Dr. Sanger's Apprentice. A computer-aided instruction to protein sequencing.
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Amino Acid Sequence↗
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Results for “Sanger sequencing”
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Advances in sequencing technology.
Faster sequencing methods will undoubtedly lead to faster single nucleotide polymorphism (SNP) discovery. The Sanger method has served as the cornerstone for genome sequence production since 1977, close to almost 30 years of tremendous utility [Sanger, F., Nicklen, S., Coulson, A.R, DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. U.S.A. 74 (1977) 5463-5467]. With the completion of the human genome sequence [Venter, J.C. et al., The sequence of the human genome, Science 291 (2001) 1304-1351; Lander, E.S. et al., Initial sequencing and analysis of the human genome, Nature 409 (2001) 860-921], there is now a focus on developing new sequencing methodologies that will enable "personal genomics", or the routine study of our individual genomes. Technologies that will lead us to this lofty goal are those that can provide improvements in three areas: read length, throughput, and cost. As progress is made in this field, large sections of genomes and then whole genomes of individuals will become increasingly more facile to sequence. SNP discovery efforts will be enhanced lock-step with these improvements. Here, the breadth of new sequencing approaches will be summarized including their status and prospects for enabling personal genomics.
DNA-Directed DNA Polymerase↗
Complete 3' noncoding region sequences of rabbit and human beta-globin messenger RNAs.
The 3' noncoding regions of rabbit and human beta-globin mRNAs, 95 and 134 nucleotides in length, have been sequenced. Furthermore, these and previous studies (Baralle, 1977; Proudfoot, 1976a, 1976b) have established about one half of the 589 nucleotide sequence of rabbit beta-globin mRNA, including all of the noncoding region. These sequence studies took advantage of the plus and minus gel-sequencing procedure originally described by Sanger and Coulson (1975) and recently adapted by G.G. Brownlee and E.M. Cartwright (manuscript in preparation). The human and rabbit 3' noncoding region sequences are extensively homologous, except that the human sequence has an addition of 39 nucleotides as compared with rabbit. This extra sequence may have arisen by gene duplication.
Animals↗
Nucleotide sequences in bacteriophage ribonucleic acid. The eighth hopkins memorial lecture.
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Amino Acid Sequence↗
Gene expression microarray analysis and genome databases facilitate the characterization of a chromosome 22 derived homogeneously staining region.
Karyotype and fluorescence in situ hybridization (FISH) analyses previously identified a homogeneously staining region (HSR) derived from chromosome 22 in OV90, an epithelial ovarian cancer (EOC) cell line. Affymetrix expression microarrays in combination with the UniGene and Human Genome Browser databases were used to identify the candidate genes comprising the amplicon of the HSR, based on comparison of expression profiles of OV90, EOC cell lines lacking HSRs and primary cultures of normal ovarian surface epithelial (NOSE) cells. A group of probe sets displaying a minimum 3-fold overexpression with a high reliability score (P-call) in OV90 were identified which represented genes that mapped within a 1-2 Mb interval on chromosome 22. A large number of probe sets, some of which represent the same genes, displayed no evidence of overexpression and/or low reliability scores (A-call). An investigation of the probe set sequences with the Affymetrix and Sanger Institute Chromosome 22 Group databases revealed that some of the probe sets displaying discordant results for the same gene were complementary to intronic sequences and/or the antisense strand. Microarray results were validated by RT-PCR. Genomic analysis suggests that the HSR was derived from the amplification of a 1.1 Mb interval defined by the chromosomal map positions of ZNF74 and Hs.372662, at 22q11.21. The deduced amplicon is derived from a complex region of chromosome 22 that harbors low-copy repeats (LCRs). The amplicon contains 18 genes as likely targets for gene amplification. This study illustrates that large-scale expression microarray analysis in combination with genome databases is sufficient for deducing target genes associated with amplicons and stresses the importance of investigating probe set design before engaging in validation studies.
Cell Line, Tumor↗
Cloning and sequencing of the cDNA for S-acyl fatty acid synthase thioesterase from the uropygial gland of mallard duck.
In vitro translation of poly(A)+ RNA from the uropygial glands of mallard ducks (Anas platyrhynchos) generated a 29-kDa protein which cross-reacted with rabbit antibodies prepared against S-acyl fatty acid synthase thioesterase (Kolattukudy, P. E., Rogers, L., and Flurkey, W. (1985) J. Biol. Chem., 260, 10789-10793). A poly(A)+ RNA fraction enriched in this thioesterase mRNA, isolated by sucrose density gradient centrifugation, was used to prepare cDNA which was cloned in Escherichia coli using the plasmid pUC9. Using hybrid-selected translation and colony hybridization, 17 clones were selected which contained the cDNA for S-acyl fatty acid synthase thioesterase. Northern blot analysis showed that the mature mRNA for this thioesterase contained 1350 nucleotides whereas the cloned cDNA inserts contained 1150-1200 base pairs. Five of the 6 clones tested for 5'-sequence had identical sequences, and the three tested for 3'-end showed the same sequence with poly(A) tails. Two clones, pTE1 and pTE3, representing nearly the full length of mRNA, were selected for sequencing. Maxam-Gilbert and Sanger dideoxy chain termination methods were used on the cloned cDNA and on restriction fragments subcloned in M13 in order to determine the complete nucleotide sequence of the cloned cDNA. The nucleotide sequence showed an open reading frame coding for a peptide of 28.8 kDa. Two peptides isolated from the tryptic digest of the thioesterase purified from the gland showed amino acid sequences which matched with two segments of the sequence deduced from the nucleotide sequence. Another segment containing a serine residue showed an amino acid sequence homologous to the active serine-containing segment of the thioesterase domain of fatty acid synthase. Thus, the clones represent cDNA for S-acyl fatty acid synthase thioesterase. The present results constitute the first case of a complete sequence of a thioesterase.
Amino Acid Sequence↗
Features of bacteriophage lambda: analysis of the complete nucleotide sequence.
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Bacteriophage lambda↗
The genome-linked protein of picornaviruses. VII. Genetic mapping of poliovirus VPg by protein and RNA sequence studies.
The poliovirus genome-linked protein (VPg) has been subjected to radiochemical microsequence analysis. Sequence studies of virion RNA by a modification of Sanger's dideoxy method have revealed a base sequence corresponding to the amino acid analysis. This result proves that VPg is virus-encoded. The RNA sequence has allowed us to predict the total amino acid sequence of VPg and part of its precursor. VPg is, at most, 27 amino acids long. It maps within the 3' terminal segment of the viral genome that encodes the precursor polypeptide NCVP1b for the virus-specific RNA polymerase NCVP4.
Amino Acid Sequence↗
Comparison of nucleotide sequences in HeLa cell mRNA and hnRNA.
Nearest neighbour analysis of nucleotides in vertebrate DNA has revealed a marked deficiency in the frequency of occurrence of the sequence CpGp. By use of the fingerprinting technique of Sanger et al. (1965) to characterise the nucleotide sequences of HeLa cell hnRNA after T1 RNAase digestion, we have shown that this deficiency also occurs in hnRNA and that it appears to be a general transcript of DNA. This is also true of the hnRNA that contains poly(A). Whilst a deficiency of CpGp occurs also in poly(A) containing mRNA from HeLa cells the deficiency is less marked than in the case of total hnRNA, or poly(A) containing hnRNA. Another difference between mRNA and hnRNA lies in the level of occurrence of the sequence UpApGp. These results are discussed with reference to the possible relationship of hnRNA to mRNA.
Base Sequence↗
[A new primer for sequencing DNA fragments cloned in M13mp vectors].
Tridecadeoxyribonucleotide d(CCAGGGTTTTCCC) was prepared by solid phase crown-ether-catalyzed phosphotriester method and proposed a new sequencing primer. This primer expands capacities of the Sanger dideoxy-chain-terminating method to solve various sequencing problems as compared to well-known universal primers. Criteria of the choice of an oligonucleotide primer without computer analysis of nucleotide sequence are described.
Base Sequence↗
Specific-primer-directed DNA sequencing.
A simple and rapid strategy for DNA sequence analysis based on the Sanger chain-termination method is described. This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors. After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides. Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined. General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed. The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single-stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA.
Bacteriophages↗
The amino acid sequence around the reactive serine of elastase.
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Amino Acid Sequence↗
Cloning and sequencing of the gene that codes for the Klebsiella pneumoniae GroEL-like protein associated with ankylosing spondylitis.
Ankylosing spondylitis (AS) is a chronic inflammatory disease of the sacroiliac joints and vertebral column of unknown aetiology, but strongly related to the presence of the HLA-B27 antigen. The participation of bacterial infections as triggering factors have also been suggested. We have associated the 60 kDa heat shock protein of Klebsiella pneumoniae (HSP60Kp) with AS since we have previously demonstrated that most of the patients have IgG antibodies and active T cells that recognize preferentially this protein, but we have not yet identified the epitopes involved in the recognition. In order to know the amino acid sequence of HSP60Kp, and to be able to analyse in the future the relevant epitopes; we amplified by PCR and cloned the gene coding for this protein into the SmaI site of pUC19. The nucleotide sequence of the gene was obtained by the Sanger method using both manual and automatic techniques. Amino acid sequence of the HSP60Kp was deduced by translating the nucleotide sequence of the gene. The antigenic analysis of this sequence was compared to the antigenic analysis of the reported sequences of Escherichia coli GroEL and Yersinia enterocolitica HSP60. Using a software to predict HLA class I motifs, the nonapeptide (KRGIDKAVL) residues 117-125 of HSP60Kp showed a much higher affinity for HLA-B27 than the similar nonapeptide of E. coli GroEL and Y. enterocolitica HSP60. The only difference between the three peptides was in position nine. This finding could explain the association of AS only with the HSP60 of Klebsiella pneumoniae. On the other hand, hydrophilicity analysis, which indicates B cell epitopes, showed three similar strongly antigenic regions in the three proteins.
Amino Acid Sequence↗
A system for on-line detection and resolution of radiolabeled DNA molecules and its application to automated DNA sequence analysis.
We describe a system for the real-time detection of radioactively labeled DNA molecules in gel matrix, and we demonstrate the application of this system to DNA sequence analysis. DNA sequencing reactions prepared by the Sanger chain termination method are resolved by electrophoresis on 8% polyacrylamide gels. During electrophoresis the 32P-labeled DNA fragments are detected by solid state detectors positioned 22 cm from the top surface of the gel. This system is able to resolve a DNA sequence of 300 bases or greater. Optimized protocols that allow sequence information to be obtained from single stranded and double-stranded templates are described. A linear relationship exists between the input dpm and the integrated peak values over a 20-fold range indicating that accurate DNA quantitation is also possible using this system.
Automation↗
How the worm was won. The C. elegans genome sequencing project.
The genome sequence of the free-living nematode Caenorhabditis elegans is nearly complete, with resolution of the final difficult regions expected over the next few months. This will represent the first genome of a multicellular organism to be sequenced to completion. The genome is approximately 97 Mb in total, and encodes more than 19,099 proteins, considerably more than expected before sequencing began. The sequencing project--a collaboration between the Genome Sequencing Center in St Louis and the Sanger Centre in Hinxton--has lasted eight years, with the majority of the sequence generated in the past four years. Analysis of the genome sequence is just beginning and represents an effort that will undoubtedly last more than another decade. However, some interesting findings are already apparent, indicating that the scope of the project, the approach taken, and the usefulness of having the genetic blueprint for this small organism have been well worth the effort.
Animals↗
[Cloning and sequencing of variable region genes of HAb25 McAb against hepatocellular carcinoma].
OBJECTIVE: To clone the variable genes of HAb25 McAb against hepatocellular carcinoma. METHODS: The HAb25 variable region genes were isolated by RT-PCR technique from the HAb25 hybridoma, and its nucleic acid sequences were analyzed by the sanger's dideoxy-mediated chain-termination method. RESULTS: The gene of VH was 360 bp long encoding 120 amino acids and the gene of VL was 330 bp long encoding 110 amino acids. Only one open reading frame could be found in each of the variable genes. A comparison of the sequences of VH and VL domatins derived from the HAb25 antibody with those of the published mouse Ig genes in GenBank by computer revealed that the VH gene was homologous to the VH186.2 germline gene family (84.00%) and VL gene was homologous to the MMIgGKAVAG(82.00%). CONCLUSION: Both of the two genes are rearranged variable region genes, which are VHDJH3 for the VH and VK J K4 for the VL.
Amino Acid Sequence↗
Construction of eukaryotic expression vector of human CC10 gene and expression of CC10 protein in lung adenocarcinoma A549 cell line.
A mammalian expression plasmid pcDNA3. 1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3. 1, and the recombinant plasmid was identified by employing double digestion restriction enzymes Hind III and BamH I and the cDNA sequence was assayed by the Sanger dideoxy-mediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot. Our results showed that the cDNA fragment included the entire coding region (273 bp). The recombinant eukaryotic cell expression vector of pcDNA3. 1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3. 1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1-hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment.
Adenocarcinoma↗
A new highly discriminatory typing scheme for Treponema pallidum reveals similar levels of genetic variability across lineages.
UNLABELLED: The global resurgence of treponematoses, particularly syphilis, poses a growing public health challenge. Despite advances in sequencing technologies, obtaining complete Treponema pallidum genome sequences for epidemiological studies remains challenging due to clinical sampling and methodological constraints. There is, therefore, a need for rapid, cost-effective, and accessible typing methods. Based on the analysis of 121 T. pallidum genomes spanning all three subspecies (TPA, TPE, and TEN) from diverse regions, we selected seven highly variable genes (tp0136, tp0326, tp0548, tp0705, tp0858, tp0865, and tp1031) to form a new typing system, combined with analysis of macrolide resistance mutations in the 23S rRNA gene. The scheme was validated on 542 global T. pallidum samples, using either Sanger reads or whole genome sequence data, obtaining 82 sequence types (STs) among the 415 fully typed samples. Macrolide resistance mutations were frequently detected, highlighting the need for ongoing epidemiological surveillance. Phylogenetic analyses based on concatenated multilocus typing (MLST) loci recovered the expected subspecies and lineage structure. Consistently, almost all sequence types formed monophyletic groups, indicating strong concordance between MLST-based classification and whole-genome phylogenies. In addition, population genetic analyses revealed comparable levels of within-lineage diversity across subspecies and lineages, despite pronounced differences in geographic distribution, and identified distinct regional genetic clusters consistent with localized transmission dynamics. Importantly, the scheme employs a single-step PCR for all seven targets, facilitating implementation in standard laboratories and is publicly accessible through PubMLST. Overall, our novel MLST scheme offers a rapid, cost-effective tool to advance molecular epidemiology of T. pallidum, facilitate transmission and resistance tracking, and support global surveillance to strengthen public health interventions for syphilis and endemic treponematoses control. IMPORTANCE: We have developed a new multilocus typing (MLST) scheme useful for all Treponema pallidum lineages after the analysis of 121 complete genome sequences of this species. The new scheme can be used directly with uncultured clinical samples, thus providing an excellent contribution to the molecular surveillance of syphilis and other treponematoses. The application of this MLST scheme to over 500 samples from all lineages and main geographical regions has revealed similar levels of genetic variation within them. Furthermore, the analyses show a complex pattern of spread, with global and local contributions to the observed distribution of genetic variation in the syphilis-producing sublineages. The new scheme represents a significant improvement over previous proposals and also reveals unsuspected levels of variability in T. pallidum lineages.
Treponema pallidum↗