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PubMed2005

Model for assessment of proficiency of human immunodeficiency virus type 1 sequencing-based genotypic antiretroviral assays.

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of > or =98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.

Anti-HIV Agents↗

PubMed1995

DNA fingerprinting of pathogenic bacteria by fluorophore-enhanced repetitive sequence-based polymerase chain reaction.

Fluorophore-labeled oligonucleotide primers complementary to defined interspersed repetitive sequences conserved in diverse bacteria were used in the polymerase chain reaction to generate DNA fingerprint patterns from selected pathogenic bacteria. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction allowed discrimination between unrelated isolates of penicillin-resistant Streptococcus pneumoniae recovered from pediatric patients and Mycobacterium avium cultured from patients with acquired immunodeficiency syndrome. Combinations of oligonucleotide primers labeled with distinct fluorescent dyes enabled simultaneous DNA fingerprinting and Shiga-like toxin gene detection in enterohemorrhagic Escherichia coli isolates. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction was performed with either purified DNA or intact cells that were lysed during the polymerase chain reaction. Fluorophore-enhanced, repetitive sequence-based polymerase chain reaction successfully combines polymerase chain reaction amplification and fluorescent label detection for DNA fingerprinting of cultured bacterial pathogens.

Base Sequence↗

PubMed1993

Influence of DNA base sequence on the binding energetics of actinomycin D.

The influences of base sequence on the thermodynamic properties associated with the interaction of actinomycin D with DNA are examined. It has been previously established that GpC steps of double-helical DNAs are highly preferred binding sites for actinomycin D. In this study, a series of oligonucleotides was designed and synthesized to probe the effects of flanking base sequence (adjacent to the GpC step) and novel non-GpC binding sites on the binding of actinomycin D. The use of these oligonucleotides provides a direct method for quantitating sequence specificities and actinomycin D binding energetics. Effects of different 5' and 3' flanking nucleotides on the interactions of actinomycin with the core GpC binding sites were examined using UV-visible spectrophotometric methods, and changes in binding energetics were quantitated. These studies demonstrate strong actinomycin D binding affinities to both classical GpC and an atypical non-GpC site. Enthalpy and entropy components of the DNA binding energetics for the GpC binding sites are compared and correlated with those determined for actinomycin D binding to the high-affinity non-GpC site of an 11-mer containing TGGGT as the central sequence. This TGGGT site, first suggested to be a high-affinity sequence in our earlier photoaffinity labeling studies, exhibits binding of actinomycin D comparable in strength to that of traditional actinomycin D binding sites (i.e., GpC steps). From these studies, the overall affinity and specific thermodynamic contributions (delta H degree, delta S degree) to binding of actinomycin D are demonstrated to be highly influenced both by the sequence at the intercalation site and by neighboring bases which flank the intercalation site.

Amino Acid Sequence↗

PubMed1985

The kinetic properties of cruciform extrusion are determined by DNA base-sequence.

The extrusion kinetics of two cruciforms derived from unrelated DNA sequences differ markedly. Kinetic barriers exist for both reactions, necessitating elevated temperatures before extrusion proceeds at measureable speeds, but the dependence upon temperature and ionic strength is quite different for the two sequences. One, the ColE1 inverted repeat, exhibits a remarkably great temperature dependence of reaction rate and is suppressed by moderate amounts of NaCl or MgCl2. In contrast, the other, a synthetic inverted repeat present in pIRbke8, shows more modest temperature dependence and has a requirement for the presence of salt, with optimal concentrations being 50 mM NaCl or 100 microM MgCl2. Under optimal conditions, cruciform extrusion rates are fast (t1/2 less than 60m) at 37 degrees C for both sequences at native superhelix densities. In 50 mM NaCl the pIRbke8 inverted repeat is characterised by an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole -1. The differences in kinetic properties between the two sequences indicate that DNA base sequence is itself an important factor in determining cruciform kinetics, and possibly even in the selection of the mechanistic pathway.

Base Sequence↗

PubMed2002

The influence of chain length and base sequence on the pharmacokinetic behavior of 99mTc-morpholinos in mice.

BACKGROUND: Despite in vivo use now over several years, in particular for nuclear medicine imaging, the influences on pharmacokinetics of chain length and base sequence of radiolabeled oligomers has not been investigated. METHODS: As test oligomer, morpholinos (MORFs), a DNA analogue, were radiolabeled with 99mTc via MAG3 and the pharmacokinetics in normal mice determined for 3 chain lengths (15, 18 and 25 mer) and 2 base sequences (MORF and its complement cMORF). In addition, LS174T-tumor bearing nude mice received the anti-CEA antibody MN14 (Immunomedics) conjugated either with MORF15 or MORF18 and subsequently received 99mTc-labeled cMORF15 or cMORF18 respectively in a pretargeting strategy. RESULTS: In normal mice, after 1 hr, regardless of chain length or sequence, all labeled MORFs and cMORFs accumulated only slightly in all tissues (e.g. at 3 hr <0.15 ID%/g) except in kidneys. Besides being excessive, the kidneys were the only tissue with levels dependent upon chain length (e.g. at 1 hr, 5, 7 and 22 ID%/g for MORF15, 18 and 25, respectively) and sequence (e.g. at 3 hrs 9 ID%/g for MORF25 and 21 ID%/g for cMORF25). Identical biodistribution trends were observed in tumored mice with all tissues including tumor showing levels independent of chain length or base sequence except for kidneys. Furthermore, while all other tissues cleared in the interval from 1-3 hrs, kidney levels remained constant in both normal and tumored animals. Largely because of these differences in kidneys, images obtained by pretargeting with 99mTc-cMORF15 were superior compared to 99mTc-cMORF18 (images of control animals not receiving the antibody showed no tumor at all). CONCLUSIONS: Judged by radiolabel accumulations in tissue, the pharmacokinetics of 99mTc labeled morpholinos were independent of chain length and base sequence. The only obvious exception was kidneys in which accumulations were significantly higher for the longer chain lengths and significantly different for cMORF vs MORF. These results show that chain length and base sequences may be varied to alter the pharmacokinetics of radiolabeled oligomers in nuclear medicine imaging studies.

Animals↗

PubMedDate not supplied

Using genomic databases for sequence-based biological discovery.

The inherent potential underlying the sequence data produced by the International Human Genome Sequencing Consortium and other systematic sequencing projects is, obviously, tremendous. As such, it becomes increasingly important that all biologists have the ability to navigate through and cull important information from key publicly available databases. The continued rapid rise in available sequence information, particularly as model organism data is generated at breakneck speed, also underscores the necessity for all biologists to learn how to effectively make their way through the expanding "sequence information space." This review discusses some of the more commonly used tools for sequence discovery; tools have been developed for the effective and efficient mining of sequence information. These include LocusLink, which provides a gene-centric view of sequence-based information, as well as the 3 major genome browsers: the National Center for Biotechnology Information Map Viewer, the University of California Santa Cruz Genome Browser, and the European Bioinformatics Institute's Ensembl system. An overview of the types of information available through each of these front-ends is given, as well as information on tutorials and other documentation intended to increase the reader's familiarity with these tools.

Animals↗

PubMed2002

Development and evaluation of nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the NucliSens Basic Kit.

BACKGROUND: Molecular methods based on RNA amplification are needed for sensitive detection of enteroviruses in clinical samples. Many 'in house' methods based on reverse-transcribed PCR (RT-PCR) could be difficult to use in the routine diagnostic laboratory since they tend to be time-consuming, use reagents from many different suppliers and include non-routine procedures. OBJECTIVES: The aim of this study was to develop and evaluate methods based on nucleic acid sequence based amplification (NASBA) for detection of enterovirus sequences. STUDY DESIGN: 'In house' prepared and commercially available reagents were utilised to develop enterovirus-specific NASBA assays. Optimised methods were evaluated using clinical samples (cerebrospinal fluid, respiratory and stool samples), titred virus controls and in vitro produced synthetic RNA. Results for NASBA were compared with RT-PCR and virus culture. RESULTS: Kit-based reagents gave an equivalent sensitivity to the more laborious 'in house' molecular assays (NASBA and RT-PCR) on clinical material and controls. All molecular methods picked up enterovirus positive clinical samples that were not identified by culture. End point detection sensitivity for the NASBA assay based on the NucliSens Basic Kit was <or=1 tissue culture infective dose 50% of a range of enteroviruses or <100 copies RNA input. The assay was specific for enteroviruses and did not pick up high titre rhinovirus preparations. Enterovirus Basic Kit NASBA results for clinical samples were easily obtained within a single working day. CONCLUSIONS: NASBA is a suitable alternative to RT-PCR for sensitive amplification and detection of enterovirus sequences in a range of clinical specimens. The use of kit-based reagents will enable a wide range of laboratories to undertake molecular-based diagnostic procedures for RNA viruses and provide results within a time frame relevant to patient management.

Enterovirus↗

PubMed1992

On the base sequence of the promoter of Alzheimer's disease amyloid A4 precursor gene in transcription initiation.

The base sequence of a specific DNA region of the APP gene identified as the promoter was analyzed by means of the quantity Sr corresponding to superdelocalizability of oxygen ion of each phosphate for the ten DNA dimer units (XY/Y' X') and the six [(XY/Y'X')+H+] complexes. The base sequence of the -35 signal was determined to be 5'-CAGCTG-3' (-49 to -44). On the other hand, the base sequence of the -10 signal was found several ones. Therefore, transcription is to initiate at multiple sites. The mechanisum how RNA polymerases can recognize each transcription site, was proposed. The result explains fairly well some characteristics of the promoter of the APP gene experimentally found so far.

Alzheimer Disease↗

PubMed2005

Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila.

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.

Bacterial Typing Techniques↗

PubMed2004

Sequence-based typing confirmed a novel B40 allele, B4046, which was identified through sequence-specific oligonucleotide hybridization routine typing.

In this paper, we report the identification of a new human leucocyte antigen-B (HLA-B) allele in a sample which has participated in a search for unrelated bone marrow donors which was initiated by the Aktion Knochenmarkspende Bayern. This novel allele officially designed B*4046 was detected by different low-resolution sequence-specific oligonucleotide typings not matching to known allele combinations in a female Caucasoid individual. Confirming the presence of a novel allele by sequence-based typing, the closest-related allele B*400103 differs from the new B*4046 allele by three mutations. This results in an amino acid substitution from threonine to alanine in B*4046 at codon 65, while codon 68 remains conserved and codon 69 "AAG" lysine is replaced by "GAG" glutamic acid in B*4046.

Alleles↗

PubMed2004

Detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (NASBA).

Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of 3 enzymes: AMV reverse transcriptase, RNase H, and T7 RNA polymerase. We have developed a real-time NASBA procedure for detection of piscine nodaviruses, which have emerged as major pathogens of marine fish. Viral RNA was isolated by guanidine thiocyanate lysis followed by purification on silica particles. Primers were designed to target sequences in the nodavirus capsid protein gene, yielding an amplification product of 120 nucleotides. Amplification products were detected in real-time with a molecular beacon (FAM labelled/methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41 degrees C for 90 min in a Corbett Research Rotorgene. Based on the detection of cell culture-derived nodavirus, and a synthetic RNA target, the real-time NASBA procedure was approximately 100-fold more sensitive than single-tube RT-PCR. When used to test a panel of 37 clinical samples (negative, n = 18; positive, n = 19), the real-time NASBA assay correctly identified all 18 negative and 19 positive samples. In comparison, the RT-PCR procedure identified all 18 negative samples, but only 16 of the positive samples. These results suggest that real-time NASBA may represent a sensitive and specific diagnostic procedure for piscine nodaviruses.

Animals↗

PubMed1998

High-resolution HLA typing for the DQB1 gene by sequence-based typing.

The ideal high-resolution typing strategy for polymorphic genes is sequence-based typing. SBT of genomic DNA has been developed for the HLA class II genes DRB1, DRB3/4/5 and DPB1. For the DQB1 gene the sequence-based typing method was shown to cause a number of problems. To resolve those problems, different primers to amplify and sequence exon 2 of DQB1 were designed and tested. With several primer combinations, preferential amplification was observed in individuals heterozygous for DQB1*02/*03 and DQB1*02/*04. The preference was for DQB1*02 in many instances but could also be demonstrated for DQB1*03 or *04 and resulted occasionally in allelic drop-out. The best primer combination was selected and successfully used to type individuals heterozygous for DQB1*02, *03 and *04. To distinguish DQB1*0201 and *0202, primers for amplification and sequencing of exon 3 were developed and correct subtyping was obtained. The ambiguous typing DQB1*0301/*0302 and DQB1*0303/*0304 was resolved by allele-specific amplification and sequencing. A total of 258 individuals were fully typed for their DQB1 subtypes. All samples had been previously typed by PCR-SSP and serology. Concordant typing results were obtained for all individuals tested. The DQB1 alleles detected included *0501, *0502, *0503, *0601, *0602, *0603, *0604, *0609, *0201, *0202, *0301, *0302, *0303, *0304, *0401 and *0402. Sequence-based typing of the DQB1 gene proved a reliable typing strategy for assignment of the different DQB1 alleles after intensive selection of primers and test conditions.

DNA Primers↗

PubMed2005

Comparison of sequence-based and structure-based energy functions for the reversible folding of a peptide.

We used computer simulations to compare the reversible folding of a 20-residue peptide, as described by sequence-based and structure-based energy functions. Sequence-based energy functions are transferable and can be used to describe the behavior of different proteins, since interactions are defined between atomic species. Conversely, structure-based energy functions are not transferable, since the interactions are defined relative to the native conformation, which is assumed to correspond to the global minimum of the energy. Our results indicate that the sequence-based and the structure-based descriptions are in qualitative agreement in characterizing the two-state behavior of the peptide that we studied. We also found, however, that several equilibrium properties, including the free-energy landscape, can be significantly different in the various models. These results suggest that the fact that a model describes the native state of a polypeptide chain does not necessarily imply that the thermodynamic and kinetic properties will also be reproduced correctly.

Algorithms↗

PubMed2004

Detection and quantification of the red tide dinoflagellate Karenia brevis by real-time nucleic acid sequence-based amplification.

Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment.

Animals↗

PubMed2000

Diagnostic implications of human cytomegalovirus immediate early-1 and pp67 mRNA detection in whole-blood samples from liver transplant patients using nucleic acid sequence-based amplification.

Nucleic acid sequence-based amplification (NASBA) was used for detection of the human cytomegalovirus (CMV) immediate early-1 (IE) and the late pp67 mRNA in 353 blood samples collected from 34 liver transplant patients. The diagnostic value of these assays was compared to that of the pp65 antigenemia assay. Overall, 95 and 42% of the antigenemia-positive samples were IE NASBA and pp67 NASBA positive, respectively. Although the results from pp67 NASBA and the antigenemia assay appeared to correspond poorly, a clear correlation was seen between pp67 NASBA-negative results and low numbers of pp65 antigen-positive cells. Twenty patients (59%) were treated with ganciclovir after the diagnosis of symptomatic CMV infection. Before initiation of the antiviral therapy, the antigenemia assay detected the onset of symptomatic infection in all patients, whereas 95 and 60% of these patients were IE NASBA and pp67 NASBA positive, respectively. Although the sensitivity of IE NASBA was very high, the positive predictive value (PPV) of this assay for the onset of a symptomatic infection was only 63%. The PPV of the antigenemia assay as well as pp67 NASBA was considerably higher (80 and 86%, respectively). Thus, the detection of IE mRNA using NASBA appears to be particularly useful as a marker for early initiation of antiviral therapy in patients at high risk for the development of a symptomatic infection. Also, IE NASBA was found to be more sensitive than the antigenemia assay for monitoring CMV infection during antiviral therapy. On the contrary, pp67 NASBA did not appear to have additional diagnostic value compared to the antigenemia assay.

Adult↗

PubMed2001

Omiga: a PC-based sequence analysis tool.

Computer-based sequence analysis, notation, and manipulation are a necessity for all molecular biologists working with any but the most simple DNA sequences. As sequence data become increasingly available, tools that can be used to manipulate and annotate individual sequences and sequence elements will become an even more vital implement in the molecular biologist's arsenal. The Omiga DNA and Protein Sequence Analysis Software tool, version 2.0 provides an effective and comprehensive tool for the analysis of both nucleic acid and protein sequences that runs on a standard PC available in every molecular biology laboratory. Omiga allows the import of sequences in several common formats. Upon importing sequences and assigning them to various projects, Omiga allows the user to produce, analyze, and edit sequence alignments. Sequences may also be queried for the presence of restriction sites, sequence motifs, and other sequence features, all of which can be added into the notations accompanying each sequence. This newest version of Omiga also allows for sequencing and polymerase chain reaction (PCR) primer prediction, a functionality missing in earlier versions. Finally, Omiga allows rapid searches for putative coding regions, and Basic Local Alignment Search Tool (BLAST) queries against public databases at the National Center for Biotechnology Information (NCBI).

Humans↗

PubMed2003

The base sequence dependent flexibility of linear single-stranded oligoribonucleotides correlates with the reactivity of the phosphodiester bond.

The effect of base sequence on the structure and flexibility of linear single-stranded RNA molecules and the influence of the base sequence on phosphodiester bond reactivity have been studied. Molecular dynamics simulations of 2.1 ns were carried out for nine chimeric oligonucleotides containing only one unsubstituted ribo unit, all the rest of sugars being 2'-O-methylated. The base sequence has recently been reported to make a big contribution to the reactivity of these compounds. A detailed examination of the interaction energies between the base moieties shows that base stacking is strongly context-dependent and cooperative. The strength of stacking at the site susceptible to chain cleavage by intramolecular transesterification was observed to be dependent on both the flanking bases of the cleavage site and those further apart in the molecule. The interaction energies between the bases in the vicinity of the scissile linkage were found to correlate well with the experimental phosphodiester bond cleavage rates: the stronger the bases close to the cleavage site are stacked, the slower the cleavage rate is.

Base Sequence↗

PubMed1990

Chicken growth hormone: cDNA-synthesis and base sequence.

1. Growth hormone (GH)-cDNA was synthesized from poly A(+)-mRNA extracts of chicken pituitary glands. 2. Chicken-cDNA library was cloned into E. coli. 3. Base sequence analysis of chicken GH-cDNA revealed only 70% similarity compared with duck GH-cDNA, and 97% similarity with a previously published chicken GH-cDNA sequence. 4. Dissimilarities in base sequences are primarily observed in the 3'-non-coding region of GH-cDNAs (chicken and duck). 5. Comparisons of amino acid sequences of chicken and duck GH exhibit only three substitutions, while the amino acid sequences of GHs of chicken are identical.

Amino Acid Sequence↗