Screening mutants by a modified Sanger's dideoxy sequencing method in 96-well microtitre trays.
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Base Sequence↗
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Chemiluminescent DNA sequencing with multiplex labeling.
Chemiluminescent detection techniques provide a sensitive, nonradioactive method for DNA sequencing. Standard Sanger dideoxy DNA sequencing reactions are initiated with biotinylated primers, separated by gel electrophoresis, transferred to nylon membrane and detected utilizing chemiluminescent 1,2-dioxetane substrates for alkaline phosphatase. A multiplex-labeling method was developed to permit detection of several overlapping sets of DNA sequence information on a single membrane, thereby increasing the productivity of a single gel electrophoretic separation. Primers labeled with different haptens at the 5' end were used to perform separate sequencing reactions. These were mixed together prior to electrophoresis, and the individual sequencing products sequentially detected using hapten-specific reagents. We incorporated primers labeled with biotin, digoxigenin, 2,4-dinitrophenyl or fluorescein, each consecutively detected with a hapten-specific alkaline phosphatase conjugate and CSPD 1,2-dioxetane chemiluminescent substrate. To further increase the amount of DNA sequence data that can be obtained from a single membrane, a direct transfer electrophoresis apparatus was used for simultaneous separation of the DNA sequencing reactions and membrane transfer. The resulting increased separation of the high molecular weight fragments yields 350-450 bp of readable DNA sequence data from each template. Chemiluminescent detection of overlapping sets of DNA sequencing reactions utilizing multiplex labeling, combined with direct transfer electrophoresis, provides an efficient, nonradioactive method for DNA sequencing.
2,4-Dinitrophenol↗
The early days of DNA sequences.
Fred Sanger was awarded the rare distinction of two Nobel Prizes for Chemistry, in 1958 and 1980. The first was for his work on the structure of proteins, particularly that of insulin, the latter for his contribution concerning the determination of nucleic acid sequences, the foundation work that ultimately led to The Human Genome Project.
History, 20th Century↗
A strategy for rapid and efficient DNA sequencing by mass spectrometry.
Two methods of solid-phase Sanger DNA sequencing followed by detection with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are demonstrated. In one method, sequencing ladders generated on an immobilized synthetic template were resolved up to the 63-mer including the primer. Detection sensitivity and resolution were sufficient for sequence analysis in the given range. This approach is particularly suitable for comparative (diagnostic) DNA sequencing. A second method that has the potential for high throughput de novo DNA sequencing is also presented; it uses immobilized duplex probes with five-base single-stranded overhangs to capture an unknown DNA template serving as primers for Sanger DNA sequencing. The power of mass spectrometry is demonstrated not only by its very high speed, but also by its ability to identify sequences that are not readable using gel electrophoresis.
Base Sequence↗
Transfer of Kingella indologenes (Snell and Lapage 1976) to the genus Suttonella gen. nov. as Suttonella indologenes comb. nov.; transfer of Bacteroides nodosus (Beveridge 1941) to the genus Dichelobacter gen. nov. as Dichelobacter nodosus comb. nov.; and assignment of the genera Cardiobacterium, Dichelobacter, and Suttonella to Cardiobacteriaceae fam. nov. in the gamma division of Proteobacteria on the basis of 16S rRNA sequence comparisons.
The 16S rRNA sequences of Kingella indologenes, Cardiobacterium hominis, and Bacteroides nodosus were determined by direct RNA sequencing, using a modified Sanger method. Sequence comparisons indicated that these three species represent a novel family in the gamma division of Proteobacteria. On the basis of these data, K. indologenes and B. nodosus cannot retain their current generic status as they are not closely related to other members of their assigned genera. Therefore, we propose transfer of K. indologenes to the new genus Suttonella as Suttonella indologenes and transfer of B. nodosus to the new genus Dichelobacter as Dichelobacter nodosus and assign the genera Cardiobacterium, Suttonella, and Dichelobacter to a new family, Cardiobacteriaceae, in the gamma division of Proteobacteria.
Bacteroides↗
[Cloning, expression of the gI gene of pseudorabies virus ea strain and its effection to viral replication].
The complete gI was cloned from the genomic DNA of Pseudorabies virus (PRV) Ea strain and the DNA sequence was determined by Sanger's sequencing technique. The nucleotide and deduced animo acids sequences indicated that the gI gene of PRV Ea strain is composed of 1101 base pairs and could encode 366 animo acids residues. The result of predicted II class structure showed that gI is a typical I class envelope protein. When compared with PRV Rice strain, there were multiple mutations in the gI gene of PRV Ea strain, and the diversity of amino acid residues also existed, especially the deletion of two bases which results in the change of the reading frame and there were additional 16 animo acids residues in the potential cytoplasmic domains of Ea strain. The fragment containing the complete gI gene was further sub-cloned into the downstream of CMV promoter of eukaryotic expression vector pcDNA3.1+, resulting in a recombinant expression plasmid pSB209. pSB209 was transfected into PK-15 cell lines and the cells expressing gI were obtained in the selection of G418. Expressed protein was further detected by Indirect Immunofluorescence Assay(IFA). In order to explore the function of gI in the viral replication, the attenuated live vaccine strain, Bartha in which the gI is deleted, was chosen to determine the plaques forming units (pfu) and the tissue culture infectious dose(TCID50) in PK-15 cell lines expressing the gI(PKgI). The data showed that the pfu and TCID50 in PKgI were 164% and 200% of those in control cell lines respectively. The above results indicated that the gI involve in and accelerate the viral replication.
Amino Acid Sequence↗
Method enabling fast partial sequencing of cDNA clones.
Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.
Base Sequence↗
DNA sequence analysis of spontaneous and beta-methoxy-acrolein-induced mutations in Salmonella typhimurium hisD3052.
The histidine auxotroph hisD3052 results from a single base-pair deletion (C:G) from the 298th codon (alanine) in the D gene of the histidine operon of S. typhimurium LT-2. Bacteria carrying this mutation revert to histidine prototrophy by frameshift mutations (additions or deletions) that restore the correct reading frame. Among the compounds that revert hisD3052 are the naturally occurring dicarbonyl compound malondialdehyde and a structural analog, beta-methoxy-acrolein. To determine the sequence changes responsible for reversion, spontaneous and beta-methoxy-acrolein induced revertants were isolated, male derivatives constructed, and infected with M13Ho167.18, a phage carrying partial O, D, and C genes of the histidine operon. M13hisD+ recombinants were selected by complementation in TA2890, single-stranded DNA was isolated, and the reverted D genes were sequenced using Sanger dideoxy chain-termination sequencing with a synthetic oligonucleotide primer. Analysis of 37 spontaneous revertants revealed 41% additions and 59% deletions with 22% of the mutations occurring as two base-pair (-CG-) deletions in a -CG- rich 'hot spot'. The profile of additions was +1 (30%), +4 (8%), +7 (3%); the profile of deletions was -2 (32%), -5 (11%), -8 (8%), and -11 (8%). Analysis of 27 beta-methoxy-acrolein-induced revertants revealed 96% additions and 4% deletions. The profile of induced additions was +1 (70%), +4 (22%), +7 (4%). No additions occurred in the -CG- rich hot-spot. Frameshift reversions of the hisD3052 gene demonstrate a surprising degree of sequence diversity and reveal the ability of the hisD3052 tester strain to detect a wide variety of frameshift reversion events. In addition, the results demonstrate that beta-methoxy-acrolein induces a high percentage of additions in this reversion system.
Acrolein↗
[Cloning and sequence analysis of rOmpA gene fragment of spotted fever group Rickettsiae isolated in China].
Rr 190.70 p-602 n primers were used to amplify the rOmpA gene fragment of prototype strains (Rickettsia sibirica 246 strain and R. parkeri) and Chinese strains (BJ-90, Ha-91 and HLJ-054) of spotted fever group rickettsiae (SFGR). The PCR products were cloned into pGEM-T vector respectively and sequenced by Sanger's dideoxy method. The sequences of the tested strains were compared with the corresponding regions of prototype and reference strains of SFGR. The results showed that the homology of rOmpA gene fragments of prototype strains varied from 90.06%-96.62% in nucleotide and 83.05%-94.35% in putative amino acid respectively. Compared Chinese strains with prototype and reference strains of SFGR, it was found that the homology of BJ-90 strain and Ha-91 strain with R. sibirica 246 strain was the highest in nucleotide (99.06% and 98.31%, respectively) and in putative amino acid (98.87% and 96.61%, respectively), whereas HL-93 strain and HLJ-054 strain showed higher homology with R. japonica (96.62% and 95.68% in nucleotide, respectively) than others. Among all the Chinese strains, the homology between BJ-90 strain and Ha-91 strain was the highest, 99.25% in uncleotide and 97.74% in putative amino acid. The homology of nucleotide and putative amino acid between HL-93 strain and HLJ-054 strain was 98.12% and 94.92% respectively. The above results indicated that all SFGR so far isolated from different sources seem to be identical or closely related to R. sibirica, except for HL-93 and HLJ-054 strains, which could be considered as new members of SFGR.
Amino Acid Sequence↗
[Default of KIAA0353 gene in varicose great saphenous vein accompanying primary deep vein valve insufficiency].
OBJECTIVE: To screen and identify the genes related to the occurrence and development of varicose great saphenous vein in the patients with primary deep vein valve insufficiency (PDVI). METHODS: mRNA fluorescent differential display (FDD) technique was used to compare the different cDNA fragments originated from differentially expressed mRNAs from the venous tissues of 10 patients with varicose great saphenous vein complicated with PDVI. Ten specimens of normal venous tissue from 10 patients dying from other diseases were used as controls. The differently expressed cDNA fragments were then re-amplified and labeled with DIG to prepare probes for later Northern blotting. Positive fragments confirmed by Northern blotting were cloned into pGEM-T easy vector and sequenced using Sanger's method. Then the sequences were compared with the data in GeneBank by BLASTN software to search for their genetic origin. RESULTS: Altogether 37 differentially expressed cDNA fragments were discovered from the 2 groups, among which 30 were confirmed by Northern blotting. There was a notable 540 bp-long cDNA fragment, which was only presented in the control group, sharing 99% homology with part of the mRNA sequence of human KIAA0353 gene. CONCLUSION: The varicose great saphenous vein of PDVI patients is a process with the involvement of multiple genes and the default of KIAA0353 gene may play a role in the occurrence and development of varicose great saphenous vein in PDVI patients.
Base Sequence↗
[Genes coding for RNA polymerase in bacteria. III. The use of modified Sanger's method for sequencing the C-terminal region of rpoB gene, N-terminal region of rpoC gene and intercistron region of RNA polymerase in Pseudomonas putida].
The Sanger method was modified and the primary structure of the SalI-C fragment of the Pseudomonas putida rpoBC operon was elucidated.
Base Sequence↗
Increased stability of nucleic acids containing 7-deaza-guanosine and 7-deaza-adenosine may enable rapid DNA sequencing by matrix-assisted laser desorption mass spectrometry.
The use of matrix-assisted laser desorption mass spectrometry (MALDI-MS) has been suggested as an ultrafast readout of Sanger DNA sequencing ladders in a manner analogous to that used with sequencing gels. Currently, a serious limitation of MALDI-MS for the analysis of DNA results from the tendency for oligonucleotides to undergo facile fragmentation in the gas phase. The present study was undertaken to gain an understanding of the influence of various chemical structural features of purine bases on the stability of oligodeoxynucleotide ions produced by MALDI. The study focused on the stability of model compounds of the type d(TTTTTTTTTTXTTTTTTTTT TTTT), where T designates deoxythymidine and X a purine-containing 2'-deoxynucleotide. A variety of different purine derivatives were chosen as the base in the nucleotide X. The mass spectra of the model compounds containing 7-deaza analogues of guanine and adenine reveal a significantly increased stability compared to the 7-aza analogues under the conditions of MALDI-MS. The previously reported incorporation of the 7-deaza-2'-deoxy-adenosine triphosphate and the 7-deaza-2'-deoxy-guanosine triphosphate into DNA by polymerases suggests their use in a Sanger dideoxy sequencing experiment. The dideoxy termination products with the 7-deaza-purines instead of the 7-aza-purines might be sufficiently stable to allow separation and detection of the sequencing ladder by MALDI-MS. Thus, an ultrafast (seconds) read-out of DNA sequence may become feasible.
Base Sequence↗
DNA sequencing of the MHC class II region and the chromosome 6 sequencing effort at the Sanger Centre.
The human Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6 (6p21.3) and spans about 4 Mb. According to different gene families the MHC is subdivided into a class I, class II and class III region and many of its gene products are associated with the immune system and the susceptibility to various diseases. To date, we have sequenced about 40% (400 kb) of the class II region between HLA-DP and HLA-DQ and a coordinated effort to sequence the entire MHC is well underway. Analysis of the sequence revealed several novel genes and provides new insights into the molecular organisation and evolution of the MHC. All our data are publicly available via the MHC database (MHCDB) which allows rapid access, retrieval and display in the context of other MHC associated data. MHCDB is online available at (http:(/)/www.hgmp.mrc.ac.uk/) and, together with all our sequences also via anonymous ftp (ftp.icnet.uk/icrf-public).
Animals↗
Multiple-primer DNA sequencing method.
A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.
DNA Primers↗
Multiple group-specific sequencing primers for reliable and rapid DNA sequencing.
Pyrosequencing technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.
Bacteria↗
Toward the 1,000 dollars human genome.
Revolutionary new technologies, capable of transforming the economics of sequencing, are providing an unparalleled opportunity to analyze human genetic variation comprehensively at the whole-genome level within a realistic timeframe and at affordable costs. Current estimates suggest that it would cost somewhere in the region of 30 million US dollars to sequence an entire human genome using Sanger-based sequencing, and on one machine it would take about 60 years. Solexa is widely regarded as a company with the necessary disruptive technology to be the first to achieve the ultimate goal of the so-called 1,000 dollars human genome - the conceptual cost-point needed for routine analysis of individual genomes. Solexa's technology is based on completely novel sequencing chemistry capable of sequencing billions of individual DNA molecules simultaneously, a base at a time, to enable highly accurate, low cost analysis of an entire human genome in a single experiment. When applied over a large enough genomic region, these new approaches to resequencing will enable the simultaneous detection and typing of known, as well as unknown, polymorphisms, and will also offer information about patterns of linkage disequilibrium in the population being studied. Technological progress, leading to the advent of single-molecule-based approaches, is beginning to dramatically drive down costs and increase throughput to unprecedented levels, each being several orders of magnitude better than that which is currently available. A new sequencing paradigm based on single molecules will be faster, cheaper and more sensitive, and will permit routine analysis at the whole-genome level.
Animals↗
The chromosome 6 sequencing project at the Sanger Centre.
Chromosome 6 is probably best known for encoding the major histocompatibility complex (MHC) which is essential to the human immune response. In addition, it has been shown to be associated with many diseases such Schizophrenia, Diabetes, Arthritis, Haemochromatosis, Narcolepsy, Epilepsy, Retinitis Pigmentosa, Deafness, Ovarian Cancer, and many more. Chromosome 6 is about 180 Mb in size and is estimated to encode around 3500 genes of which only about 10% are currently known. It is our aim to map, sequence and annotate the entire chromosome in close collaboration with the chromosome 6 community.
Chromosomes, Human, Pair 6↗
[Molecular cloning and expression of E2 gene of the Chinese classical swine fever virus(shimen strain) and preliminary studies of its DNA vaccine].
A 1.1 bp fragment of E2 gene of Chinese classical swine fever virus(CSFV) Shimen strain, a standard virulent strain, was amplified by RT-PCR from total RNA of cell cultures infected by CSFV, and cloned into pGEM T vector. The nucleotide sequence of this fragment was sequenced by Sanger's method and the amino acid sequence was deduced. Compared with the corresponding region of Alfort, Brescia and C strain of CSFV, the nucleotide sequence homology is 84.7%, 92.6% and 95.2% respectively, and the amino acid sequence 89.4%, 92.6% and 94.6%, respectively, we subcloned 1.1 bp of E2 gene cDNA into baculovirus transfer vector and successfully constructed two recombinant baculoviruses expressing GST-E2 and GST-GFP-E2 fusion protein respectively by homologous recombination in sf-9 cell. Furthermore, we also constructed recombinant eukaryotic expression vector pcE2 containing E2 gene in frame and transfected COS-7 cell by lipofectamine, the indirect immunofluorescence assay (IFA) showed that the expressed E2 protein can be recognized by E2 specific monoclonal antibody the pcE2 DNA was directly injected into BALB/c mice intramuscular(i.m.) and the CSFV E2-specific antibodies was measured by enzyme-linked immunosorbent assay(ELISA) the ELISA results indicated the E2-specific antibodies was induced in inoculated mice and virus neutralization assays also indicate single inoculations of plasmids expressing CSFV E2 glycoprotein raised neutralizing antibody in BALB/c mice. these results will be beneficial to investigate the possibility of DNA vaccine against CSFV.
Amino Acid Sequence↗