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At least 253 records · Page 14Linked to original sources
PubMed1999

Identification of a novel HLA-DQA1 allele (DQA1*0106) by sequence-based DQA1 typing.

We report here a novel DQA1 allele (DQA1*0106) identified during sequence-based HLA-DQA1 typing. Polymerase chain reaction with proofreading pfu DNA polymerase and subsequent sequencing yielded identical results as that with Taq DNA polymerase. Molecular cloning and sequencing confirmed that the new DQA1 allele is identical to DQA1*01021/2 at exon 2 except for a single nucleotide substitution (ACT-->GCT), changing codon 44 from Thr to Ala. This is the first report of polymorphism at codon 44 of HLA-DQA1 alleles.

Alanine↗
PubMedDate not supplied

High throughput HLA sequence-based typing (SBT) utilizing the ABI Prism 3700 DNA Analyzer.

AIMS AND BACKGROUND: The genetic complexity of the human major histocompatibility complex (MHC) has required the development of various molecular typing methods. The purpose of this paper is to compare the results of two of these molecular methods: sequenced based typing (SBT) and polymerase chain reaction (PCR) using sequence specific primers (PCR-SSP). METHODS: The SBT method described utilizes an ABI Prism 3700 DNA Analyzer, which has been designed fro high throughput production of sequence data through highly automated operation with significant walk-away time. The ABI Prism 3700 DNA Analyzer is a 96-capillary electrophoresis instrument with the capability of running four 96-well plates black to back in a sixteen-hour period. Potentially, data from this machine can produce Class I sequences for A or B loci for 64 samples in this time frame. The SBT method encompassed exons 2, 3, and 4 with forward and reverse sequence orientation reactions using the PE Biosystems HLA-A and HLA-B Sequenced Based Typing Kits (PE Applied Biopsystems/Perkin-Elmer, Foster City, CA, USA). Most SBT methods previously employed only gather data from exons 2 and 3 which distinguishes most of the polymorphism necessary to identify the majority of alleles in the HLA region. However, in an effort to discern numerous null alleles in the HLA region, exon 4 data is also included. The PCR-SSP method utilized consists of one 96 well tray, with 95 primer mixes and one negative control, per sample designed to produce an intermediate/high resolution HLA-A, B typing. RESULTS: Data from one 96-well capillary run on the ABI Prism 3700 DNA Analyzer, which consists of results from 16 samples for HLA-A or HLA-B loci, was compared to data derived from sixteen HLA-A and HLA-B PCR-SSP typings. 75% of loci tested achieved a higher resolution HLA typing by the SBT method. DISCUSSION: The ability to provide allele level HLA typing results can have significant functional implications for the bone marrow transplant community and numerous vaccine studies.

DNA↗
PubMed2002

New HLA-A*11 allele, A*1112, identified by sequence-based typing.

In this report, we describe the identification of HLA-A*1112, a novel HLA-A*11 allele found in two Italian families. The new allele was detected during routine HLA typing by a polymerase chain reaction sequence-specific primer and was confirmed by high-resolution sequencing-based typing. The nucleotide sequences of HLA-A*1112 exons 2 and 3 are identical to HLA-A*11011 except for a single nucleotide substitution in codon 90 (GAC-->GCC).

Alleles↗
PubMed2004

Identification of a new HLA-C allele, Cw*0316, by sequence-based typing.

The presence of a new allele, Cw*0316, was detected in a Caucasian individual through an unusual association. Molecular typing of the individual by sequence-specific primers and sequence-specific oligonucleotides showed the presence of B*58, B*41 and Cw*17. Sequence-based typing revealed the additional presence of another human leucocyte antigen-C allele. The new allele showed four nucleotide differences with Cw*030202 at positions 559, 560, 589 and 594 in exon 3, leading to three codon changes, codons 187, 197 and 198. This resulted in two amino acid substitutions at positions 163 (L-T) and 173 (K-E) of the mature protein, which proved sufficient to abrogate serological reactivity with Cw3-specific sera.

Alleles↗
PubMed2004

Two novel HLA-DRB1 alleles identified using a sequence-based typing: HLA-DRB1*1443 and HLA-DRB1*1351*.

Two novel HLA-DRB1 alleles, DRB1*1443 and DRB1*1351, were identified using a sequence-based typing protocol. DRB1*1443 differed from DRB1*140501 by one single-nucleotide substitution in exon-2 (codon 77, ACC-->GCC), which corresponded to an amino acid change of threonine to alanine. DRB1*1351 was identical to DRB1*1301 but differed by a single-nucleotide substitution at codon 50 (GTG-->TTG), resulting in an amino acid change of valine to leucine. Both new alleles present unique polymorphisms, which have not been seen among other DRB1 alleles and which have no known effect on peptide binding.

Alleles↗
PubMed2005

Multispacer typing technique for sequence-based typing of Bartonella quintana.

Bartonella quintana is a worldwide fastidious bacterium of the Alphaproteobacteria responsible for bacillary angiomatosis, trench fever, chronic lymphadenopathy, and culture-negative endocarditis. The recent genome sequencing of a B. quintana isolate allowed us to propose a genome-wide sequence-based typing method. To ensure sequence discrimination based on highly polymorphic areas, we amplified and sequenced 34 spacers in a large collection of B. quintana isolates. Six of these exhibited polymorphisms and allowed the characterization of 4 genotypes. However, the strain variants suggested by the noncoding sequences did not correlate with the results of pulsed-field gel electrophoresis (PFGE), which suggested a higher degree of variability. Modification of the PFGE profile of one isolate after nine subcultures confirmed that rearrangement frequencies are high in this species, making PFGE unreliable for epidemiological purposes. The low extent of sequence heterogeneity in the species suggests a recent emergence of this bacterium as a human pathogen. Direct typing of natural samples allowed the identification of a fifth genotype in the DNA extracted from a human body louse collected in Burundi. We have named the typing technique herein described multispacer typing.

Animals↗
PubMed2001

Identification of a novel DRB1 allele, DRB1*1112, by sequence-based DRB typing.

We report here a novel DRB1 allele (DRB1*1112) identified during sequence-based HLA-DRB typing. Polymerase chain reaction with generic DRB primers and group-specific primers and subsequent sequencing yielded identical results. Molecular cloning and sequencing confirmed that the new DRB1 allele is identical to DRB1*11011 and 1129 at exon 2 except for a single nucleotide substitution at codon 37, changing the codon from Tyr (DRB1*11011) or Ser (DRB1*1129) to Phe.

Aged↗
PubMed2003

Establishment of a sequence-based typing system for BoLA-DRB3 exon 2.

A rapid, high-resolution sequence-based typing (SBT) system for BoLA-DRB3 exon 2 was developed. Amplification of the entire exon was achieved by a fully nested PCR with locus-specific primers and sequencing was performed directly on the PCR product. Heterozygous sequence data were obtained by automated sequence analysis of both alleles. Forward and reverse sequence data were assembled to improve identification of all heterozygous positions. Specific software (Haplofinder, Roslin Institute Software, Roslin, UK) was designed for allele assignment. Fifty-four females from a Holstein-Charolais resource herd cross, their 12 sires and five unrelated Holstein animals were used to establish the method. In parallel, these animals were typed by DRB3 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to confirm the results. Polymerase chain reaction-RFLP analysis defined 15 known types in the 71 animals, while SBT of the same animals showed 19 known alleles. Subsequently, 72 more animals from the same resource herd were typed by the established SBT method without PCR-RFLP typing. This SBT strategy and the Haplofinder software can be applied to the analysis of any polymorphic locus for which suitable locus-specific primers and allelic sequences are available.

Alleles↗
PubMed2003

Sequence-based alignment of sorghum chromosome 3 and rice chromosome 1 reveals extensive conservation of gene order and one major chromosomal rearrangement.

The completed rice genome sequence will accelerate progress on the identification and functional classification of biologically important genes and serve as an invaluable resource for the comparative analysis of grass genomes. In this study, methods were developed for sequence-based alignment of sorghum and rice chromosomes and for refining the sorghum genetic/physical map based on the rice genome sequence. A framework of 135 BAC contigs spanning approximately 33 Mbp was anchored to sorghum chromosome 3. A limited number of sequences were collected from 118 of the BACs and subjected to BLASTX analysis to identify putative genes and BLASTN analysis to identify sequence matches to the rice genome. Extensive conservation of gene content and order between sorghum chromosome 3 and the homeologous rice chromosome 1 was observed. One large-scale rearrangement was detected involving the inversion of an approximately 59 cM block of the short arm of sorghum chromosome 3. Several small-scale changes in gene collinearity were detected, indicating that single genes and/or small clusters of genes have moved since the divergence of sorghum and rice. Additionally, the alignment of the sorghum physical map to the rice genome sequence allowed sequence-assisted assembly of an approximately 1.6 Mbp sorghum BAC contig. This streamlined approach to high-resolution genome alignment and map building will yield important information about the relationships between rice and sorghum genes and genomic segments and ultimately enhance our understanding of cereal genome structure and evolution.

Base Sequence↗
PubMedDate not supplied

Base sequence selectivity in the binding of 7(R),8(S)-dihydroxy-9(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to oligodeoxyribonucleotide duplexes.

The effect of nucleotide sequence on the binding of 7(R),8(S)-dihydroxy-9(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] to the exocyclic amino group of deoxyguanosine was investigated in duplexes formed by self-complementary oligodeoxyribonucleotide decamers which contained two deoxyguanosines (dGs) within unique sequences. A 35S-postlabeling procedure was developed for analysis of (+)-anti-BPDE adducts as dinucleotides containing 5'-(+)-anti-BPDE-dG adducts. This allows identification of the 3' neighbor of the reacted guanine and permits quantitation of the binding of (+)-anti-BPDE to each specific guanine in the oligodeoxyribonucleotide duplexes. Of all the central dG-containing sequences studied, dG surrounded by deoxycytidines (CGC) reacted to the greatest extent: over 4-fold more (+)-anti-BPDE bound to this central dG compared to the least reactive deoxyguanosine (AGT). (+)-anti-BPDE exhibited a preference for binding to a central deoxyguanosine when either the 5' or 3' neighbor was deoxyguanosine. The binding of (+)-anti-BPDE to oligodeoxyribonucleotide duplexes containing different numbers of consecutive dGs was analyzed in order to determine how the length of these sequences influences binding. Increases in the length of consecutive deoxyguanosine residues from 3 to 5 had little effect on the quantity of (+)-anti-BPDE bound to dG above that expected from the presence of a neighboring dG and an increase in the number of dG residues available for reaction. The results obtained with these oligodeoxyribonucleotide duplexes were consistent with the data available for the reaction of (+)-anti-BPDE with DNA, indicating that these duplexes are a valuable model for studying the effect of base sequence on the interaction of BPDE isomers with DNA. The dinucleotide postlabeling technique developed for these studies, with appropriate oligodeoxyribonucleotides and chromatographic conditions, will be useful for determining the effect of base sequence on the binding of other hydrocarbon diol epoxides as well as other reactive hydrocarbon metabolites to deoxyguanosine or deoxyadenosine in oligodeoxyribonucleotide duplexes and fragments of DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1996

Repetitive element sequence-based PCR for species and strain discrimination in the genus Listeria.

Repetitive element sequence-based PCR (rep-PCR) was used to generate DNA fingerprints for Listeria spp. Two primer sets (REP 1R-I REP 2-I and ERIC 1R ERIC 2) used in respectively REP- and ERIC-PCR revealed that bacteria of the genus Listeria possess short repetitive extragenic palindromic elements and enterobacterial repetitive intergenic consensus sequences. Specific band profiles obtained by ERIC-PCR enabled the identification of Listeria species. With both REP- and ERIC-PCR the L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3b and 4b could be clearly distinguished from each other. Within the serotype 1/2a, REP-PCR showed a higher discriminative potential than ERIC-PCR and a comparable discriminative potential as RAPD combining 3-4 primers.

Base Sequence↗
PubMed1989

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells.

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.

Animals↗
PubMed2001

Sequence-based typing of HLA class I alleles in Alaskan Yupik Eskimo.

In comparison to South America, native North Americans tend to be less diverse in their repertoire of HLA class I alleles. Based upon this observation, we hypothesized that the Yupik Eskimo would exhibit a limited number of previously identified class I HLA alleles. To test this hypothesis, sequence-based typing was performed at the HLA-A, -B and -C loci for 99 Central Yupik individuals from southwestern Alaska. Two new class I alleles, A*2423 and Cw*0806, were identified. While A*2423 was observed in only one sample, Cw*0806 was present in 26 of the 99 individuals and all of the Cw*0806 samples contained B*4801. Allele Cw*0806 differs from Cw*0803 by a single nucleotide substitution such that Cw*0803 may be the progenitor of Cw*0806. Allele Cw*0803 was originally characterized as unique to South America, but detection of Cw*0803 in the Yupik indicates that Cw*0803 was a founding allele of the Americas. The presence of new alleles and previously unrecognized founding alleles in the Yupik population show that natives of North America are more diverse than previously envisioned.

Alaska↗
PubMed1991

A trace display and editing program for data from fluorescence based sequencing machines.

'Ted' (Trace editor) is a graphical editor for sequence and trace data from automated fluorescence sequencing machines. It provides facilities for viewing sequence and trace data (in top or bottom strand orientation), for editing the base sequence, for automated or manual trimming of the head (vector) and tail (uncertain data) from the sequence, for vertical and horizontal trace scaling, for keeping a history of sequence editing, and for output of the edited sequence. Ted has been used extensively in the C.elegans genome sequencing project, both as a stand-alone program and integrated into the Staden sequence assembly package, and has greatly aided in the efficiency and accuracy of sequence editing. It runs in the X windows environment on Sun workstations and is available from the authors. Ted currently supports sequence and trace data from the ABI 373A and Pharmacia A.L.F. sequencers.

Animals↗
PubMedDate not supplied

Base sequence selectivity in the alkylation of DNA by 1,3-dialkyl-3-acyltriazenes.

The base sequence selectivity of DNA alkylation for a series of structurally related 1,3-dialkyl-3-acyltriazenes was examined with calf thymus DNA or polymers containing the sequences GGG, CGC, TGT, and AGA. The reaction products at the N7 and the O6 positions of guanine were identified, quantitated, and then correlated with the decomposition rates of the triazenes, 1-(2-chloroethyl)-3-methyl-3-carbethoxy- (CMC), 1-(2-chloroethyl)-3-methyl-3-acetyl- (CMA), 1-(2-hydroxyethyl)-3-methyl-3-carbethoxy- (HMC), 1-(2-hydroxyethyl)-3-methyl-3-acetyl- (HMA), and 1,3-dimethyl-3-acetyl- (DMA). The results of these studies revealed that DNA sequences with runs of purines were more reactive toward alkylation by all of the triazenes tested, irrespective of whether the alkylation was measured by N7, O6, or total guanine adducts. Within this generalization, the (hydroxyethyl)triazenes showed a preference for the AGA sequence, while the (chloroethyl)triazenes favored the GGG sequence. The structure of the 3-acyl group of the triazene also played a role in the extent of alkylation of a particular sequence of DNA. Both the (chloroethyl)- and the (hydroxyethyl)triazenes produced higher alkylation product yields for the 3-carbethoxytriazenes as compared with the 3-acetyl derivatives for most of the sequences examined. These overall patterns correlated well with the order of decomposition of the triazenes at 37 degrees C: HMC > DMA > HMA > CMC > CMA. This study has demonstrated how varying the structure of 1,3-dialkyl-3-acyltriazenes can modulate DNA alkylation, a finding which may be important in the design of new triazene antitumor agents.

Alkylation↗
PubMed1997

Molecular typing of Staphylococcus epidermidis and other CNS with repetitive element sequence-based PCR.

The chromosomal distribution of the repetitive DNA sequence found in Mycoplasma pneumoniae (REP-MP2) provides an ideal target for detecting DNA fragment patterns specific to individual Staphylococcus epidermidis and S. haemolyticus strains. A REP-MP2 sequence-based PCR (rep-PCR) was developed and applied to CNS isolates. We identified a 450 bp genomic DNA fragment which was common and specific to S. epidermidis isolates and not found in other CNS. In addition, S. epidermidis isolates showed several bands that could be grouped into 14 different fragment patterns. Similarly, S. haemolyticus isolates were classified into 10 groups. Significant correlations between the typing patterns of S. epidermidis and resistance to oxacillin (P< 0.05), gentamicin (P< 0.01), erythromycin (P< 0.02), and sulfamethoxazole-trimethoprim (P< 0.001) were found. The rep-PCR method is a rapid and reproducible discriminatory means for molecular typing of S. epidermidis and other CNS.

Anti-Bacterial Agents↗
PubMed2003

Detection of Mycoplasma pneumoniae by real-time nucleic acid sequence-based amplification.

Real-time isothermal nucleic acid sequence-based amplification (RT-NASBA) was applied to the detection of Mycoplasma pneumoniae. In vitro-generated M. pneumoniae RNA was used to assess the sensitivity of the assay. The 95% hit rate was 148 molecules of M. pneumoniae RNA in the amplification and 10(4) molecules of in vitro-generated RNA after nucleic acid extraction. The sensitivity of the RT-NASBA and the conventional NASBA assays corresponded to 5 color-changing units (CCU) of M. pneumoniae. In spiked throat swabs, nasopharyngeal aspirates, bronchoalveolar lavages, and sputum, the sensitivity of both NASBA assays corresponded to 5 to 50 CCU of M. pneumoniae. A total of 17 clinical specimens positive for M. pneumoniae by PCR were also positive by conventional NASBA, but one specimen was negative by RT-NASBA. These results indicate that the sensitivity of detection of M. pneumoniae by RT-NASBA in respiratory samples might be slightly reduced compared to that by conventional NASBA. However, the real-time assay is superior in speed and ease of handling.

Humans↗