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PubMed1981

A method for sequencing single-stranded cloned DNA in both directions.

A DNA sequencing method has been developed whereby DNA that has been cloned in a single-stranded bacteriophage vector can be sequenced from both ends. The method involves first making a minus-strand sense template from a single-stranded insert in the vector M13mp2 using a flanking primer, and then sequencing the synthesized template using the dideoxynucleotide termination method (Sanger et al., 1977, 1980) with a second primer. Special conditions are described under which the first primer is easily removed after making the template, and sequencing in the opposite direction can be done in the normal way (Sanger et al., 1980) without separating the double strands. This method renders it possible to read up to twice the amount of sequence data from a long insert and also to check short inserts by producing complementary sequence patterns.

Bacteriophage lambda↗
PubMed2026

MNV-aware molecular characterization of a rare homozygous TTPA complex allele in ataxia with vitamin E deficiency.

Ataxia with vitamin E deficiency (AVED) is a rare autosomal-recessive neurological disorder caused by biallelic pathogenic variants in TTPA. Early vitamin E supplementation may prevent or limit irreversible neurological damage, but diagnosis is often delayed. Multi-nucleotide variants (MNVs) in TTPA have rarely been described and may be misinterpreted when adjacent substitutions are evaluated independently. We investigated a 34-year-old woman with childhood-onset progressive ataxia using clinical, biochemical, neuroimaging, and electrophysiological assessments. Serum vitamin E levels were measured longitudinally during supplementation. Whole-exome sequencing, read-level inspection, and Sanger sequencing were used to identify and confirm a homozygous TTPA complex allele, NM_000370.3:c. 296G > A;299 A > C, predicted to result in NP_000361.1:p. Gly99_Tyr100delinsAspSer, and to assess familial segregation. Population-database review, in silico prediction, and exploratory structure-based analysis were performed to evaluate its potential clinical relevance. The patient had markedly reduced baseline serum vitamin E levels of 0.8 µg/mL, which increased to 7.5 µg/mL after 12 months of supplementation. This biochemical correction was temporally accompanied by qualitatively observed improvements in gait stability, coordination, speech, and fine motor performance. Read-level analysis supported the presence of both substitutions on the same allele, and Sanger sequencing confirmed the homozygous complex allele in the patient and heterozygous carrier status in both parents. The affected residues are conserved and located within the CRAL-TRIO domain of α-tocopherol transfer protein. Exploratory structure-based analysis suggested altered local residue interactions; however, no functional assay was performed, and effects on protein stability, α-tocopherol binding, or transfer could not be established. This report expands the molecular spectrum of AVED by describing a homozygous TTPA complex allele and highlights the importance of MNV-aware interpretation of closely spaced substitutions. Vitamin E supplementation resulted in biochemical correction and was accompanied by possible partial clinical improvement despite initiation in adulthood. Functional studies are required to determine the precise effect of this allele on α-tocopherol transfer protein function.

Humans↗
PubMed2025

Identification of pathogenic variants in six Chinese families with keratoconus of autosomal dominant inheritance: pathogenicity analysis and variable phenotype.

PURPOSE: Keratoconus (KC) is a bilateral, asymmetric disease causing corneal thinning, irregular astigmatism, and vision decline, with unclear etiology. This study aims to investigate pathogenic variants of candidate genes in Chinese KC families via whole exome sequencing (WES). METHODS: The Pentacam 3D anterior segment analysis system was applied for keratectasia detection, and the Corvis ST was used for corneal biomechanics measurement. Probands from KC families were screened via WES and further verified in other family members through Sanger sequencing. Additionally, qPCR was used to validate copy number variants and identify pathogenic gene loci. The identified variants were then classified according to the Standards and Guidelines for the Interpretation of Sequence Variants published by the American College of Medical Genetics and Genomics (ACMG). Finally, STRING protein-protein interaction (PPI) networks analysis was performed to investigate interactions among candidate gene-related proteins. RESULTS: Using WES, four heterozygous missense variants were detected in the ZNF469, KRT12, COL8A2, and COL18A1 genes: c.4384G > A: p.Asp1462Asn, c.1229T > G:p.Val410Gly, c.505A > G:p.Ile169Val, and c.1159G > A:p.Gly387Arg. Additionally, a heterozygous frameshift variant was detected in the PMS2 gene: c.1551_1572del:p.Ser517Argfs*71. The affected parents carried the same variants as the probands verified by Sanger sequencing. A copy number variant was detected in the DPP6 gene: seq[GRCh38] dup(7)(q36.2q36.2) chr7:g.153782360_ 153982491dup. According to ACMG guidelines, ZNF469, KRT12, COL8A2, and COL18A1 gene variants are Likely Pathogenic; PMS2 and DPP6 gene variants are Pathogenic. STRING analysis highlights a tightly interconnected network centered on COL8A2, involving COL18A1, FN1, ZNF469, and KRT12. DPP6 was involved in KC via affecting FN1. In four of six autosomal dominant KC (adKC) families, affected parents had the same variants as probands but milder phenotypes. CONCLUSION: In this study, six novel variants in ZNF469, KRT12, COL8A2, COL18A1, PMS2, and DPP6 were linked to adKC. Family phenotypes showed variable expressivity with irregular dominance inheritance. Abnormal KC-related gene protein expression may contribute to corneal structural instability. This study broadened KC genetic screening candidates and suggested genetic testing could aid early KC diagnosis and intervention.

Adult↗
PubMed1990

Regeneration of immunosorbent surfaces used in clinical, industrial and environmental biosensors. Role of covalent and non-covalent interactions.

The durability and regeneration of antibodies immobilized to commercial immunosorbents were investigated by monitoring Ag-Ab dissociation. Solutions consisting of 0.01 M hydrochloric acid (HCl), 10% propionic acid, 50% ethylene glycol and 10% SDS in 6 M urea were used in the evaluation of antigen dissociation from antibody covalently immobilized to glass and polystyrene beads, microtiter plates and Immobilon filters. RAH-IgG, used as a model antibody, bound strongly to all covalent surfaces. However, on adsorption to Nunc-1 microtiter plates, 25-60% of RAH-IgG was removed by all dissociating solutions. Covalent binding to Sanger beads was weakest relative to other covalent surfaces, exhibiting 30% and 65% detachment with ethylene glycol and SDS in urea, respectively. Although all four solutions dissociated antigen from surface-bound antibody, HCl and propionic acid were more effective on most surfaces. The antibody remained functional following antigen dissociation and reassociated to nearly 100% on all surfaces except Sanger beads and Nunc-1 microtiter plates. This study was initiated to evaluate regeneration and reuse of microelisa plates and emerging biosensors as a means of reducing routine laboratory analysis costs. Data are presented to demonstrate the reusability of microtiter plates in ELISAs following antigen dissociation from covalently bound antibody.

Antigen-Antibody Complex↗
PubMed2005

Entamoeba histolytica: construction and applications of subgenomic databases.

Knowledge about the influence of environmental stress such as the action of chemotherapeutic agents on gene expression in Entamoeba histolytica is limited. We plan to use oligonucleotide microarray hybridization to approach these questions. As the basis for our array, sequence data from the genome project carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute were used to annotate parts of the parasite genome. Three subgenomic databases containing enzymes, cytoskeleton genes, and stress genes were compiled with the help of the ExPASy proteomics website and the BLAST servers at the two genome project sites. The known sequences from reference species, mostly human and Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence contigs and the homologs were copied into a Microsoft Access database. In a similar way, two additional databases of cytoskeletal genes and stress genes were generated. Metabolic pathways could be assembled from our enzyme database, but sometimes they were incomplete as is the case for the sterol biosynthesis pathway. The raw databases contained a significant number of duplicate entries which were merged to obtain curated non-redundant databases. This procedure revealed that some E. histolytica genes may have several putative functions. Representative examples such as the case of the delta-aminolevulinate synthase/serine palmitoyltransferase are discussed.

5-Aminolevulinate Synthetase↗
PubMed2026

A novel PKHD1 missense variant disrupting splicing in a fetus with Caroli disease.

BACKGROUND: Caroli disease (CD) is a rare inherited disorder characterized by dilatation of intrahepatic bile ducts, and prenatal diagnosis of this disease is extremely rare. PKHD1 is the only known causative gene, yet the pathogenicity of most missense variants remains unclear. METHODS: Exome sequencing (ES) was performed on a fetus with clinical features of CD. Candidate variants were validated by Sanger sequencing in the family. The impact of the novel missense variant on pre-mRNA splicing was assessed using minigene assays, and structural modeling of the PKHD1 protein was conducted with AlphaFold 3. RESULTS: At 23 weeks of gestation, the fetus showed hepatic cysts on ultrasound and a "central dot" sign on MRI, suggesting a diagnosis of CD. The fetus also exhibited features of autosomal recessive polycystic kidney disease and oligohydramnios. ES identified and Sanger sequencing confirmed three PKHD1 variants: a paternal nonsense variant c.5323C>T; p.(Arg1775*), and two maternal missense variants c.6682G>C; p.(Glu2228Gln) and c.8012G>T; p.(Arg2671Leu). The variant c.6682G>C is novel and minigene assays demonstrated that it caused exon 40 skipping, leading to an in‑frame deletion (c.6491_6682del; p.(Gly2164_Arg2227del)). Structural modeling predicts that this deletion lies within a large β‑barrel domain and may compromise its structural stability. Conclusion We characterize a novel missense variant that causes aberrant splicing of PKHD1 in CD. This finding underscores the necessity of functional analysis for evaluating the pathogenicity of missense variants, especially those at the last nucleotide of an exon. Our study expands the mutation spectrum of PKHD1 and provides insights into genotype‑phenotype correlations.

Humans↗
PubMed2026

A novel missense mutation in tropomyosin 1 gene associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common genetic heart disorder that can lead to heart failure or sudden death. Family-based identification of rare sarcomeric variants can support molecular diagnosis and cascade screening in inherited HCM. This study aimed to identify and evaluate a novel TPM1 variant found in a Vietnamese family with HCM. The proband, a 3-year-old boy diagnosed with HCM, and eight relatives from three generations underwent clinical and genetic evaluation. A candidate variant initially identified by targeted next-generation sequencing was validated by PCR and Sanger sequencing. Familial segregation analysis was performed, and variant pathogenicity was assessed according to ACMG guidelines with support from in silico prediction and structural modeling. Sanger sequencing confirmed a heterozygous missense variant in exon 6 of TPM1 NM_001018005.2:c.576G > C, p.(Glu192Asp), in the proband, his father, and paternal grandfather, all of whom exhibited clinical signs of HCM. The variant was absent in unaffected relatives and in public population databases. Based on ACMG criteria (PM1, PM2, PM5, and PP3), the variant was classified as likely pathogenic. This novel TPM1 variant segregated with HCM in a Vietnamese family, expands the known mutational spectrum of TPM1 in hypertrophic cardiomyopathy, and warrants further functional investigation and familial genetic evaluation.

American College of Medical Genetics and Genomics ↗
PubMed2006

Human papillomavirus type 16 variant assignment by pyrosequencing.

Polymorphisms in human papillomavirus type 16 (HPV16) result in variants from the prototype sequence which can be designated according to geographic distribution and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Detection of variants has been used to distinguish persistent HPV16 infection from re-infection in natural history studies, and variants have been associated with an increased risk of cervical disease in some populations. Variant determination usually relies on conventional Sanger sequencing of regions of the viral genome, with the major variant group assignments requiring the sequencing of only seven polymorphic sites spread over a 242-bp region of the E6 gene. We applied pyrosequencing to facilitate rapid sequencing and enable the simultaneous detection of multiple variants. A single-stranded template for pyrosequencing was prepared by amplifying a 314-bp fragment (nt 75-388) with a biotin at the 5'-end of the reverse primer to facilitate strand separation and purification. Polymorphisms at the nucleotide sites 109, 131, 132, 143, 145, 178 and 350 were determined in three separate sequencing reactions, one of which was a multiplex format. Pyrosequencing of 97 HPV16-positive exfoliated cervical samples confirmed the Sanger sequencing results; however pyrosequencing identified additional variants in several samples containing mixed variants.

Base Sequence↗
PubMed2005

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↗
PubMedDate not supplied

Unintended pregnancy. Consequences and solutions for a worldwide problem.

Unintended pregnancy is a worldwide problem that affects women, their families, and society. Unintended pregnancy can result from contraceptive failure, non-use of contraceptive services, and, less commonly, rape. Abortion is a frequent consequence of unintended pregnancy and, in the developing world, can result in serious, long-term negative health effects including infertility and maternal death. In many developing countries, poverty, malnutrition, and lack of sanitation and education contribute to serious health consequences for women and their families experiencing an unintended pregnancy. Regardless of the cause, unintended pregnancy and its negative consequences can be prevented by access to contraceptive services including emergency contraception, safe and legal abortion services, and a society that allows women to determine their own reproductive choices. Addressing unintended pregnancy and its substantial human and dollar costs should be a priority in every country. The availability of reliable contraception for all, regardless of age or ability to pay, is an essential first step. Women and adolescents require access to age-appropriate and culturally sensitive reproductive health care services, including emergency contraception. Access to safe, legal abortion services is necessary to impact the staggering maternal mortality rates worldwide. Midwives throughout the world provide the majority of care for women of reproductive age. It is essential to identify those at risk for unintended pregnancy, provide the services they require, and remain diligent to ensure that those women and their families have safe options to consider when faced with an unintended pregnancy. In 1920, Magaret Sanger said, "No women can call herself free who does not control her own body." Although great strides have been made to improve the health and status of women since Ms. Sanger spoke those words, there remains much work to be done.

Contraception↗
PubMed1996

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↗
PubMed2005

A 768-lane microfabricated system for high-throughput DNA sequencing.

A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm x 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor. A total of greater than 172000 bases, 99% accuracy (corresponding to quality score 20) is achieved for each iteration of a 384 lane plate. At current operating conditions, this implies a system throughput exceeding 4 megabases of raw sequence (Phred 20) per day on the new platform. Standard operation is at "1/32x" Sanger chemistry, equal to typical genome center operation on mature capillary array machines, and a 16-fold improvement in scaling relative to previous microfabricated devices. Experiments provide evidence that sample concentration can be further reduced to 1/256x Sanger chemistry in the microdevice. Life-testing indicates a usable life of >150 hours (more than 50 runs) for the 384 lane plates. The combined advances, particularly those in read length and sample requirement, directly address the cost model requirements for adaptation of the new technology as the next step beyond capillary array instruments.

DNA↗
PubMed1979

Rapid sequence determination of late simian virus 40 16S mRNA leader by using inhibitors of reverse transcriptase.

A method for the determination of the primary structure of spliced mRNA junction and leader sequences is described. By analogy to the DNA sequencing procedure of Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci USA 74, 5463--5467], we use 2",3'-dideoxynucleoside triphosphates as chain-terminating inhibitors of the reverse transcriptase (RNA-dependent DNA polymerase) reaction. By using specific DNA restriction fragments as primers in combination with this technique, we have determined the sequence of the spliced junction between the body and the leader sequence of the 16S late mRNA of simian virus 40. The method described should be of general utility in mapping spliced mRNA regions for which the corresponding protein sequence (if any) is unknown.

Base Sequence↗
PubMed1980

Sequence of 1060 3'-terminal nucleotides of poliovirus RNA as determined by a modification of the dideoxynucleotide method.

The dideoxynucleotide method for sequencing DNA developed by Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467] was modified to allow sequence analysis of poliovirus RNA without recourse to cloning. Our method involves reverse transcription of poliovirus RNA followed by cDNA-dependent DNA synthesis in the presence of unlabeled dNTPs and 2',3'-dideoxynucleoside triphosphates, with Escherichia coli DNA polymerase I (Klenow) used to catalyze the reaction. DNA synthesis is primed by 5'-32P-labeled RNase T1- or RNase A-resistant oligonucleotides generated from poliovirus RNA. The sequence of 1060 nucleotides preceding the 3'-terminal poly(A) is presented. Based on the position of termination codons we propose that viral translation terminates at nucleotide -562.

Base Sequence↗
PubMed2004

Sequencing from compomers: using mass spectrometry for DNA de novo sequencing of 200+ nt.

One of the main endeavors in today's life science remains the efficient sequencing of long DNA molecules. Today, most de novo sequencing of DNA is still performed using the electrophoresis-based Sanger concept of 1977, in spite of certain restrictions of this method. Methods using mass spectrometry to acquire the Sanger sequencing data are limited by short sequencing lengths of 15-25 nt. We propose a new method for DNA sequencing using base-specific cleavage and mass spectrometry that appears to be a promising alternative to classical DNA sequencing approaches. A single stranded DNA or RNA molecule is cleaved by a base-specific (bio-)chemical reaction using, for example, RNAses. The cleavage reaction is modified such that not all, but only a certain percentage of bases are cleaved. The resulting mixture of fragments is then analyzed using MALDI-TOF mass spectrometry, whereby we acquire the molecular masses of fragments. For every peak in the mass spectrum, we calculate those base compositions that will potentially create a peak of the observed mass and, repeating the cleavage reaction for all four bases, finally try to uniquely reconstruct the underlying sequence from these observed spectra. This leads us to the combinatorial problem of sequencing from compomers and, finally, to the graph-theoretical problem of finding a walk in a subgraph of the de Bruijn graph. Application of this method to simulated data indicates that it might be capable of sequencing DNA molecules with 200+ nt.

Algorithms↗
PubMed1995

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↗
PubMed2005

Emerging technologies in DNA sequencing.

Demand for DNA sequence information has never been greater, yet current Sanger technology is too costly, time consuming, and labor intensive to meet this ongoing demand. Applications span numerous research interests, including sequence variation studies, comparative genomics and evolution, forensics, and diagnostic and applied therapeutics. Several emerging technologies show promise of delivering next-generation solutions for fast and affordable genome sequencing. In this review article, the DNA polymerase-dependent strategies of Sanger sequencing, single nucleotide addition, and cyclic reversible termination are discussed to highlight recent advances and potential challenges these technologies face in their development for ultrafast DNA sequencing.

Animals↗
PubMed1979

Nucleotide sequence coding for the N-terminal region of the matrix protein influenza virus.

After polyadenylation in vitro of the influenza virus RNA segment which contains the coding information for the matrix protein, a cDNA copy can be made using the primer p(dT)8-dA and reverse transcriptase. The sequence of 166 nucleotides of the cDNA was determined by a modification [Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93--117] of the plus/minus method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441--481] and adaptation of the "dideoxy" method [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463--5467] for sequencing DNA. The cDNA sequences is of the same sense as the mRNA for matrix protein and contains a potential initiating codon, d(ATG), at position 26--28. When matrix protein purified from virus particles was digested with chymotrypsin or trypsin and the amino acid compositions of separated peptides determined, one peptide containing nine amino acids found which had a composition corresponding to that predicted by the cDNA sequence following the first methionine codon, confirming that protein synthesis initiates at this position. The compositions of four other peptides matches those predicted from the nucleotide sequence. There is no processing of the N terminus of the protein before incorporation into the virus particle except for removal of the N-terminal methionine and addition of a "blocking" group on the resulting N-terminal serine residue.

Amino Acid Sequence↗