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A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterized DNA segments and repetitive DNA elements.
DNA sequencing by synthesis (SBS) offers an approach for potential high-throughput sequencing applications. In this method, the ability of an incoming nucleotide to act as a reversible terminator for a DNA polymerase reaction is an important requirement to unambiguously determine the identity of the incorporated nucleotide before the next nucleotide is added. A free 3'-OH group on the terminal nucleotide of the primer is necessary for the DNA polymerase to incorporate an incoming nucleotide. Therefore, if the 3'-OH group of an incoming nucleotide is capped by a chemical moiety, it will cause the polymerase reaction to terminate after the nucleotide is incorporated into the DNA strand. If the capping group is subsequently removed to generate a free 3'-OH, the polymerase reaction will reinitialize. We report here the design and synthesis of a 3'-modified photocleavable fluorescent nucleotide, 3'-O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleavable 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene), as a reversible terminator for SBS. This nucleotide analogue contains an allyl moiety capping the 3'-OH group and a fluorophore Bodipy-FL-510 linked to the 5 position of the uracil through a photocleavable 2-nitrobenzyl linker. Here, we have shown that this nucleotide is a good substrate for a DNA polymerase. After the nucleotide was successfully incorporated into a growing DNA strand and the fluorophore was photocleaved, the allyl group was removed by using a Pd-catalyzed reaction to reinitiate the polymerase reaction, thereby establishing the feasibility of using such nucleotide analogues as reversible terminators for SBS.
AT-rich DNA sequences other than AnTm tracts (n + m > or = 4) are known to be intrinsically curved. The AATAT-element constitutes one known example of these sequences. In this paper, the elucidation of the structural basis of the curvature induced by this sequence element was addressed. As judged by the patterns of cleavage by the hydroxyl radical and DNase I, the AATAT sequence shows a narrow minor groove. Furthermore, the 5' adenine residue of the AA dinucleotide contained within the sequence is hyperreactive to diethylpyrocarbonate. Similar structural properties are shown by several sequences inducing intrinsic DNA curvature, such as an A5-tract or the closely related ATAAT, AATATA and TAATAT sequences, which are also shown here to induce curvature. On the other hand, other related sequences, such as TATAA and ATATA, that do not induce curvature, show different structural characteristics.
A novel DNA methylation assay technique, termed bisulfite single-strand conformation polymorphism (bisulfite-SSCP), is a combination of sodium-bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. After bisulfite treatment followed by PCR amplification, methylated and unmethylated alleles can be simultaneously separated in a nondenaturing gel using an automated DNA sequencer. Using bisulfite-SSCP, methylation of hMLH1 was detected in a quantitative manner. This method is not only simple, quick, accurate, and quantitative, but detailed information about methylation is also available with less work. Methylation analysis of large numbers of samples for multiple loci will be facilitated by bisulfite-SSCP.
A DNA sequencing method is presented that utilizes capillary array electrophoresis, two-color fluorescence detection, and a two-dye labeling protocol. Sanger DNA sequencing fragments are separated on an array of capillaries and detected on-column using a two-color, laser-excited, confocal-fluorescence scanner. The four sets of DNA sequencing fragments are separated in a single capillary and then distinguished by using a binary coding scheme where each fragment set is labeled with a characteristic ratio of two dye-labeled primers. Since only two dye-labeled primers are required, it is possible to select dyes that have identical mobility shifts. It is also shown that the ratio of the signal in the two detection channels provides a reliable identification of the sequencing fragment. DNA sequencing results on a 25-capillary array are presented.
A new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state-of-the-art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with greater than 99% accuracy.
We have isolated and sequenced a cloned complementary DNA insert complementary to the messenger RNA of a cytoplasmic actin expressed in human epidermal cells. This provides the first cytoplasmic actin complementary DNA sequence for a vertebrate organism. The actin amino acid sequence predicted from this complementary DNA is identical to that of a bovine cytoplasmic actin and shows 98 and 85% homology with a Dictyostelium and a yeast actin, respectively. The complementary DNA sequence indicates that the 3' end of the mRNA contains an unusually long (greater than 400 nucleotides) 3' non-translated region. A comparison of this 3' non-coding region with those of recently determined actin complementary DNA sequences from other species reveals little or no homology among these sequences. Thus, these results indicate that although the actin amino acid sequences are extremely conserved, the non-coding regions of the mRNAs diverge rapidly.
It is generally accepted that there are 3 subspecies of Oncorhynchus masou in Japan, namely, Masu salmon (Oncorhynchus masou masou (Brevoort)), Amago salmon (O. masou ishikawae Jordan & McGregor), and Biwa salmon (O. masou rhodurus Jordan & McGregor or O. masou subsp. Kimura). Since the genetic relationship of these three taxa is not well known, there has been considerable confusion over their nomenclature. We have clarified the genetic relationship among these three taxa by partially sequencing their mitochondrial DNA. Sequences of 948 base pairs from the 3' region of the ATPase subunit 6 gene to the 5' region of the cytochrome oxidase subunit 3 gene were obtained for 20 individuals including wild Biwa salmon, wild and farmed Amago and Masu salmon. Furthermore, 2,162 base pairs from the 3' region of ATPase subunit 6 gene to the 5' region of NADH dehydrogenase subunit 4L gene were determined in 4 individuals. In total, there were 26 sites of base substitutions. The haplotypes of Masu salmon and Amago salmon were similar. On the other hand, 17 of the 26 sites had substitutions characteristic of Biwa salmon. A matrix of genetic distances and maximum parsimony analysis among the haplotypes indicated that Biwa salmon is genetically more distant from Masu and Amago salmon, than Masu salmon is from Amago salmon. This means that Biwa salmon diverged from the common ancestor of the Oncorhynchus masou complex before the divergence between Masu salmon and Amago salmon.
Mitochondrial DNA (mtDNA) evolution was investigated in skinks of the genus Chalcides found in the Canary Islands (Ch. sexlineatus, Ch. viridanus and Ch. simonyi), together with some North African congenerics (Ch. polylepis and Ch. mionecton). Several sites were included within islands to cover areas of known within-island geographical variation in morphology. Skinks from the islands of El Hierro and La Gomera appear to be sister taxa. The relationships between this clade and the Tenerife and Gran Canarian skinks were not fully resolved, although the best working hypothesis indicated monophyly with the former, with the latter forming a closely related outgroup. Ch. simonyi from Fuerteventura was more distantly related to the Western Canary Island skinks and did not show close relationships with the North African species Ch. mionecton and Ch. polylepis. Possible colonization sequences for the four most Western Canary Islands were considered. El Hierro appears to have been colonized relatively recently from La Gomera, commensurate with the recent origin of this island, while dispersal between La Gomera and Tenerife and between Gran Canaria and Tenerife or La Gomera appears to have taken place considerably earlier. Substantial within-island haplotype divergence was found in Gran Canaria and Tenerife. This may be a result of recent periods of intense volcanic activity found within these two islands. Lower levels of within-island differentiation are found in La Gomera and El Hierro and may be explained by lower levels of volcanic activity during recent geological history and a more recent colonization, respectively.
An approach for DNA sequencing is described that circumvents the need for synthetic oligonucleotide primers, which seriously restrict the progress of DNA sequencing in the commonly used protocol. The method is based on the use of short restriction fragments as primers randomly distributed along single-stranded templates. Premapping of target DNA is eliminated and subcloning manipulation is minimized. This method has been used successfully for sequencing genes in the range of 2 kb, for which about 10 restriction fragment primers per kilobase were sufficient to generate a continuous overlapping sequence in alignment. The approach has also been readily applied for an automated sequencing system with the fluorescent chain-terminating dideoxynucleotides, thus implying its potential for sequencing large genomic DNAs.
A simplified method of DNA sequencing by dideoxy chain termination is developed that approaches a single-step protocol. Utilizing the sequencing advantages contributed by a thermophilic polymerase and a guanine analog, stable sequencing reaction concentrates have been obtained that readily perform the entire sequencing reaction simply by adding prepared DNA to each of the four reaction concentrates required by this method. The mechanics and dynamics of these reactions have been investigated and the capacity of these reactions to withstand normal user variation is demonstrated. This study focuses on one form of this simplified method embodied in the FASTaq DNA sequencing kit.
We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase. The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders from unpurified PCR fragments of sufficiently high quality such that heterozygotes can be reproducibly detected and identified by software that recognizes signal-strength patterns indicative of mixed-base positions.
Purines play essential roles in many cellular functions, including DNA replication, transcription, intra- and extra-cellular signaling, energy metabolism, and as coenzymes for many biochemical reactions. The de-novo synthesis of purines requires 10 enzymatic steps for the production of inosine monophosphate (IMP). Defects in purine metabolism are associated with human diseases. Further, many anticancer agents function as inhibitors of the de-novo biosynthetic pathway. Genes or cDNAs for most of the enzymes comprising this pathway have been isolated from humans or other mammals. One notable exception is the phosphoribosylformylglycineamide amidotransferase (FGARAT) gene, which encodes the fourth step of this pathway. This gene has been cloned from numerous microorganisms and from Drosophila melanogaster and C. elegans. We report here the identification of a human cDNA containing the coding region of the FGARAT mRNA and the isolation of a P1 clone that contains an intact human FGARAT gene. The P1 clone corrects the purine auxotrophy and protein deficiency of Chinese hamster ovary (CHO) cell mutants (AdeB) deficient in both the activity and the protein for FGARAT. The P1 clone was used to regionally map the FGARAT gene to chromosome region 17p13, a location consistent with our prior assignment of this gene to chromosome 17. A comparison of the DNA sequence of the human FGARAT and FGARAT DNA sequence from 17 other organisms is reported. The isolation of this gene means that DNA clones for all the 10 steps of IMP synthesis have been isolated from humans or other mammals.
The resonances of most of the nonexchangeable protons of both + and - strands of the consensus Pribnow dodecamer d( CGTTATAATGCG ) have been assigned by two-dimensional nuclear magnetic resonance methods. Application of the two-dimensional nuclear Overhauser effect ( NOESY ) sequential connectivity method, combined with two-dimensional autocorrelated ( COSY ) spectra to reveal scalar-coupled protons, results in assignment of virtually all of the base and sugar protons, except the sugar C5 protons which are inadequately resolved. Analysis of the nuclear Overhauser data indicates that the helix assumes a fairly uniform B form conformation.
Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the "boiling-stable" polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed.
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MOTIVATION: DNA sequence alignment, an important genomic task, involves assigning short DNA reads to the most probable locations on an extensive reference genome. Conventional methods tackle this challenge in two steps: genome indexing followed by efficient search to locate likely positions for given reads. Building on the success of Large Language Models in encoding text into embeddings, where the distance metric captures semantic similarity, recent efforts have encoded DNA sequences into vectors using Transformers and have shown promising results in tasks involving classification of short DNA sequences. Performance at sequence classification tasks does not, however, guarantee sequence alignment, where it is necessary to conduct a genome-wide search to align every read successfully, a significantly longer-range task by comparison. RESULTS: We bridge this gap by developing a "Embed-Search-Align" (ESA) framework, where a novel Reference-Free DNA Embedding (RDE) Transformer model generates vector embeddings of reads and fragments of the reference in a shared vector space; read-fragment distance metric is then used as a surrogate for sequence similarity. ESA introduces: (i) Contrastive loss for self-supervised training of DNA sequence representations, facilitating rich reference-free, sequence-level embeddings, and (ii) a DNA vector store to enable search across fragments on a global scale. RDE is 99% accurate when aligning 250-length reads onto a human reference genome of 3 gigabases (single-haploid), rivaling conventional algorithmic sequence alignment methods such as Bowtie and BWA-Mem. RDE far exceeds the performance of six recent DNA-Transformer model baselines such as Nucleotide Transformer, Hyena-DNA, and shows task transfer across chromosomes and species. AVAILABILITY AND IMPLEMENTATION: Please see https://anonymous.4open.science/r/dna2vec-7E4E/readme.md.
There is considerable interest in the development of sequence-selective DNA drugs. Chemical agents able to interfere with DNA topoisomerases - essential nuclear enzymes- are widespread in nature, and some of them have outstanding therapeutic efficacy in human cancer and infectious diseases. Several classes of antineoplastic drugs, such as amsacrine, daunorubicin, etoposide (acting on type II topoisomerases), camptothecin and indolocarbazole derivatives of the antibiotic rebeccamycin (acting on type IB topoisomerases), have been shown to stimulate DNA cleavage by topoisomerases leading to cell death. However, these molecules exhibit little sequence preference. A convenient strategy to confer sequence specificity consists in the attachment of these topoisomerase poisons to sequence-specific DNA binding elements. Among sequence-specific DNA ligands, oligonucleotides can bind with high specificity of recognition to the major groove of double-helical DNA, resulting in triple helix formation. In this context, derivatives of camptothecin, indolocarbazole, anthracycline and acridine poisons have been covalently tethered to triple helix-forming oligonucleotides. The use of triple-helical DNA structures offers an efficient system to target topoisomerase I and II-mediated DNA cleavage to specific sequences and to increase the drug efficacy at these sites. Chemical optimization of the conjugates is essential to the efficacy of drug targeting. Consequently, the rational design of this new class of anti-cancer agents, conceived from topoisomerase poisons and triplex-forming oligonucleotides, may be exploited to improve the efficacy and selectivity of the DNA damage induced by topoisomerases.