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At least 163 records · Page 9Linked to original sources
PubMed2003

High resolution sequence-based DPB1 typing identified two novel DPB1 alleles, DPB1*9401 and DPB1*9501, from a Kenyan population.

Two novel DPB1 alleles, DPB1*9401 and DPB1*9501, were identified from a Kenyan population during sequence-based HLA-DPB1 typing. Molecular cloning and sequencing of multiple clones confirmed that one of the new DPB1 alleles is identical to DPB1*0402 at exon 2 except for a single nucleotide substitution (CGG -->TGG), changing codon 70 from Arg to Trp. The new allele has been named DPB1*9401. This is the first report of polymorphism at codon 70 of HLA-DPB1 alleles. New codon combinations have been identified in another novel DPB1 allele named DPB1*9501. The extensive diversity at DPB1 locus of this East African population is being revealed by high resolution sequence-based DPB1 typing.

Amino Acid Sequence↗
PubMed2003

Role of base sequence context in conformational equilibria and nucleotide excision repair of benzo[a]pyrene diol epoxide-adenine adducts.

We investigate the influence of base sequence context on the conformations of the 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts through molecular dynamics (MD) simulations with free energy calculations, and relate the structural findings to results of nucleotide excision repair (NER) assays in human cell extracts. In previous studies, these adducts were studied in the CA*A sequence context, and here we report results for the CA*C sequence. Our simulations indicate that the base sequence context affects the syn-anti conformational equilibrium in the 10S (+) adduct by modulating the barrier heights between these states on the energy surface, with a higher barrier in the CA*C case. Our nucleotide excision repair assay finds greater NER susceptibilities in the 10S (+) adduct for the CA*C sequence context. A structural rationale ties together these results. A sequence specific hydrogen bond, accompanied by a significantly increased roll and consequent bending in the 10S (+) adduct, has been found in our simulations for the CA*C sequence, which could account for the enhanced nucleotide excision repair as well as the syn-anti equilibrium difference we observe in this isomer and sequence. Such sequence specific differential repair could contribute to the existence of mutational hotspots and thereby contribute to the complexity of cancer initiation.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Energetics of interaction of oligopeptide (lac 53-57) with DNA base sequences and origin of sequence-specific recognition.

Computer model building with a dynamic energy minimization procedure is used here to study the interaction of a pentapeptide sequence from the lac repressor headpiece (lac 53-57) with different base sequences of DNA. The peptide fragment for this purpose was considered in the classical beta-antiparallel as well as the beta-associated conformation. The model of its interaction with DNA was optimised for various binding positions and base sequences. Partitioning of energy is analysed for different dielectric constant values and the main contributing factors to sequence-specific binding are discussed.

Base Sequence↗
PubMed1997

Comparison of DRB sequence-based typing using different strategies.

Sequence-based typing (SBT) has become an important tool in the identification of HLA alleles. In this study a comparison was made between SBT of DRB1/3/4/5 alleles performed in two laboratories each using a different strategy for SBT. The laboratories in Utrecht and in Maastricht performed direct sequencing of PCR amplified genomic DNA from 30 selected samples. Primers and conditions for PCR amplification were different. Sequencing was either performed with T7 polymerase, using internal sequencing primers, or with cycle sequencing using an M13 tailed system. Two different automated DNA sequencers were used; the ALFexpress from Pharmacia and Applied Biosystems 373A. We concluded that nor the method of sequencing nor the sequencing machine influences typing results. However the PCR reaction used for generating template DNA is the most critical step. Different primers and different conditions can lead to false negative reactions. The fact that these false negative reactions can occur with different alleles in different combinations but not in all, implicates that extensive quality control is needed to assure correct typing results.

HLA-DR Antigens↗
PubMed1995

Sequencing-based typing reveals new insight in HLA-DPA1 polymorphism.

An HLA-DPA1 sequencing-based typing (SBT) system has been developed to identify DPA1 alleles. Up to now eight DPA1 alleles have been defined. Six can be discriminated based upon exon 2 polymorphism. The three subtypes of DPA1*01: DPA1*0101, DPA1*0102 and DPA1*0103, have identical exon 2 sequences but show differences in exon 4. Exon 4 sequences were known for only the three DPA1*01 subtypes and for DPA1*0201. We now present additional sequence information for exon 4 and the unknown segments at the 3' end of exon 2. Additionally with the use of this sequencing technique it is also possible to identify previously unidentified polymorphism. We have studied the exon 2 and exon 4 polymorphism of DPA1 in 40 samples which include all known DPA1 alleles. A new allele, DPA1*01 new, was identified which differs by one nucleotide in exon 2 from DPA1*0103, resulting in an aspartic acid at codon 28. The DPA1*01 subtypes DPA1*0101 and DPA1*0102 could not be confirmed in samples which previously were used to define these subtypes, and consequently they do not exist. The exon 4 sequence of DPA1*0201 is corrected based on sequence data of DAUDI, the cell line in which DPA1*0202 was originally defined. The exon 4 regions of the remaining four alleles were resolved: the exon 4 regions of the alleles DPA1*02021 and DPA1*02022 were found to be identical to the--corrected--DPA1*0201 whereas the exon 4 region of DPA1*0301 differs by one nucleotide compared to DPA1*0103. The DPA1*0401 exon 4 region differs by one nucleotide compared to the corrected DPA1*0201.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2004

Unzipping of DNA with correlated base sequence.

We consider force-induced unzipping transition for a heterogeneous DNA model with a correlated base sequence. Both finite-range and long-range correlated situations are considered. It is shown that finite-range correlations increase stability of DNA with respect to the external unzipping force. Due to long-range correlations the number of unzipped base pairs displays two widely different scenarios depending on the details of the base sequence: either there is no unzipping phase transition at all, or the transition is realized via a sequence of jumps with magnitude comparable to the size of the system. Both scenarios are different from the behavior of the average number of unzipped base pairs (non-self-averaging). The results can be relevant for explaining the biological purpose of correlated structures in DNA.

Base Pairing↗
PubMed2003

Strand breaks produced in X-írradiated crystalline DNA: influence of base sequence.

This study reports the radiation-chemical yields for DNA single-strand breaks (SSBs) in crystals of CGCACG:CGTGCG (I) and CACGCG:CGCGTG (II) duplexes induced by direct ionization using X rays. The DNA fragmentation products, consisting of 3'- and 5'-phosphate-terminated fragments, were quantified by ion-exchange chromatography using a set of reference compounds. The yields of single-strand breaks in I and II are 0.16 +/- 0.03 micro mol/J and 0.07 +/- 0.02 micro mol/J, respectively. The probability of cleavage at a given site is relatively independent of which of the four bases is at that site. For the very small sample of base sequences studied to date, there is no obvious dependence on base sequence. However, there appears to be an increased frequency of strand breaks at the non-phosphorylated termini of the oligodeoxynucleotides. These results show that direct ionization is efficient at producing single-strand breaks in DNA and that its action is relatively indiscriminate with respect to base sequence.

Base Sequence↗
PubMed2002

Characteristics and applications of nucleic acid sequence-based amplification (NASBA).

Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. In some NASBA systems, DNA is also amplified though very inefficiently and only in the absence of the corresponding RNA target or in case of an excess (>1,000-fold) of target DNA over RNA. As NASBA is primer-dependent and amplicon detection is based on probe binding, primer and probe design rules are included. An overview of various target nucleic acids that have been amplified successfully using NASBA is presented. For the isolation of nucleic acids prior to NASBA, the "Boom" method, based on the denaturing properties of guanidine isothiocyanate and binding of nucleic acid to silica particles, is preferred. Currently, electro-chemiluminescence (ECL) is recommended for the detection of the amplicon at the end of amplification. In the near future, molecular beacons will be introduced enabling "real-time detection," i.e., amplicon detection during amplification. Quantitative HIV-1 NASBA and detection of up to 48 samples can then be performed in only 90 min.

DNA↗
PubMed1992

DNA base sequence changes in spontaneous and ethyl methanesulfonate-induced mutations of a chromosomally-integrated gene in Chinese hamster ovary cells.

A series of spontaneous and ethyl methanesulfonate-induced 6-thioguanine-resistant mutants were isolated in the CHO-10T5 cell line. This cell line was constructed by the introduction of a shuttle vector containing the Escherichia coli gpt gene into a hypoxanthine-guanine phosphoribosyltransferase deficient derivative of the Chinese hamster cell line CHO-K1. Shuttle vector sequences were recovered from many of the mutant cell lines by the COS cell fusion technique and the DNA base sequence of the gpt genes was determined whenever possible. The base sequences were determined for gpt genes recovered from 29 spontaneous mutants. Of these 29 mutants, 9 have single base substitutions, 1 has a small duplication, 17 have simple deletions, 1 has a deletion with additional bases inserted at the deletion site, and 1 has no change in the gpt coding sequence. Many of the deletions were less than 20 basepairs in length and several occurred in a region previously observed to be a hotspot for spontaneous deletions. The generation of the deletion/insertion mutation may have involved a quasi-palindromic intermediate. A total of 59 ethyl methansesulfonate-induced mutants were isolated and vector sequences were recovered from 50 mutants. All 50 mutants sequenced had single base substitutions and most (45) were G:C to A:T transitions. While there were no strong hotspots in this collection of mutations, the site distribution was obviously nonrandom. Many of the G:C to A:T transitions either produced a nonsense codon or occurred at glycine codons.

Animals↗
PubMed2004

DNA nucleic acid sequence-based amplification-based genotyping for polymorphism analysis.

Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method known to be a suitable tool for RNA research. We demonstrate that NASBA technology can be applied to single nucleotide polymorphism (SNP) analysis using human genomic DNA as a template. Combination of DNA NASBA with multiplex hybridization of specific molecular beacons makes it possible to unambiguously discriminate the presence of the SNP of interest. This protocol is easy-to-use, robust, and makes it possible to rapidly detect single nucleotide substitutions in clinical or cell line DNA sequences using a large range of DNA input. Such a real-time genotyping DNA NASBA assay can find broad application in clinical diagnostics.

Base Sequence↗
PubMed1997

Comparison of major groove hydration in isomorphous A-DNA octamers and dependence on base sequence and local helix geometry.

The family of ten isomorphous tetragonal A-DNA octamers provides a unique opportunity to examine major groove hydration in terms of base sequence and local parameter effects. The presence of a severely underwound central py.pu base step (average = 24.1 degrees), which lies on a crystallographic 2-fold in the unit cell, provides a sharp change in the local environment in which to study and separate the effects of base sequence and local helix geometry on major groove hydration. For this reason, and to avoid bias secondary to end effects, hydration analysis was restricted to the central four dyad-related base paris. This study finds that d(CG) base pairs are better hydrated than d(TA) base pairs, 2.5 H2O vs 1.3 H2O; steps with high twist angles are better hydrated than steps with low twist angles, 6.9 H2O vs 0 H2O; negative roll angles are better hydrated than positive roll angles, 2.8 H2O vs 1.8 H2O; and flanking base pairs are better hydrated than central base pairs, 2.6 H2O vs 2.0 H2O, a phenomenon which is sequence independent, occurring for both d(CG) and d(TA) base pairs. The twist angle and base roll combine to significantly affect the pattern and degree of major groove hydration in this family of octamers. A previous study of A-DNA octamers and their helix parameters established a strong dependency on crystal packing forces with little or no dependence on the base sequence [Ramakrishnan & Sundaralingam (1993) J. Biomol. Struct. Dyn. 11. 11-26]. We find that the degree and pattern of major groove hydration are strongly influenced by the local helix parameters, implying an indirect, but significant, relationship between major groove hydration and environmental forces, i.e., crystal packing, drug binding, and protein-DNA interactions.

Crystallography, X-Ray↗
PubMed2002

Detection of Mycoplasma pneumoniae in spiked clinical samples by nucleic acid sequence-based amplification.

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, bronchoalveolar lavage specimens, and sputum specimens, the sensitivity of the NASBA assay in the presence of the internal control was 2 x 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.

Humans↗
PubMed2002

Rational design of DNA sequence-based strategies for subtyping Listeria monocytogenes.

The ability to differentiate bacteria beyond the species level is essential for identifying and tracking infectious disease outbreaks and to improve our knowledge of the population genetics, epidemiology, and ecology of bacterial pathogens. Commonly used subtyping methods, such as serotyping, phage typing, ribotyping, and pulsed-field gel electrophoresis, can yield ambiguous results that are difficult to standardize and share among laboratories. DNA sequence-based subtyping strategies can reduce interpretation ambiguity. We report the development of a rational approach for designing sequence-based subtyping methods. Listeria monocytogenes was selected as the model organism for testing the efficacy of this approach. Two housekeeping genes (recA and prs), one stress response gene (sigB), two virulence genes (actA and inlA), and two intergenic regions (hly-mpl and plcA-hly) were sequenced for 15 L. monocytogenes isolates. Isolates were chosen from a representative collection of more than 1,000 L. monocytogenes isolates to reflect the genetic diversity of this species. DNA sequences were aligned, and sliding window analyses were performed for each gene to define 600-bp-long regions that were (i) most polymorphic (using ProSeq) or (ii) most discriminatory (using a new algorithm implemented in WINDOWMIN). Complete gene sequences for actA (1,929 bp) and inlA (2,235 bp) provided the highest discrimination (identifying 15 and 14 allelic types, respectively). WINDOWMIN allowed identification of 600-bp regions within these genes that provided similar discriminatory power (yielding 15 and 13 allelic types, respectively). The most discriminatory 600-bp fragments identified in the housekeeping and stress response genes differentiated the isolates into 8 to 10 subtypes; intergenic region sequences yielded 8 and 12 allelic types based on 335- and 242-bp sequences for hly-mpl and plcA-hly, respectively. Regions identified as most polymorphic were not necessarily most discriminatory; therefore, application of the WINDOWMIN algorithm provided a powerful tool for determining the best target regions for DNA sequence-based subtyping. Our specific results also show that inclusion of virulence gene target sequences in a DNA sequence-based subtyping scheme for L. monocytogenes is necessary to achieve maximum subtype differentiation.

Algorithms↗
PubMed2004

Unexpected detection of DNA by nucleic acid sequence-based amplification technique.

Nucleic acid sequence-based amplification (NASBA) is a technique that has been previously shown to selectively mediate the detection of RNA in microbial cells. In a series of tests, nucleic acids were extracted from Salmonella enterica serotype Typhimurium and Mycobacterium avium subsp. paratuberculosis, and subjected to four enzymatic treatments prior to NASBA. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase/S1 nuclease. The results obtained were different for the two bacteria. With S. enterica serotype Typhimurium, RNase and RNase/S1 nuclease abolished the NASBA signal, as expected. But with M. avium subsp. paratuberculosis RNase, S1 nuclease, and RNase/S1 nuclease had no effect on the NASBA signal, whereas DNase treatment abolished it. This indicates that in the latter bacterium, NASBA can detect DNA, and demonstrates the necessity of verifying the nucleic acid origin of a NASBA signal if detection of RNA is objective.

DNA, Bacterial↗
PubMed2000

Identification of a new DRB3*02 allelic variant (DRB3*0209) by high-resolution sequence-based typing.

The HLA-DRB3/B4/B5 sequence-based typing method developed in this study in combination with PCR-SSP, enabled us to identify a new DRB3*02 allele, that was named as DRB3*0209 (GenBank accession number AF148518). This name has been officially assigned by the WHO Nomenclature Committee in May 1999. The new allele differs from DRB3*0207 by one substitution in codon 51 from AGG to ACG and another in codon 60 from TAC to TCC, resulting in aminoacid changes from Arg-->Thr (codon 51) and from Tyr-->Ser (codon 60). The DRB3*0209 allele was discovered in two related North Italian families. The fact that it was present in an hemizygous situation in three members of the paternal family and in one member of the secondary related family enabled us to isolate and sequence the new DRB3 allele without cloning, to identify its association with the DRB1 locus, and to generate an Epstein-Barr virus (EBV)-transformed cell line, now present in our ECBR (European Collection for Biomedical Research) Cell Line Bank.

Alleles↗
PubMed1991

On the base sequences of the promoters in transcription initiation.

The base sequence of a specific DNA region identified as the promoter is investigated 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. A mechanisum that the RNA polymerase can recognize its transcription site (phosphate), is proposed and applied to Escherichia coli promoters, lacUV5, recAp, rrnEp1, rrnEp2 (experimental facts).

Base Sequence↗