RESUMEN
The number of different fluorescent colors that can be distinguished in a PCR screening assay restricts the number of different targets that can be detected. If only six colors can be distinguished, and the probe for each target is labeled with a unique color, then only six different targets can be identified. Yet, it is often desirable to identify more targets. For instance, the rapid identification of which bacterial species (if any) is present in a patient's normally sterile blood sample, out of a long list of species, would enable appropriate actions to be taken to prevent sepsis. We realized that the number of different targets that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colors for the identification of each target species. We prepared a demonstration assay in which 15 different molecular beacon probe pairs were present, each pair specific for the same identifying sequence in the 16S ribosomal RNA gene of a different bacterial species, and each pair labeled with a unique combination of two fluorophores out of the six differently colored fluorophores that our PCR instrument could distinguish. In a set of PCR assays, each containing all 30 color-coded molecular beacons, and each containing DNA from a different bacterial species, the only two colors that arose in each real-time assay identified the species-specific target sequence that was present. Due to the intrinsic low background of molecular beacon probes, these reactions were specific and extremely sensitive, and the threshold cycle reflected the abundance of the target sequence present in each sample.
Asunto(s)
Reacción en Cadena de la Polimerasa Multiplex/métodos , Bacterias/genética , Infecciones Bacterianas/microbiología , Color , Colorantes Fluorescentes , Genes Bacterianos , Humanos , Tamizaje Masivo , Sondas Moleculares/genética , ARN Ribosómico 16S/genética , Sepsis/microbiología , Especificidad de la EspecieRESUMEN
Conventional cytogenetic techniques can distinguish homologous chromosomes in a qualitative manner based upon obvious morphological features or using in situ hybridization methods that yield qualitative data. We have developed a method for quantitative genotyping of single-nucleotide variants in situ using circularizable DNA probes, so-called padlock probes, targeting two different alpha satellite repeat variants present in human chromosome 7 centromeres, and a single-nucleotide variation in alpha satellite repeats on human chromosome 15 centromeres. By using these PCR-generated padlock probes, we could quantitatively distinguish homologous chromosomes and follow the transmission of the chromosomes by in situ analysis during three consecutive generations.
Asunto(s)
Análisis Citogenético/métodos , Sondas de ADN , Hibridación Fluorescente in Situ/métodos , Reacción en Cadena de la Polimerasa , Centrómero/genética , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 7 , ADN Satélite , Femenino , Marcadores Genéticos , Humanos , Masculino , Linaje , Polimorfismo de Nucleótido SimpleRESUMEN
Padlock probes are molecular tools that combine highly specific target sequence recognition with the potential for multiplexed analysis of large sets of target DNA or RNA sequences. In this brief review, we exemplify the ability of these probes to distinguish single-nucleotide target sequence variants. We further discuss means to detect the location of target sequences in situ, and to amplify reacted padlock probes via rolling-circle replication, as well as to sort reaction products on tag-arrays. We argue that the probes have the potential to render high-throughput genetic analyses precise and affordable.
Asunto(s)
Sondas de ADN/química , Sondas de ADN/genética , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , ADN/biosíntesis , ADN/química , ADN/genética , ADN Ligasas/metabolismo , Sondas de ADN/metabolismo , Replicación del ADN , Modelos Moleculares , ARN/química , ARN/genética , Sensibilidad y Especificidad , Especificidad por SustratoRESUMEN
DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation. This mechanism has been exploited to distinguish DNA sequence variants in situ using so-called padlock probes. Padlock probes are linear oligonucleotides with target-complementary sequences at both ends, and an on-target-complementary segment in between. The end sequences are brought next to each other upon hybridization to the target DNA sequence, and if the ends are perfectly matched to the target sequence, they can be joined by a DNA ligase. Padlock probes detect target sequences with very high specificity, because both probe segments must hybridize to the target for circularization to occur. This unit presents a protocol for discrimination between closely similar DNA sequences in situ using padlock probes. A discussion of methods for greatly amplifying the signal from circularized probes is also included.DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation.
