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PubMed2004

Multiparametric duplex real-time nucleic acid sequence-based amplification assay for mRNA profiling.

Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method. We have developed real-time NASBA assays to detect mRNA coding for the estrogen receptor alpha (ESR1) and the progesterone receptor (PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB) as the normalizing gene. Both the ESR1/PPIB and PGR/PPIB duplex NASBA assays are highly sensitive, specific, and reproducible. Quantification is determined using external standard calibration curves and the ratio between the number of target and housekeeping gene mRNA copies. Amplification of the target gene in the duplex NASBA assay was disrupted when this latter was mixed with a large amount of the housekeeping PPIB gene, suggesting that it is preferable for the normalizing gene chosen to have an expression level comparable to the target gene. Sensitivity and robustness of the duplex NASBA assays were assessed in breast cancer cell lines. Such a rapid and easy-to-use multiparametric duplex real-time NASBA assay could also advantageously be set up for other mRNA profiling applications.

Biological Assay↗

PubMed2000

Identification of an HLA-B7 serological variant and its characterization by sequencing based typing.

We have identified an HLA-B*07 variant allele, B*0716, in a Caucasoid cadaver kidney donor. The HLA class I type by polymerase chain reaction using sequence-specific primers (PCR-SSP) was A*01, 32; B*07, 08; Cw*07. Serological typing, using monoclonal and polyclonal anti-HLA antisera, gave disparate results for the B antigens. Monoclonal antibodies identified B7 and B8 antigens but polyclonal antisera recognised only the B8 antigen. PCR using sequencing based typing (PCR-SBT) confirmed the presence of both B*0703 and B*0801 alleles but with a mutation in one of the alleles. The HLA-B*07 allele was isolated by allele-specific PCR and was shown to have a mutation, G-->T, at 292 in exon 2. This mutation changes codon 74, which encodes aspartic acid (GAC) present in all previously identified B*07 alleles, to tyrosine (TAC) in the variant. The serological results suggest that codon 74 is a crucial part of a B7 antigen-specific epitope recognised by tissue typing polyclonal antisera.

Alleles↗

PubMed1988

DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian cells.

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected. Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined. The great majority of the BrdUrd-induced mutations involving single-base changes were found to be G.C----A.T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrd-induced G.C----A.T transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore, in approximately equal to 90% of the cases, the guanine residue involved in mutation was the one in the more 3' position.

Base Composition↗

PubMed2005

Description and characterization of two new HLA alleles, B4051 and DRB11364, identified by sequence-based typing.

Human leukocyte antigen (HLA)-B and HLA-DRB1 typing in two female individuals revealed reaction patterns that did not correspond to any known HLA-B specificity and appeared to identify a very rare HLA-DRB1 allele, respectively. Sequence-based analysis of these samples revealed two new HLA alleles, one similar to B*4023 and the other to DRB1*1308. The new HLA-B allele, which was assigned the name HLA-B*4051, could have been generated by a double crossing over recombination between B*4001 and B*1401 or 1402, whereas DRB1*1364, the new DRB1 allele, could have been generated either by a double crossing over recombination between DRB1*1308 and DRB1*1201, 1202, or 1203 or by two independent crossing over events between DRB1*1401, DRB1*1201, 1202, or 1203 and DRB1*1301.

Alleles↗

PubMed2002

Map error reduction: using genetic and sequence-based physical maps to order closely linked markers.

The Marshfield comprehensive genetic maps are frequently used for linkage and association studies, however, for some regions of these maps the marker order has low level of likelihood ratio support. In order to investigate the level of statistical support and the accuracy of the genetic maps compared to sequence-based physical maps, two approximately 30 cM autosomal regions were selected. The first region was selected from chromosome 3 and consisted predominately of draft sequence. The second region was selected from chromosome 21 and consisted of finished sequence data. The physical order of these markers was based upon their position on Celera (CEL) and Human Genome Project-Santa Cruz (HGP-sc) sequence-based physical maps. The chromosome 3 and 21 regions contained 100 and 61 markers, respectively, on the Marshfield genetic map. The genetic and physical map order was consistent for 88.9 and 89.2% of the markers in the region on chromosome 3 and 21, respectively. Using a novel scoring criterion to assess inconsistent marker order between genetic and physical maps, it was determined that the physical order was likely the correct order for 3.3 and 7.1% of the markers in the chromosome 3 and 21 regions, respectively. To increase the accuracy of the order of markers selected for fine mapping a method is presented which combines information from genetic and sequence-based physical maps.

Genetic Linkage↗

PubMed2001

Base sequence effects in bending induced by bulky carcinogen-DNA adducts: experimental and computational analysis.

The covalent binding of bulky mutagenic or carcinogenic compounds to DNA can lead to bending, which could significantly alter the interactions of DNA with critical replication and transcription proteins. The impact of adducts derived from the highly reactive bay region enantiomeric (+)- and (-)-anti-7,8-diol-9,10-epoxide derivatives of benzo[a]pyrene (BPDE) are of interest because the (+)-7R,8S,9S,10R-anti-BPDE enantiomer is highly tumorigenic in rodents, while the (-)-7S,8R,9R,10S-anti-BPDE enantiomer is not. Both (+)- and (-)-anti-BPDE bind covalently with DNA predominantly by trans addition at the exocyclic amino group of guanine to yield 10S (+)- and 10R (-)-trans-anti-[BP]-N(2)-dG adducts. We have synthesized a number of different oligonucleotides with single (+)- and (-)-trans-anti-[BP]-N(2)-dG adducts (G) in the base sequence context XG*Y, where X and Y are different DNA bases. The G* residues were positioned at or close to the center of 11 base pair ( approximately 1 helical turn) or 16 base pair ( approximately 1.5 turns) duplexes. All bases, except for X and Y and their partners, were identical. These sequences were self-ligated with T4 ligase to form multimers that yield a ladder of bands upon electrophoresis in native polyacrylamide gels. The extent of bending in each oligonucleotide was assessed by monitoring the decrease in gel mobilities of these linear, self-ligated oligomers, relative to unmodified oligonucleotides of the same base sequence. The extent of global bending was then estimated using a sequence-specific three-dimensional model from which the values of the base-pair step parameter roll adjacent to the lesion site could be extracted. We find that (+)-trans-anti-[BP]-N(2)-dG adducts are considerably more bent than the (-) isomers regardless of sequence and that A-T base pairs flanking the [BP]-N(2)-dG lesion site allow for local flexibility consistent with adduct conformational heterogeneity. Interestingly, the fit of computed versus observed gel mobilities using classical reptation treatments requires enhancement of unmodified DNA flexibility in gels, compared to aqueous salt solution. The differences in bending between the two stereoisomeric adduct duplexes and the observed base sequence context effects may play a significant role in the differential processing of these lesions by cellular replication, transcription, and repair enzymes.

Algorithms↗

PubMed1996

Genetic diversity of Neisseria gonorrhoeae IB-2 and IB-6 isolates revealed by whole-cell repetitive element sequence-based PCR.

Phenotypic characterization of 19 Neisseria gonorrhoeae serovar IB-2 and 8 serovar IB-6 isolates by the combined use of auxotypes, serological characterization, and penicillin susceptibility testing indicated intraserovar genetic diversity. In the present study, we applied whole-cell repetitive element sequence-based PCR (rep-PCR) analysis which allows a rapid assessment of the clonal relationships of IB-2 and IB-6 isolates. DNA templates were prepared by boiling cells harvested directly from plate cultures, eliminating the need for time-consuming phenol extraction. Six different rep-PCR profiles were established among the 19 IB-2 isolates. Rep-PCR typing results had a good correlation with pulsed-field gel electrophoresis patterns. It is slightly less discriminatory than BglII-generated macrorestriction pattern analysis by pulsed-field gel electrophoresis. It is capable of discriminating epidemiologically related from epidemiologically unrelated IB-2 isolates. It should serve as a rapid and useful subtyping tool for epidemiologic investigations in which there is a predominance of major serovar groups.

Bacterial Typing Techniques↗

PubMed1997

A new DRB115 allele (DRB11506) identified by sequence-based typing.

A new DRB1*15 allele (DRB1*1506) was detected during the studies of the 12th International Histocompatibility Workshop within the Allele and Haplotype Society #11 which studied the DR2 and DR51 antigens. The new allele was found in four unrelated Asian Indian individuals by sequence-based typing. It has a base substitution from T to C in codon 50 at a previously considered conserved position.

Alleles↗

PubMed1999

Comparative analysis of HLA-B 8101 by serology, isoelectric focusing, and sequence-based typing.

We describe a bone marrow donor evaluation on a twenty-four-year-old patient from Honduras, Central America. The patient's phenotype included a HLA-B7v, with serologic reactivity to HLA- B7, 8101, and 48, and was consistent with, HLA-B*8101. One-dimensional isoelectric focusing (IEF) identified HLA-B 7.1, a low frequency isotype that has previously been associated with the HLA-B7 variant, B*0705. To further classify this allele, sequence based typing (SBT) was performed, confirming HLA-B*8101 sequence homology. The diagnostic evaluation of this case illustrates the correlation of sequenced based typing and isoelectric focusing in defining HLA Class I antigen characteristics and the use of IEF in screening for rare and novel alleles.

HLA-B Antigens↗

PubMed1999

The influence of base sequence on the immunostimulatory properties of DNA.

DNA is a complex macromolecule whose immunological properties vary with base sequences. As shown with synthetic oligonucleotides, potent immune stimulation results from six base motifs called CpG motifs or immunostimulatory sequences (ISS). These sequences center on an unmethylated CpG dinucleotide and occur much more commonly in bacterial DNA than mammalian DNA. As such, CpG motifs may function as a danger signal to stimulate B cell activation and cytokine production. In addition to CpG motifs, runs of deoxyguanosine (dG) residues in DNA can induce B cell activation and promote macrophage cytokine expression by adjacent CpG motifs. The array of these sequences may determine the overall immune activity of a DNA molecule and affect such processes as host defense against infection as well as the use of plasmids and synthetic oligonucleotides to treat disease.

Animals↗

PubMed2003

DNA sequence-based "bar codes" for tracking the origins of expressed sequence tags from a maize cDNA library constructed using multiple mRNA sources.

To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence "bar codes" were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects.

Base Sequence↗

PubMed2003

Mitochondrial DNA sequence-based population genetic structure of the firefly, Pyrocoelia rufa (Coleoptera: Lampyridae).

The genetic divergence, population genetic structure, and possible speciation of the Korean firefly, Pyrocoelia rufa, were investigated on the midsouthern Korean mainland, coastal islets, a remote offshore island, Jedu-do, and Tsushima Island in Japan. Analysis of DNA sequences from the mitochondrial COI protein-coding gene revealed 20 mtDNA-sequence-based haplotypes with a maximum divergence of 5.5%. Phylogenetic analyses using PAUP, PHYLIP, and networks subdivided the P. rufa into two clades (termed clade A and B) and the minimum nucleotide divergence between them was 3.7%. Clade A occurred throughout the Korean mainland and the coastal islets and Tsushima Island in Japan, whereas clade B was exclusively found on Jeju-do Island. In the analysis of the population genetic structure, clade B formed an independent phylogeographic group, but clade A was further subdivided into three groups: two covering western and eastern parts of the Korean peninsula, respectively, and the other occupying one eastern coastal islet and Japanese Tsushima Island. Considering both phylogeny and population structure of P. rufa, the Jeju-do Island population is obviously differentiated from other P. rufa populations, but the Tsushima Island population was a subset of the Korean coastal islet, Geoje. We interpreted the isolation of the Jeju-do population and the grouping of Tsushima Island with Korean coastal islets in terms of Late Pleistocene-Holocene events. The eastern-western subdivision on the Korean mainland was interpreted partially by the presence of a large major mountain range, which bisects the midpart of the Korean peninsula into western and eastern parts.

Animals↗

PubMed2004

Development of a nucleic acid sequence-based amplification assay that uses gag-based molecular beacons to distinguish between human immunodeficiency virus type 1 subtype C and C' infections in Ethiopia.

A gag-based molecular beacon assay utilizing real-time nucleic acid sequence-based amplification technology has been developed to differentiate between the two genetic subclusters of human immunodeficiency virus type 1 (HIV-1) subtype C (C and C') circulating in Ethiopia. Of 41 samples, 36 could be classified as C or C' by sequencing of the gag gene. All 36 isolates were correctly identified by the gag beacon test. Three isolates with genomes that were recombinant in gag were unambiguously typed as belonging to the C' subcluster. Further analysis revealed that these contained the most sequence homology with a reference subcluster C' sequence in the target region of the beacon and hence were correct for the analyzed region. For one sample, sequencing and gag molecular beacon results did not match, while another isolate could not be detected at all by the beacon assay. Overall, high levels of sensitivity and specificity were achieved for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C' beacon). The availability of a diagnostic test which can quickly and reliably discriminate between C and C' HIV-1 infections in Ethiopia is an important first step toward studying their respective biological characteristics. As the assay is specific to the Ethiopian HIV-1 subtype C epidemic, it will contribute to characterizing the circulating viruses in this population, thereby generating the information necessary for the development of a potential efficacious HIV-1 vaccine appropriate for the Ethiopian context.

Base Sequence↗

PubMed2003

Sequence-based typing identifies a novel HLA-DPB1 allele, DPB1*9601.

In this report we describe the identification of a novel HLA-DPB1 allele, DPB1*9601, found in a Caucasian individual sample named ucla#356. The new allele was detected in the DNA of ucla#356 during routine HLA sequence-based typing (SBT) of samples participating in the UCLA International HLA DNA Exchange (number 55) for HLA DNA Proficiency Testing. DPB1*9601 was identical to DPB1*3901 except for a single nucleotide substitution 'G'-->'C' in previously constant position 277 (position 177, respectively, counting only exon 2). This nucleotide change causes an amino acid substitution from aspartic acid in DPB1*3901 to histidine at codon 64 in the novel allele. This new allele has been submitted to the EMBL database and has been assigned the accession number AJ514871. The WHO Nomenclature Committee has officially assigned the name DPB1*9601.

Base Sequence↗

PubMed2001

[Sequencing-based typing of HLA-DPB1 and DQB1 exon 2 in patients with type 1 diabetes mellitus].

OBJECTIVE: To investigate the correlation between type 1 diabetes mellitus susceptibility and HLA-DPB1 and DQB1 genes in Shandong Han population. METHODS: HLA-DPB1 and DQB1 genes analysis were performed by sequencing-based typing in 52 type 1 diabetes mellitus patients and 38 normal controls. RESULTS: The frequency of DPB1*2201 in diabetics was significantly higher than that in controls(26.92% vs 5.26%, P<0.01), but the frequency of DPB1*0402 in diabetics was significantly lower than that in controls(11.54% vs 34.21%, P<0.01). The frequencies of DQB1*0201, *0303 and *0604 in diabetics were higher than those in controls (24.04% vs 10.53%, P< 0.01; 22.12% vs 6.58%, P<0.05; 19.23% vs 7.89%, P<0.05), while the frequency of DQB1*0301 in diabetics was significantly lower than that in controls (4.81% vs 25.00%, P<0.01). CONCLUSION: DPB1*2201, DQB1*0201, *0303 and *0604 may be susceptible alleles, and DPB1*0402 and DQB1*0301 may be protective alleles in patients with type 1 diabetes mellitus.

Adolescent↗

PubMed1994

Complete base sequence for the mitochondrial large subunit ribosomal RNA of the gypsy moth Lymantria dispar (L.).

A 1355 bp sequence (accession number L32141) isolated from a gypsy moth (Lymantria dispar) cDNA library showed 68-74% sequence identity to mitochondrial large subunit ribosomal RNA (mt IrRNA) sequences of Locusta migratoria, Apis mellifera, Aedes albopictus, Anopheles gambiae and two Drosophila species. A comparison of the primary sequences of the mt IrRNAs from the above insects in four orders and from Esherichia coli demonstrated regions of conservation which presumably correspond to regions of functional and/or structural homology. A secondary structure for the gypsy moth mt IrRNA sequence was derived based on the proposed secondary structures of Drosophila yakuba and Aedes albopictus mt IrRNAs (Gutell & Fox, Nucleic Acid Res 16 (Suppl.), r175-r269, 1988). This sequence was found to hybridize to about 10-15% of the clones in several (eleven) gypsy moth cDNA libraries.

Animals↗

PubMed1999

The effect of base sequence on the stability of RNA and DNA single base bulges.

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.

Base Sequence↗

PubMed1998

Sequencing-based typing reveals six novel MHC class I chain-related gene B (MICB) alleles.

The MHC class I chain-related gene A (MICA) is highly polymorphic. In this paper we demonstrate polymorphism in the other expressing member of the MIC family of genes: MICB. Using a sequencing-based typing approach on cDNA, analysis of exons 2 through 6 revealed eight polymorphic sites resulting in six unique MICB sequences. Although MICB has high nucleotide homology with MICA, its polymorphism is restricted and at different sites compared to those in MICA.

Alleles↗