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Results for “Base Sequence”

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At least 19 recordsLinked to original sources
PubMed2001

Two-step high resolution sequence-based HLA-DRB typing of exon 2 DNA with taxonomy-based sequence analysis allele assignment.

A two-step high resolution sequence-based DRB typing method was developed. The system needs only one polymerase chain reaction (PCR) to type all functional DRB alleles of a given individual. It uses a pair of generic PCR primers to amplify exon 2 DNA of all functional DRB genes and a first-step taxonomy-based sequence analysis (FSTBSA) method to assign allele groups after sequencing the PCR products with a generic primer. In the second step, group-specific primers are used to sequence the same PCR products and a taxonomy-based sequence analysis (TBSA) is used to assign alleles. Thus, both low and high resolution DRB typing can be done with PCR amplified exon 2 DNA from a single PCR reaction. Correct allele group assignment by FSTBSA was confirmed by sequencing the PCR products with group-specific primers and correctly assigned all 158 DNA samples including 34 samples pre-typed by PCR-sequence-specific primer or PCR-sequence-specific oligonucleotide probe. FSTBSA correctly assigned 116 heterozygous combinations of 81 DRB1-DRB3/4/5 haplotypes. Sixty-seven DRB1, 6 DRB3, 1 DRB4, and 3 DRB5 alleles were identified in this study. TBSA successfully resolved all heterozygous allele combinations including 31 heterozygous combinations of 33 alleles of DRB1*03, 08, 11, 12, 13, and 14 allele groups, and six heterozygous combinations of six DRB3 alleles.

Alleles↗
PubMed1994

The DEF data base of sequence based protein fold class predictions.

A new method for predicting protein fold-classes and protein domains from sequence data is constructed and used for generating a data base of protein fold-class assignments. Any given sequence of amino acids is assigned a specific prediction of one out of 45 typical protein fold-classes, a prediction of one out of 4 super fold-classes for the content of secondary structures and a profile of fold-class predictions along the sequence. The prediction accuracy for the super fold-classes is around 91% correct and 82% correct for the specific fold-classes. This accuracy is maintained down to a few percent of sequence identity.

Amino Acid Sequence↗
PubMed2001

Automated non-culture-based sequence typing of meningococci from body fluids.

In recent years, the polymerase chain reaction has been used for the non-culture diagnosis of meningococcal disease, and sequence-based typing takes this further by providing the full characterisation normally only available by culture. In this study, porA gene sequencing was used to perform non-culture-based sequence typing of Neisseria meningitidis strains direct from body fluids. Non-culture porA gene sequencing provided the serosubtype of the infecting organism, and proved to be a useful method as N. meningitidis was not isolated from any of the patients in this study. In conclusion, porA gene sequencing is a very useful tool for the non-culture characterisation of meningococci and provides important information for public health management of cases and contacts.

Bacterial Typing Techniques↗
PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article↗
PubMed2024

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article↗
PubMed1992

Sheep 6-phosphogluconate dehydrogenase. Revised protein sequence based upon the sequences of cDNA clones obtained with the polymerase chain reaction.

Sheep liver 6-phosphogluconate dehydrogenase (6-PGDH) is an enzyme of the pentose phosphate pathway. Evidence has appeared which suggests that the 6-PGDH protein sequence determined previously by direct analysis of the protein isolated from ovine liver is incorrect. Determining the enzyme's DNA sequence was considered to be the best way of solving the problem. In the first instance, a degenerate forward and a degenerate reverse primer were designed on the basis of the known protein sequence, and a partial-length cDNA clone was isolated from total sheep liver cDNA using the polymerase chain reaction. The clone encoded the expected part of the protein sequence. The clone was unsuccessfully used as a prime-cut probe to screen a sheep liver library and a bovine heart library. As a result, the polymerase chain reaction was utilized again to successfully generate a family of overlapping cDNA clones encoding a mature protein of 482 amino acids. The mature protein sequence encoded by the cDNA differs significantly from the sequence derived by direct analysis of the protein, but on closer examination the fundamental difference is caused by the incorrect placement of three enzyme fragments obtained by cyanogen bromide cleavage during the direct sequence analysis of the protein. Placing the fragments in the correct order results in the two sequences being virtually identical except for some minor amino acid changes between the amide and acid forms, and a small number of deletions and insertions.

Amino Acid Sequence↗
PubMed2004

Spatially varying steady state longitudinal magnetization in distant dipolar field-based sequences.

Sequences based on the distant dipolar field (DDF) have shown great promise for novel spectroscopy and imaging. Unless spatial variation in the longitudinal magnetization, Mz(s), is eliminated by relaxation, diffusion, or spoiling techniques by the end of a single repetition, unexpected results can be obtained due to spatial harmonics in the steady state MzSS(s) profile. This is true even in a homogeneous single-component sample. We have developed an analytical expression for the MzSS(s) profile that occurs in DDF sequences when smearing by diffusion is negligible in the TR period. The expression has been verified by directly imaging the MzSS(s) profile after establishing the steady state.

Magnetic Resonance Imaging↗
PubMed1980

The effects of base sequence and dangling bases on the stability of short ribonucleic acid duplexes.

Variable temperature proton nuclear magnetic resonance spectroscopy is used to establish that both sequence and dangling bases affect stability of short RNA double helices. Tetraribonucleotides, CAUG, UGCA and AGCU form reference duplexes (Tm 25 degrees, 33 degrees, 34 degrees, respectively) which contain an equal number of G . C and A . U base pairs and which show primary sequence is important. Pentaribonucleotides GAUGA, ACAUG, UGCAA and AGCUA, form duplexes (Tm 36 degrees, 35 degrees, 46 degrees, 45 degrees, respectively) with dangling adenines. Average Tm difference from the reference is +11 degrees for two 3' or 5'-dangling adenines. Pentaribonucleotides, CAUGU and UCAUG, form duplexes (Tm both 30 degrees) with dangling uracils. Average Tm difference from the reference is +5 degrees for two 3' or 5'-dangling uracils. Fraying was detected only in duplexes from the AGCU series. Presence of a dangling adenine, in duplex, AGCUA, caused a reduction in fraying.

Base Composition↗
PubMed2003

Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5' noncoding regions of additional rhinovirus strains.

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5' noncoding region (5' NCR) of the viral genome. The nucleotide sequence of the 5' NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.

5' Untranslated Regions↗
PubMed1996

Sequenase sequence profiles used for HLA-DPB1 sequencing-based typing.

Sequencing-based HLA typing (SBT) is a PCR based high resolution HLA typing method in which polymorphic regions of the gene are sequenced and directly used for typing. Currently, for class II SBT, alleles are identified by comparison of the exon 2 sequence with their corresponding allele sequence library. Routine SBT requires reliable identification of heterozygosity, and automated assignment of the alleles. In sequencing strategies different enzymes can be used for primer extension. The most characteristic difference between sequences obtained by two protocols using Sequenaseregistered, or Taq-cycle sequencing, respectively, is a difference in incorporation of nucleotides in the primer extension leading to different sequence profiles. In Taq-cycling sequencing variable nucleotide incorporation results in irregular, but reproducible peak patterns, whereas Sequenase incorporates nucleotides in nearly equal amounts, resulting in more even peak patterns. In a previously published multi-center study we evaluated HLA-DPB1 SBT using Taq-cycle sequencing, and showed that typing can reliably be performed, considering the specific sequence profiles. In this study the applicability of Sequenase for HLA-DPB1 SBT was tested. A panel of samples were typed by SBT at five test sites which participate in the Sequencing Based Typing component of the 12th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. The assignment of homozygosity and heterozygosity was validated by Multi-Sequence Analysis, performing cluster analysis of chromatographic data of all sequences at each position. Sequence characteristics were examined and considered for appropriate assignment. Data reveals that Sequenase sequencing can also reliably be used for HLA-DPB1 typing.

Bacteriophage T7↗
PubMed2003

Typing of nonencapsulated haemophilus strains by repetitive-element sequence-based PCR using intergenic dyad sequences.

Intergenic dyad sequences (IDS) are short repeated elements that have been described for several Haemophilus genomes and for only two other bacterial genera. We developed a repetitive-element sequence-based PCR using an IDS-specific primer as a typing method (IDS-PCR) for nonencapsulated Haemophilus strains and compared this technique with pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. IDS-PCR was rapid, easy to perform, and reproducible, with a high discriminatory capacity for nontypeable Haemophilus influenzae (NTHI) strains. The 69 NTHI strains tested generated 65 different banding patterns. Epidemiologically related strains gave similar or identical fingerprints, and all of the unrelated strains except two showed different patterns. These results were in agreement with those obtained by PFGE. For 20 genital strains usually identified as being biotype IV NTHI and belonging to a cryptic genospecies of Haemophilus with remarkable genetic homogeneity, four bands were significantly present and six bands were significantly absent from the fingerprints. The 20 strains were gathered in 11 closely related profiles, whereas PFGE provided no band when DNA was treated with SmaI. IDS-PCR improved the differentiation previously obtained within this species by ribotyping and multilocus enzyme electrophoresis. Our findings suggest that IDS-PCR is a rapid, reliable, and discriminatory method for typing NTHI strains and is currently the most efficient method for distinguishing strains within the cryptic genospecies of HAEMOPHILUS:

Base Sequence↗
PubMed1993

The amino acid sequence of neocarzinostatin apoprotein deduced from the base sequence of the gene.

A segment of the neocarzinostatin apoprotein gene corresponding to T30 to A91 of the protein was amplified using a polymerase chain reaction (PCR) with total DNA from Streptomyces carzinostaticus subsp. neocarzinostaticus E-793 (ATCC 15944) as the template and with 5'- and 3'-primers synthesized in consideration of the codon usage of streptomyces. The PCR product was cloned, sequenced and confirmed to direct an amino acid sequence reasonably well matching that reported. Using the PCR product as a probe, we cloned a DNA segment (2580 bp) spanning an open reading frame (ORF) for preapoprotein (leader peptide plus apoprotein) and its upstream and downstream flanking regions. The amino acid sequence deduced from the base sequence of the DNA clearly identified those amino acid residues which had remained inconsistent among different research groups. The base sequence homology with other apoprotein genes of related antibiotics was analyzed and was found to be limited within the structural gene.

Amino Acid Sequence↗
PubMed1996

Is there an error correcting code in the base sequence in DNA?

Modern methods of encoding information into digital form include error check digits that are functions of the other information digits. When digital information is transmitted, the values of the error check digits can be computed from the information digits to determine whether the information has been received accurately. These error correcting codes make it possible to detect and correct common errors in transmission. The sequence of bases in DNA is also a digital code consisting of four symbols: A, C, G, and T. Does DNA also contain an error correcting code? Such a code would allow repair enzymes to protect the fidelity of nonreplicating DNA and increase the accuracy of replication. If a linear block error correcting code is present in DNA then some bases would be a linear function of the other bases in each set of bases. We developed an efficient procedure to determine whether such an error correcting code is present in the base sequence. We illustrate the use of this procedure by using it to analyze the lac operon and the gene for cytochrome c. These genes do not appear to contain such a simple error correcting code.

Base Sequence↗
PubMed1988

The translation of DNA primary base sequence into three-dimensional structure.

A procedure is outlined to obtain a reliable computer-generated representation of the DNA duplex from its primary sequence of base pairs. The calculations are based on the potential energies of interaction of adjacent side groups. The methods are, however, completely general and can be adapted to any set of base sequence dependent conformational rules. Static representations of the DNA are compared with the distributions of conformations obtained from Monte Carlo simulation studies. Direct matrix generator calculations of the average (equilibrium) extension and orientation of various sequences and numerical estimates of the flexibility of the chains as a whole are also reported. The methods are applied to three short fragments of kinetoplast DNA from Crithidia fasciculata which exhibit dramatically different behavior on non-denaturing polyacrylamide gels.

Base Sequence↗
PubMed2004

Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification.

Sequence-based typing (SBT) is one of the most comprehensive methods utilized for HLA typing. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations which occur when two or more different allele combinations produce identical sequences. The purpose of this study is to investigate the probability of this occurrence. We performed HLA-A,-B SBT for Exons 2 and 3 on 676 donors. Samples were analyzed with a capillary sequencer. The racial distribution of the donors was as follows: 615-Caucasian, 13-Asian, 23-African American, 17-Hispanic and 8-Unknown. 672 donors were analyzed for HLA-A locus ambiguities and 666 donors were analyzed for HLA-B locus ambiguities. At the HLA-A locus a total of 548 total ambiguous allele combinations were identified (548/1344 = 41%). Most (278/548 = 51%) of these ambiguities were due to the fact that Exon 4 analysis was not performed. At the HLA-B locus 322 total ambiguous allele combinations were found (322/1332 = 24%). The HLA-B*07/08/15/27/35/44 antigens, common in Caucasians, produced a large portion of the ambiguities (279/322 = 87%). A large portion of HLA-A and B ambiguous allele combinations can be addressed by utilizing a group-specific primary amplification approach to produce an unambiguous homozygous sequence. Therefore, although the prevalence of ambiguous allele combinations is high, if the resolution of these ambiguities is clinically warranted, methods exist to compensate for this problem.

Journal Article↗