Rogelio Maldonado-Rodriguez

Hybridization of glass-tethered oligonucleotide probes to target strands preannealed with labeled auxiliary oligonucleotides

AbstractIn this article we introduce a strategy of preanncaling 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 δF508 and heterozygous δF508/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 l0 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, δI507, δF508, 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 allcles. 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 with32P. 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 slacking interactions between the “capture probe” and the auxiliary oligonucleotide. The presence of the δF508 mutation was delected with this system, including discrimination between homozygous and heterozygous conditions. Base mismatch discrimination using the arrayed 9-mcr probes was improved by increasing the temperature of hybridization from 15 to 25‡C. Auxiliary oligonucleotides, preannealed to the single-stranded template, may serve several purposes to enable a more robust genosensor-based DNA sequence analysis:1A convenient means of introducing label into the target DNA molecule.2Disruption of interfering short-range secondary structure in the region of analysis.3Covering up of redundant binding sites in the target strand (i.e., where a given probe has more than one complement within the target).4Tandem 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 oligonucleolides. all of the above benefits can be derived simultaneously.

Mutagenic consequences of the incorporation of 6-thioguanine into DNA

6-Thioguanine (S6G) has been used in the treatment of acute leukemias because of its cytotoxic effect on proliferating leukemic cells. The cytotoxicity of S6G is thought to derive from its incorporation into DNA in place of guanine. The deoxyribonucleoside triphosphate of S6G, SdGTP, is a good substrate for bacterial and human DNA polymerases (Ling et al., Mol Pharmacol 40: 508-514, 1991). Since SdGTP was observed to misincorporate in place of adenine at a greater frequency than did dGTP, it appeared plausible that this analog could produce more subtle effects (mutations) due to mispairing with thymine. To assess whether such mutations occur, SdGTP was incorporated into the lacI gene of phage M13lacISaXb in reactions that omitted dGTP (-G) or dATP (-A). LacI mutation frequency was determined by beta-galactosidase colorimetric staining (inactivation of the lac repressor results in blue plaques in the absence of inducer). When a high concentration of SdGTP (24 microM) was used in the DNA polymerase reaction, phage infectivity was inhibited. When a relatively low concentration (2.4 nM) was added to the -G and -A reactions, mutagenic effects were observed. DNA sequencing of mutant progeny arising from the -G + S6G reaction revealed C-to-T base transitions and some C-to-A transversions. Similarly, the presence of SdGTP in the -A reactions led to mutants with T-to-C transitions. No insertions or deletions were observed. These data indicate that mispairing of S6G with thymine leads to mutagenic effects in this assay.

Low density DNA microarray for detection of most frequent TP53 missense point mutations

BackgroundWe have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNAs were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes.Results18 codon substitutions were found by DNA sequencing. In 13 of them a perfect correlation with the pattern of hybridization was seen (In 5 no signal was seen with the wt probe while a new signal was seen with the appropriate mutant probe, and in 8 more, as expected, no signal was seen with any probe due to the absence of the corresponding probe in the array). In 3 other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. In the other 2 cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. In both cases (false mutation or no mutation detected) relatively stable mismatched target/probe duplexes should be formed. These problems could be avoided by the addition of probes to improve the performance of the array.ConclusionOur results demonstrate that a simple TP53 microarray employing short (7-mer) probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common TP53 missense mutations from a variety of DNA sources with good specificity.

Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization

We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5′-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.

LifePrint: a novel k-tuple distance method for construction of phylogenetic trees

Purpose Here we describe LifePrint, a sequence alignment-independent k-tuple distance method to estimate relatedness between complete genomes. Methods We designed a representative sample of all possible DNA tuples of length 9 (9-tuples). The final sample comprises 1878 tuples (called the LifePrint set of 9-tuples; LPS9) that are distinct from each other by at least two internal and noncontiguous nucleotide differences. For validation of our k-tuple distance method, we analyzed several real and simulated viroid genomes. Using different distance metrics, we scrutinized diverse viroid genomes to estimate the k-tuple distances between these genomic sequences. Then we used the estimated genomic k-tuple distances to construct phylogenetic trees using the neighbor-joining algorithm. A comparison of the accuracy of LPS9 and the previously reported 5-tuple method was made using symmetric differences between the trees estimated from each method and a simulated “true” phylogenetic tree. Results The identified optimal search scheme for LPS9 allows only up to two nucleotide differences between each 9-tuple and the scrutinized genome. Similarity search results of simulated viroid genomes indicate that, in most cases, LPS9 is able to detect single-base substitutions between genomes efficiently. Analysis of simulated genomic variants with a high proportion of base substitutions indicates that LPS9 is able to discern relationships between genomic variants with up to 40% of nucleotide substitution. Conclusion Our LPS9 method generates more accurate phylogenetic reconstructions than the previously proposed 5-tuples strategy. LPS9-reconstructed trees show higher bootstrap proportion values than distance trees derived from the 5-tuple method.

In Silico Genomic Fingerprints of the Bacillus anthracis Group Obtained by Virtual Hybridization.

In this study we evaluate the capacity of Virtual Hybridization to identify between highly related bacterial strains. Eight genomic fingerprints were obtained by virtual hybridization for the Bacillus anthracis genome set, and a set of 15,264 13-nucleotide short probes designed to produce genomic fingerprints unique for each organism. The data obtained from each genomic fingerprint were used to obtain hybridization patterns simulating a DNA microarray. Two virtual hybridization methods were used: the Direct and the Extended method to identify the number of potential hybridization sites and thus determine the minimum sensitivity value to discriminate between genomes with 99.9% similarity. Genomic fingerprints were compared using both methods and phylogenomic trees were constructed to verify that the minimum detection value is 0.000017. Results obtained from the genomic fingerprints suggest that the distribution in the trees is correct, as compared to other taxonomic methods. Specific virtual hybridization sites for each of the genomes studied were also identified.

Construction and expression of a chimeric gene encoding human terminal deoxynucleotidyltransferase and DNA polymerase β

Abstract A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Polβ and TdT to investigate the region of Polβ required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154–212 of TdT was replaced by the corresponding region encoding aa 1–60 of Polβ. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of Polβ represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Polβ protein possessed significant template-dependent polymerase activity suggests that aa 1–60 of Polβ are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Polβ possesses ssDNA-binding activity [Kumar ., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar ., Biochemistry 29 (1990b) 7156-7159; Prasad ., J. Biol. Chem. 268 (1993) 22746-22755].

Design of a set of probes with high potential for influenza virus epidemiological surveillance

An Influenza Probe Set (IPS) consisting in 1,249 9-mer probes for genomic fingerprinting of closely and distantly related Influenza Virus strains was designed and tested in silico. The IPS was derived from alignments of Influenza genomes. The RNA segments of 5,133 influenza strains having diverse degree of relatedness were concatenated and aligned. After alignment, 9-mer sites having high Shannon entropy were searched. Additional criteria such as: G+C content between 35 to 65%, absence of dimer or trimer consecutive repeats, a minimum of 2 differences between 9mers and selecting only sequences with Tm values between 34.5 and 36.5oC were applied for selecting probes with high sequential entropy. Virtual Hybridization was used to predict Genomic Fingerprints to assess the capability of the IPS to discriminate between influenza and related strains. Distance scores between pairs of Influenza Genomic Fingerprints were calculated, and used for estimating Taxonomic Trees. Visual examination of both Genomic Fingerprints and Taxonomic Trees suggest that the IPS is able to discriminate between distant and closely related Influenza strains. It is proposed that the IPS can be used to investigate, by virtual or experimental hybridization, any new, and potentially virulent, strain.

Universal fingerprinting chip server.

The Virtual Hybridization approach predicts the most probable hybridization sites across a target nucleic acid of known sequence, including both perfect and mismatched pairings. Potential hybridization sites, having a user-defined minimum number of bases that are paired with the oligonucleotide probe, are first identified. Then free energy values are evaluated for each potential hybridization site, and if it has a calculated free energy of equal or higher negative value than a user-defined free energy cut-off value, it is considered as a site of high probability of hybridization. The Universal Fingerprinting Chip Applications Server contains the software for visualizing predicted hybridization patterns, which yields a simulated hybridization fingerprint that can be compared with experimentally derived fingerprints or with a virtual fingerprint arising from a different sample. Availability http://bioinformatica.homelinux.org/UFCVH/