RESUMO
In this article we introduce a strategy of preannealing labeled auxiliary oligonucleotides to single-stranded target DNA, prior to hybridization of the DNA target to oligonucleotide arrays (genosensors) formed on glass slides for the purpose of mutation analysis. Human genomic DNA samples from normal individuals and cystic fibrosis (CF) patients (including homozygous delta F508 and heterozygous delta F508/wild type (wt) in the region examined) were used. A PCR fragment of length 138 bp (wt) or 135 bp (mutant) was produced from exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, using a new pair of polymerase chain reaction (PCR) primers. This fragment contains four of the most frequent mutation sites causing the disease (Q493X, delta I507, delta F508, and V520F). Each of these mutations was tested using a pair of nonamer (9-mer) probes covalently attached to glass slides, representing the normal (wt) and the mutant alleles. Single-stranded target DNA was isolated from the PCR fragment using one PCR primer labeled with biotin and a streptavidin minicolumn to capture the biotin-labeled strand. Prior to hybridization to the 9-mer array on a glass slide, the unlabeled target strand was preannealed with one, three, or four auxiliary oligonucleotides, at least one being labeled with 32P. As observed previously in several laboratories, the discrimination between normal (wt) and mutant alleles at each site using oligonucleotide array hybridization ranged from very good to poor, depending on the number and location of mismatches between probe and target. Terminal mismatches along the probe were difficult to discriminate, internal mismatches were more easily discriminated, and multiple mismatches were very well discriminated. An exceptionally intense hybridization signal was obtained with a 9-mer probe that hybridized contiguously (in tandem) with one auxiliary oligonucleotide preannealed to the target DNA. The increased stability is apparently caused by strong base stacking interactions between the "capture probe" and the auxiliary oligonucleotide. The presence of the delta F508 mutation was detected with this system, including discrimination between homozygous and heterozygous conditions. Base mismatch discrimination using the arrayed 9-mer probes was improved by increasing the temperature of hybridization from 15 to 25 degrees C. Auxiliary oligonucleotides, preannealed to the single-stranded template, may serve several purposes to enable a more robust genosensor-based DNA sequence analysis: 1. A convenient means of introducing label into the target DNA molecule. 2. Disruption of interfering short-range secondary structure in the region of analysis. 3. Covering up of redundant binding sites in the target strand (i.e., where a given probe has more than one complement within the target). 4. Tandem hybridization with the capture probe (providing contiguous stacking) as a means for achieving efficient mismatch discrimination at the terminal position of the capture probe (adjacent to the auxiliary oligonucleotide). By use of multiple auxiliary oligonucleotides, all of the above benefits can be derived simultaneously.
Assuntos
DNA/química , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Sequência de Bases , Estudos de Casos e Controles , Fibrose Cística/genética , Vidro , Haplótipos , Heterozigoto , Homozigoto , Humanos , Reação em Cadeia da Polimerase , TemperaturaRESUMO
The mutagenic role of 1-N6-Ethenodeoxydenosine (epsilon A) was assessed by a genetic assay of mutations in the 5' coding region of the lacl gene of Escherichia coli. 1-N6-Etheno-2-deoxydadenosine 5'-triphosphate (epsilon dATP) was substituted for dATP during in vitro DNA synthesis on M13 recombinant uracil single stranded DNA bearing the lacl gene, catalyzed by the large fragment of E. coli DNA polymerase I. DNA products were transfected into a strain of E. coli lacking a chromosomal copy of the lacl gene, and i- phenotypic mutants were seen as blue plaques in the absence of inducer. Mutant progeny were characterized by dideoxy sequencing in the N-terminal region of the lacl gene where epsilon A had originally replaced A, and were found to have T-->C transitions. The frequency of base substitution mutation was different in each of three target sites tested. Taking into account the sequence changes and the coding properties at target sites, we conclude that in general, epsilon A increases the mutation frequency when compared with the control (transfection with unsubstituted DNA). This increase was similar to that produced by in vitro primer elongation in absence of dATP. The combined results of the electrophoretic assay of primer elongation, measurements of mutation frequency, and sequence analysis of mutant phage progeny support the proposal that epsilon A in template DNA can be mutagenic through epsilon AC mispairing during in vivo replication.
Assuntos
DNA Bacteriano/genética , Desoxiadenosinas/genética , Mutagênese , Composição de Bases , Sequência de Bases , Códon/genética , Primers do DNA , DNA Bacteriano/química , Escherichia coli/genética , Genes Bacterianos , Dados de Sequência Molecular , Moldes GenéticosRESUMO
The importance of the DNA-damage repair mechanisms comes from the fact that any chemical modification to the DNA molecule, no matter how small it is, very often causes cell death and sometimes it can produce mutations whose biological effects are unpredictable. Taking into account that in the last decade great advances have been reported for the understanding of these mechanisms, and this knowledge can be essential to reach the control of the mutagenesis, we give a current review of the main DNA-damage repair pathways, with special emphasis in its classification by the kind of mechanisms and in their main features. As a particular case, the error prone repair mechanism is included (SOS-REPAIR). Additionally the three inducible DNA repair systems are described. That is to say, those cases in which the cell treatment with sublethal doses of some physical or chemical agents, induces a cell response that increases its capacity to repair the DNA-damage causes by higher doses of the same agent. Finally some small general comments are made.
Assuntos
Reparo do DNA/genéticaRESUMO
Actualmente contamos con una mutante de Escherichia coli sin actividad de fosforilasa de la timidina, por consecuencia, este microorganismo crece con timidina y no con timina siendo posible en esta cepa hacer los estudios del efecto de BrUdR y de otro analogo de la timidina la hmUdR sobre el crecimiento de E. coli. La adicion de cantidades limitadas de timidina en un medio minimo dio lugar a la aparicion de "muerte por falta de timidina". Los analogos se agregaron solos o mezclados en diferentes proporciones con timidina, observandose que hmUdR solo, no estimulo el crecimiento, pero al agregarse junto con timidina disminuyo la muerte de la bacteria por falta de timidina. La BrUdR por su parte aumento la viabilidad tanto cuando se agrego sola como cuando se anadio mezclada con timidina. Estos estudios sugirieron que la cepa utiliza de preferencia timidina