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1.  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
2.  Specific mutation screening of TP53 gene by low-density DNA microarray 
TP53 is the most commonly mutated gene in human cancers. Approximately 90% of mutations in this gene are localized between domains encoding exons 5 to 8. The aim of this investigation was to examine the ability of the low density DNA microarray with the assistance of double tandem hybridization platform to characterize TP53 mutational hotspots in exons 5, 7, and 8 of the TP53. Nineteen capture probes specific to each potential mutation site were designed to hybridize to specific site. Virtual hybridization was used to predict the stability of hybridization of each capture probe with the target. Thirty-three DNA samples from different sources were analyzed for mutants in these exons. A total of 32 codon substitutions were found by DNA sequencing. 24 of them a showed a perfect correlation with the hybridization pattern system and DNA sequencing analysis of the regions scanned. Although in this work we directed our attention to some of the most representative mutations of the TP53 gene, the results suggest that this microarray system proved to be a rapid, reliable, and effective method for screening all the mutations in TP53 gene.
PMCID: PMC3781765  PMID: 24198462
oligonucleotide microarray; TP53 gene; point mutations
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
5.  Transformation Between Haemophilus influenzae and Haemophilus parainfluenzae 
Journal of Bacteriology  1970;104(1):390-400.
Heterospecific transformation between Haemophilus influenzae and H. parainfluenzae is from one to more than six orders of magnitude lower than homospecific transformation, depending on the marker assayed. However, the physical integration of deoxyribonucleic acid (DNA) in heterospecific compared with homospecific transformation is only slightly decreased. Measurement of integration of ultraviolet-irradiated heterospecific transforming DNA suggests that compared with homospecific DNA a longer piece of heterospecific transforming DNA must undergo pairing for integration to occur. Heterospecific transforming DNA behaves towards ultraviolet inactivation of biological activity as though it had undergone some previous inactivation. The efficiency of heterospecific transformation can be improved by light sonic treatment of the DNA or by the use of DNA containing markers which originated from the heterospecific recipient. The presence of an excision mechanism in the recipient cell does not affect killing or marker efficiency in heterospecific transformation. The data indicate that the low frequency of transformation between H. influenzae and H. parainfluenzae results mostly from lethality. It is proposed that integration of heterospecific transforming DNA results in alterations in the base sequence of the recipient genome which cannot be repaired. Transcription and translation of the altered DNA could result in synthesis of nonfunctional essential proteins.
PMCID: PMC248225  PMID: 5312117
6.  Repair of Ultraviolet-Irradiated Transforming Deoxyribonucleic Acid in Haemophilus influenzae 
Journal of Bacteriology  1970;101(3):808-812.
Ultraviolet-sensitive and wild-type Haemophilus influenzae cells were exposed to irradiated and unirradiated transforming deoxyribonucleic acid (DNA) containing a marker which can be linked to another marker in the cells. Lysates were made after various times of incubation and assayed for transforming activity on an excisionless recipient. Repair can be noted as an increase in activity from the irradiated donor DNA after its linkage to the recipient DNA. No repair can be observed in a mutant which is unable to integrate transforming DNA. There is a little repair in another mutant which is unable to excise pyrimidine dimers. H. influenzae cells also repair nondimer damage, as judged by the increase in activity observed in lysates made with irradiated and maximally photoreactivated DNA.
PMCID: PMC250395  PMID: 5309577
7.  Killing of Haemophilus influenzae Cells by Integrated Ultraviolet-Induced Lesions from Transforming Deoxyribonucleic Acid 
Journal of Bacteriology  1969;100(3):1284-1288.
Highly competent cultures of Haemophilus influenzae are inactivated by exposure to transforming deoxyribonucleic acid (DNA) irradiated with ultraviolet light (UV). As a function of UV dose to the DNA, the killing goes to a maximum and then decreases. The killing of H. influenzae by unirradiated H. parainfluenzae DNA, reported by other workers, is enhanced by low doses of UV, but drops off at high doses. Since there are no such lethal effects in a strain of H. influenzae that takes up DNA normally but does not integrate it, it is concluded that the killing is associated with integrated UV lesions. All the killing of wild-type cells due to irradiated DNA is eliminated by photoreactivation of the DNA. The killing of an excisionless strain of H. influenzae, however, is not eliminated by maximal photoreactivation of the irradiated transforming DNA. The nonphotoreactivable fraction of killing in the excisionless strain increases with increasing dose. The kinetics of the killing-dose curves may be explained only partially in terms of UV-induced loss of integration. It is postulated that the number of pyrimidine dimers relative to other DNA components integrated decreases at higher UV doses.
PMCID: PMC250315  PMID: 5308034

Results 1-7 (7)