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1.  Design of a set of probes with high potential for influenza virus epidemiological surveillance 
Bioinformation  2013;9(8):414-420.
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.
PMCID: PMC3670124  PMID: 23750091
IPS; fingerprinting; Virtual Hybridization; Shannon Entropy; Microarray; Influenza virus
2.  LifePrint: a novel k-tuple distance method for construction of phylogenetic trees 
Here we describe LifePrint, a sequence alignment-independent k-tuple distance method to estimate relatedness between complete genomes.
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.
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.
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.
PMCID: PMC3169951  PMID: 21918634
phylogeny; sequence alignment; similarity search; tuple; viroid
3.  Low density DNA microarray for detection of most frequent TP53 missense point mutations 
BMC Biotechnology  2005;5:8.
We 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 ssDNA's 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.
18 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.
Our 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.
PMCID: PMC553977  PMID: 15713227
4.  Fingerprinting of prokaryotic 16S rRNA genes using oligodeoxyribonucleotide microarrays and virtual hybridization 
Nucleic Acids Research  2003;31(2):779-789.
An oligonucleotide microarray hybridization system to differentiate microbial species was designed and tested. Seven microbial species were studied, including one Bacillus and six Pseudomonas strains. DNA sequences near the 5′ end of 16S rRNA genes were aligned and two contiguous regions of high variability, flanked by highly conserved sequences, were found. The conserved sequences were used to design PCR primers which efficiently amplified these polymorphic regions from all seven species. The amplicon sequences were used to design 88 9mer hybridization probes which were arrayed onto glass slides. Single-stranded, fluorescence-tagged PCR products were hybridized to the microarrays at 15°C. The experimental results were compared with the ΔG° values for all matched and mismatched duplexes possible between the synthetic probes and the 16S target sequences of the seven test species, calculated using a ‘virtual hybridization’ software program. Although the observed hybridization patterns differed significantly from patterns predicted solely on the basis of perfect sequence matches, a unique hybridization fingerprint was obtained for each of the species, including closely related Pseudomonas species, and there was a reasonable correlation between the intensity of observed hybridization signals and the calculated ΔG° values. The results suggest that both perfect and mismatched pairings can contribute to microbial identification by hybridization fingerprinting.
PMCID: PMC140498  PMID: 12527788

Results 1-4 (4)