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4. In the present study, the purified P4 proteins from two other cystoviruses, phi 8 and phi 13, were characterized and compared with phi 6 P4. All three proteins are hexameric, single-stranded RNA-stimulated NTPases with alpha/beta folds. Using a direct motor assay, we found that phi 8 and phi 13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of phi 6 P4 requires association with the procapsid. This difference is explained by the intrinsically high affinity of phi 8 and phi 13 P4s for nucleic acids. The unidirectional translocation results in RNA helicase activity. Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.
Asunto(s)
ARN Bicatenario/química , ARN/química , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Bacteriófago phi 6/metabolismo , Cápside , Microscopía por Crioelectrón , Cystoviridae/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Cinética , Luz , Datos de Secuencia Molecular , Neutrones , ARN/metabolismo , ARN Helicasas/química , ARN Bicatenario/metabolismo , ARN Viral/química , Dispersión de Radiación , Espectrometría Raman , Factores de Tiempo , Uridina Trifosfato/farmacología , Ensamble de VirusRESUMEN
Bacteriophage phi6 has a segmented double-stranded RNA genome. The genomic single-stranded RNA (ssRNA) precursors are packaged into a preformed protein capsid, the polymerase complex, composed of viral proteins P1, P2, P4, and P7. Packaging of the genomic precursors is an energy-dependent process requiring nucleoside triphosphates. Protein P4, a nonspecific nucleoside triphosphatase, has previously been suggested to be the prime candidate for the viral packaging engine, based on its location at the vertices of the viral capsid and its biochemical characteristics. In this study we were able to obtain stable polymerase complex particles that are completely devoid of P4. Such particles were not able to package ssRNA segments and did not display RNA polymerase (either minus- or plus-strand synthesis) activity. Surprisingly, a mutation in P4, S250Q, which reduced the level of P4 in the particles to about 10% of the wild-type level, did not affect RNA packaging activity or change the kinetics of packaging. Moreover, such particles displayed minus-strand synthesis activity. However, no plus-strand synthesis was observed, suggesting that P4 has a role in the plus-strand synthesis reaction also.