Top PDF Algorithms for nucleic acid sequence design

Algorithms for nucleic acid sequence design

Algorithms for nucleic acid sequence design

Chapter 5 Summary and outlook This thesis focuses on making thermodynamic nucleic acid design a high-quality, versatile, and low-cost technology that is easy to use and easily accessible. We demonstrated how to achieve high-quality sequences via ensemble defect optimization for only 4/3 the cost of the objective function for large structures. We have further increased design versatility by developing an algorithm for the design of multi-state systems of nucleic acids that also achieves high-quality sequence for low cost with ensemble defect optimization. Finally, we have presented a web server that makes parallel implementations of these and other algorithms easy to use and accessible with the use of a specialized front-end to a high-performance compute cluster. The highly interactive features and visualization tools of NUPACK allow for users to explore and communicate their results with greater understanding and further reach than was previously possible. Nevertheless, improving cost, scope, and availability remains an open area of research.
Show more

97 Read more

iTriplet, a rule-based nucleic acid sequence motif finder

iTriplet, a rule-based nucleic acid sequence motif finder

otherwise no conclusion can be made. As we have dis- cussed in the inter-sequence algorithm section, if mem- bers of the triplet are very similar to each other, the intersection will become big, i.e. high numbers of com- mon motifs. Based on this property, we derived a straight- forward scoring system based on the numbers of common motifs uncovered to support whether the finding is statis- tically significant. Due to this, the 5' and 3' overlapping neighbors of the true motif are often included as part of the prediction as well. Therefore in some cases of the genes listed in Table 3, the predicted motif is longer than the model specified. Each prediction is a consensus of a number of common motifs. The method of constructing the consensus is similar to the frequency plot of Weblogo [19]. Nucleotides with frequency at a position greater than 30% will be included in the consensus sequence. As can be seen from Table 3, our predictions for promoter and 5' UTR sequences, and 3' UTR regulatory elements are largely consistent with published experimental data.
Show more

14 Read more

Nucleic Acid Sequence Based Amplification (Nasba)-Prospects And Applications

Nucleic Acid Sequence Based Amplification (Nasba)-Prospects And Applications

(MAP) (Rodríguez-Lázaro et al. 2004a) was very disappointing. It would have been very advantageous to have a rapid NASBA-based viability assay. Other organisms for which a rapid viability assay would be useful include Legionella, Listeria, Campylobacter, etc. NASBA could also be used to test viable-but non-culturable (VBNC) state in several bacteria, e.g., Campylobacter. NASBA is now an established diagnostic tool in clinical use (Rodríguez-Lázaro et al. 2006), but it does not appear to be progressing toward implementation in food analysis. To be optimally applicable to the detection of viable microorganisms in foods, NASBA will need to be applied directly, i.e., with no prior enrichment of the samples in nutrient media to increase the number of target cells and to allow sampling of small volumes. The assay should be applied to nucleic acids extracted directly from the food sample. As NASBA is an isothermal process, it does not require an expensive thermocycling apparatus. However, detection of the amplified RNA signal can require complex equipment (Cook, 2003). Biosensor based assays (Baeumner et al., 2003) although sophisticated in their working principle, could appear relatively easy to use and could be developed into a format suitable for operation by nonskilled personnel. They may also mediate quantification of the target, through measurement of the signal intensity (Baeumner et al. 2003). Quantification may also be achieved by employing a NASBA assay in real-time format, with signal detection mediated by, e.g., molecular beacons (Rodríguez-Lázaro et al. 2004b). NASBA, as a more complex process than PCR, is unlikely to match the progress of PCR toward adoption as a routine diagnostic tool for food and environmental samples unless committed developmental effort is made to explore and capitalize on its potential to detect viable microbial cells, which PCR does not in itself possess.
Show more

16 Read more

Multiple sequence alignments of partially coding nucleic acid sequences

Multiple sequence alignments of partially coding nucleic acid sequences

served also on the level of nucleic acid, while exon 1 has a well-conserved amino acid sequence but ex- hibits high variability at the nucleic acid level. The non-coding sequence in the intron and the flanking sequences are highly variable. Thus, this example is a hard test case for our approach. Figure 3 sum- marizes the gap lengths in the Hox4 alignments. A comparison of the number of gaps with a length di- visible by 3 with the other gaps of other lengths is a useful indicator whether coding regions are rea- sonably aligned: Base triplets preferentially should not be disrupted as amino acids within a protein se- quence cannot be disrupted. While codaln correctly aligns the coding sequences in both exons, ClustalW only treats exon 2 correctly, which is highly con- served on the level of nucleic acids. The nucleic acid alignment for the more variable exon 1, in contrast, is much more divergent.
Show more

8 Read more

Analysis, Design, and Construction of Nucleic Acid Devices

Analysis, Design, and Construction of Nucleic Acid Devices

Abstract Nucleic acids present great promise as building blocks for nanoscale devices. To achieve this poten- tial, methods for the analysis and design of DNA and RNA need to be improved. In this thesis, traditional algorithms for analyzing nucleic acids at equilibrium are extended to handle a class of pseudoknots, with examples provided relevant to biologists and bioengineers. With these analyti- cal tools in hand, nucleic acid sequences are designed to maximize the equilibrium probability of a desired fold. Upon analysis, it is concluded that both affinity and specificity are important when choosing a sequence; this conclusion holds for a wide range of target structures and is robust to ran- dom perturbations to the energy model. Applying the intuition gained from these studies, a process called hybridization chain reaction (HCR) is invented, and sequences are chosen that experimentally verify this phenomenon. In HCR, a small number of DNA or RNA molecules trigger a system wide configurational change, allowing the amplification and detection of specific, nucleic acid sequences.
Show more

172 Read more

Experimental characterization of the human non sequence specific nucleic acid interactome

Experimental characterization of the human non sequence specific nucleic acid interactome

Low sequence complexity has the potential to induce the identification of numerous abundant proteins that could have low affinity for nucleic acids - for example, sequence-specific NABPs that would retain low nucleic acid affinity for some of the baits we used. Although this phenomenon certainly exists, convergent and indepen- dent observations show that it does not contribute to an important level. In the ‘Protein identification and filter- ing’ section we noted that, while the proportion of known NABPs rose from 21% in the core proteomes to 70% in the pulldowns, 252 NABPs of the core proteomes - hence abundant - were not identified in the affinity- purified samples, thus indicating affinity purification spe- cificity. Extending this analysis to transcription factors, which are sequence-specific predominantly, we observed that general NABPs were much more enriched in pull- downs compared to transcription factors (Figures 1b,d), further showing the absence of a strong nucleic acid low affinity-driven bias on this class of proteins. Moreover, carefully realized pulldown experiments with non-specific interactions removed (for example, comparing with proper negative controls as was done in this study) have a long history of revealing relevant protein interactions - for example, with oligonucleotide baits [16,17]. In line with this, inspection of Supplementary Table S5 in Addi- tional file 2 for DNA- or RNA-specific NABPs reveals numbers of well known DNA- and RNA-associated pro- teins with a functional role.
Show more

17 Read more

Computational design of nucleic acid feedback control circuits

Computational design of nucleic acid feedback control circuits

Several future additions to the methodology and case studies reported in this paper are possible. For example, the Visual DSD extensions we proposed allowed us to express rich dynamical laws beyond mass action kinetics, for example to capture Michaelis − Menten enzyme kinetics or additional competition e ff ects. Our current PI controller models involved fi rst order reaction approximations, consistent with previous work in the fi eld, 15,37 but detailed models capturing e ff ects such as enzyme concentrations, processivity and competition 50 could enable the study of such systems in more general contexts. In this paper, we only considered the performance of the proposed controllers when coupled to a simple plant system implemented using ideal chemical reactions − the applicability of such circuits to realistic in vivo or in vitro biochemical processes will be investigated in the future as a strategy toward designing practical molecular control circuits. Furthermore, since the approaches we investigated are applicable to the engineering of a broad range of control and signal processing systems, our methodology could be used to design a range of systems for regulating biochemical processes, beyond the PI controllers we studied here. The design procedures and tools developed as part of this work are a step toward making the physical realization of such complex
Show more

18 Read more

Molecular Beacons of Xeno-Nucleic Acid for Detecting Nucleic Acid

Molecular Beacons of Xeno-Nucleic Acid for Detecting Nucleic Acid

Considering the chemical and biological stabil- ity, PNAs could be used to design gene therapeutic drugs [48,49,50] and other molecular biology and functional genomics [51,52]. Besides, PNA probes are extremely useful in situ hybridization and provide very good chromosome images [53,54,55,56,57,58]. A few early reports have examined the properties of PNAs as both specific [59,60,61] as well as general [62] nucleic acid capture probes. Recently, PNA-based MBs, optoelectronic [63], microarray or electrochem- ical probes [64] have been developed for different biochemical and biotechnological applications [65]. Fang et al. developed an electrochemical nucleic acids probe made of PNA which exhibits high sensitivity and specificity when challenged with heterogeneous samples of RNA. They further used the probes to de- tect a newly identified cancer biomarker-a gene fusion associated with prostate cancer. The system could detect specific mRNAs in unamplified patient sam- ples in as little as 10 ng [66]. PNA was also success- fully used to label-free DNA/PNA hybridization de- tection when combined with silicon-based platform [67].
Show more

14 Read more

Development of novel fluorescent oligonucleotide probes for use in nucleic acid sequence analysis

Development of novel fluorescent oligonucleotide probes for use in nucleic acid sequence analysis

The solvents were removed in vacuo and the resulting oil dissolved in CH2Ch 200mL, washed with water 200mL, saturated sodium bicarbonate solution 200mL, brine 150mL and then dried Na2S04[r]

216 Read more

The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient massive parallel sequencing

The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient massive parallel sequencing

1 Operational Directorate of Virology, Veterinary and Agrochemical Research Centre, Ukkel, Brussels, Belgium, 2 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany Abstract Sequence Independent Single Primer Amplification is one of the most widely used random amplification approaches in virology for sequencing template preparation. This technique relies on oligonucleotides consisting of a 39 random part used to prime complementary DNA synthesis and a 59 defined tag sequence for subsequent amplification. Recently, this amplification method was combined with next generation sequencing to obtain viral sequences. However, these studies showed a biased distribution of the resulting sequence reads over the analyzed genomes. The aim of this study was to elucidate the mechanisms that lead to biased sequence depth when using random amplification. Avian paramyxovirus type 8 was used as a model RNA virus to investigate these mechanisms. We showed, based on in silico analysis of the sequence depth in relation to GC-content, predicted RNA secondary structure and sequence complementarity to the 39 part of the tag sequence, that the tag sequence has the main contribution to the observed bias in sequence depth. We confirmed this finding experimentally using both fragmented and non-fragmented viral RNAs as well as primers differing in random oligomer length (6 or 12 nucleotides) and in the sequence of the amplification tag. The observed oligonucleotide annealing bias can be reduced by extending the random oligomer sequence and by in silico combining sequence data from SISPA experiments using different 59 defined tag sequences. These findings contribute to the optimization of random nucleic acid amplification protocols that are currently required for downstream applications such as viral metagenomics and microarray analysis.
Show more

9 Read more

Detection of Dengue Viral RNA Using a Nucleic Acid Sequence Based Amplification Assay

Detection of Dengue Viral RNA Using a Nucleic Acid Sequence Based Amplification Assay

Jakarta, Indonesia 5 ; Naval Medical Research Center Detachment, AMEMB-NAMRID, APO AA 34031 6 ; and Institute of Epidemiology, National Taiwan University, Taipei, Taiwan, Republic of China 7 Received 7 February 2001/Returned for modification 3 April 2001/Accepted 26 May 2001 Faster techniques are needed for the early diagnosis of dengue fever and dengue hemorrhagic fever during the acute viremic phase of infection. An isothermal nucleic acid sequence-based amplification (NASBA) assay was optimized to amplify viral RNA of all four dengue virus serotypes by a set of universal primers and to type the amplified products by serotype-specific capture probes. The NASBA assay involved the use of silica to extract viral nucleic acid, which was amplified without thermocycling. The amplified product was detected by a probe-hybridization method that utilized electrochemiluminescence. Using normal human plasma spiked with dengue viruses, the NASBA assay had a detection threshold of 1 to 10 PFU/ml. The sensitivity and specificity of the assay were determined by testing 67 dengue virus-positive and 21 dengue virus-negative human serum or plasma samples. The “gold standard” used for comparison and evaluation was the mosquito C6/36 cell culture assay followed by an immunofluorescent assay. Viral infectivity titers in test samples were also determined by a direct plaque assay in Vero cells. The NASBA assay was able to detect dengue viral RNA in the clinical samples at plaque titers below 25 PFU/ml (the detection limit of the plaque assay). Of the 67 samples found positive by the C6/36 assay, 66 were found positive by the NASBA assay, for a sensitivity of 98.5%. The NASBA assay had a specificity of 100% based on the negative test results for the 21 normal human serum or plasma samples. These results indicate that the NASBA assay is a promising assay for the early diagnosis of dengue infections.
Show more

5 Read more

Detection of Mycoplasma pneumoniae in Spiked Clinical Samples by Nucleic Acid Sequence Based Amplification

Detection of Mycoplasma pneumoniae in Spiked Clinical Samples by Nucleic Acid Sequence Based Amplification

Department of Microbiology, University of Antwerp UIA, Antwerp, Belgium, 1 and Organon Teknika BV, Boxtel, The Netherlands 2 Received 20 August 2001/Returned for modification 11 October 2001/Accepted 24 January 2002 Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (CCU) of M. pneumoniae. An appropriate specimen preparation procedure was developed: after protease treatment of the respiratory specimens, guanidine thiocyanate lysis solution (4.7 M guanidine thiocyanate [Sigma-Aldrich NV], 46 mM Tris-HCl [Merck, Darmstadt, Germany], 20 mM EDTA [Sigma-Aldrich NV], 1.2% [wt/vol] Triton X-100 [Sigma-Aldrich NV], pH 6.2.) was added. With spiked throats, nasopharyngeal aspirates, bronchoal- veolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 ⴛ 10 4 molecules of in vitro-generated RNA or 5 CCU of M. pneumoniae. The sensitivity of the NASBA assay was comparable to that of a PCR targeted to the P1 adhesin gene. Fifteen clinical specimens positive for M. pneumoniae by PCR were also positive by NASBA. These results indicate that the sensitivity of detection of M. pneumoniae in spiked respiratory samples by NASBA is high. Together with the use of the internal control, the assay merits evaluation as a diagnostic tool.
Show more

7 Read more

Nucleic Acid Sequence Based Amplification with Oligochromatography for Detection of Trypanosoma brucei in Clinical Samples

Nucleic Acid Sequence Based Amplification with Oligochromatography for Detection of Trypanosoma brucei in Clinical Samples

the CSF PCR, there is still an urgent need for a test that can be reliably used in staging and treating HAT. In this study, the T. brucei NASBA-OC test that was devel- oped was used to detect parasite RNA in CSF from HAT patients and had a reproducibility of 100%, making this assay reliable and valuable for deciding the course of treatment. The T. brucei NASBA-OC assay had a sensitivity of 88.6% and detected parasites in six microscopically negative samples but failed to detect them in five samples that were positive by microscopy. However, the results of the T. brucei NASBA-OC assay are promising given the difference in the volumes of CSF samples processed by the two methods under comparison. For microscopic detection, 4 ml of CSF was centrifuged and the resulting sediment examined under the microscope. On the other hand, only 200 ␮ l of CSF was used to extract nucleic acid for NASBA.
Show more

6 Read more

Sensitivity and Specificity of Nucleic Acid Sequence-Based Amplification Method for Diagnosis of Cutaneous Leishmaniasis

Sensitivity and Specificity of Nucleic Acid Sequence-Based Amplification Method for Diagnosis of Cutaneous Leishmaniasis

Sensitivity and Specificity of Nucleic Acid Sequence-Based Amplification Method for Diagnosis of Cutaneous Leishmaniasis Abstract Background and Objective : Culture, microscopic method is a gold standard method for identification of Lishmania parasite. The use of Molecular methods such as RT- PCR compared to microscopic methods has a higher sensitivity and specificity; however, it is not widely used due to its expensive equipment and the time requested. The use of nucleic acid sequence based amplification (NASBA) method is highly valuable for diagnosis of live parasite because there is no need for to use Thermo cycler. We aimed to assess sensitivity and specificity of NASBA for molecular detection of cutaneous Leishmaniasis.
Show more

7 Read more

Development of a Computer Algorithm for Generation of Primers for Nucleic Acid Sequence Based Amplification (NASBA)

Development of a Computer Algorithm for Generation of Primers for Nucleic Acid Sequence Based Amplification (NASBA)

11 Biopython is an extensive Python based bioinformatics package that works to incorporate many useful tools in a single repository. One such useful tool is BLAST (Basic local alignment search tool), an algorithm for finding the statistical significance in the similarity between a query and a database of choice. Figure 6 shows in greater detail how BLAST works to create an alignment. A score is assigned by the number of matches and mismatches for a set of three nucleotides or amino acids, called a query word, through a local alignment. The maximum score for the combinations of local alignments is used to identify the correct alignment between the queries and subject. BLAST will be useful in creating an alignment of multiple genomes to create a multiple alignment object (a data type found that orders a number of queries against a subject). Figure 7 shows a visualization of a multiple alignment object, which allows one to compare a wild type genome or protein against multiple mutant genomes to amino acid sequences. Biopython also includes the Entrez module which is able to access the NCBI database to retrieve a genome or amino acid sequence of choice with a tag associated with each one, called an accession number.
Show more

39 Read more

Peptide Nucleic Acid Microarrays

Peptide Nucleic Acid Microarrays

Fig. 3. Detection of a SNP. PNA 12-mers of identical sequence but for a G or A nucleotide at their sixth or seventh position (marked bold), respectively, were spotted in duplicate on succinimidyl activated glass slides, with one row of buffer spots placed in-between. Slide A was hybridized with a polymerase chain reaction product of a heterozygous sample and slide B with a polymerase chain reaction product of a homozygous sample.

11 Read more

How To Make A Nucleic Acid

How To Make A Nucleic Acid

The concentration of the solutions of expensive PNA monomers, activator (HATU), and base (DIEA) was adjusted on the basis of the loading capacity and amount of used resin and the solutions were filled in 10 ml vials. HATU and DIEA had to be included in the pro- gramming of the synthetic sequence. For example, if a construct like ACGT has to be made, the sequence of the synthetic steps will be 651652653654 (starting from right corresponding to the C-terminus) where 1, 2, 3, 4, 5, and 6 represent A, C, G, T, HATU, and DIEA, respectively (Figure 2). The programmed method has been developed in a way that after the delivery of a building block, HATU is added and mixed for 3 min followed by DIEA delivery and mixing for 1 h. In the fully automated scheme, it was performed in a contin- uous way until the desired sequence was obtained. However, in the semi-automated mode, the single-shot delivery feature of the used peptide synthesizer was applied for the PNA monomers, so that the entire content of the PNA vials was delivered to the reaction vessel without priming. Thus, the reagent consumption was reduced further on but the synthetic scheme becomes discontinuous. If a construct like ACGTACGT has to be made, the system is continuous till the first four building blocks are coupled and then requires a refilling of the vials to be used for the next time. Positions of PNA monomers, HATU, and DIEA as well as the sequence programming and method developments were similar to those in the fully au- tomatic mode. Importantly, HATU and DIEA were also placed in 10 ml vials and delivered in 0.3 ml aliquots without the single-shot
Show more

6 Read more

Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved pa- tient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and vari- ous nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based compari- sons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power for C.
Show more

11 Read more

Detection and identification of human Plasmodium species with real time quantitative nucleic acid sequence based amplification

Detection and identification of human Plasmodium species with real time quantitative nucleic acid sequence based amplification

Methods: This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA) assays, based on the small- subunit 18S rRNA gene, to identify the four human Plasmodium species. Results: The lower detection limit of the assay is 100 – 1000 molecules in vitro RNA for all species, which corresponds to 0.01 – 0.1 parasite per diagnostic sample (i.e. 50 μ l of processed blood). The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re- reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay.
Show more

6 Read more

Analytical Performance and Clinical Utility of a Nucleic Acid Sequence Based Amplification Assay for Detection of Cytomegalovirus Infection

Analytical Performance and Clinical Utility of a Nucleic Acid Sequence Based Amplification Assay for Detection of Cytomegalovirus Infection

Using a panel of specimens containing known quantities of CMV mRNA, the reproducibility of the NucliSens CMV pp67 assay’s performance was evaluated at three different laboratories with three kit lots over multiple days. CMV mRNA spike. For reproducibility and specificity studies, an RNA spike was synthesized using standard techniques to obtain an exact sequence of 1,125 nucleotides corresponding to authentic CMV pp67 mRNA. Following purifica- tion with an anion-exchange column (Qiagen, Leusden, The Netherlands), the RNA concentration was determined by measuring the absorbance at 260 nm and then diluted to yield an estimated number of input copies for subsequent exper-
Show more

6 Read more

Show all 10000 documents...