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PubMed1990

DNA sequencing, automation, and the human genome.

DNA sequencing is one of the key analytical operations of modern molecular biology and a crucial element of biotechnology. The principles of DNA sequencing and details of the technologies of both manual, radioisotope-based and automated, fluorescence-based approaches are described. The goals and rationale of the Human Genome Initiative are discussed along with implications for future sequencing technologies. Finally, a glimpse of emerging DNA sequencing technologies is offered.

Autoanalysis↗
PubMed2005

Improved alignment of nucleosome DNA sequences using a mixture model.

DNA sequences that are present in nucleosomes have a preferential approximately 10 bp periodicity of certain dinucleotide signals, but the overall sequence similarity of the nucleosomal DNA is weak, and traditional multiple sequence alignment tools fail to yield meaningful alignments. We develop a mixture model that characterizes the known dinucleotide periodicity probabilistically to improve the alignment of nucleosomal DNAs. We assume that a periodic dinucleotide signal of any type emits according to a probability distribution around a series of 'hot spots' that are equally spaced along nucleosomal DNA with 10 bp period, but with a 1 bp phase shift across the middle of the nucleosome. We model the three statistically most significant dinucleotide signals, AA/TT, GC and TA, simultaneously, while allowing phase shifts between the signals. The alignment is obtained by maximizing the likelihood of both Watson and Crick strands simultaneously. The resulting alignment of 177 chicken nucleosomal DNA sequences revealed that all 10 distinct dinucleotides are periodic, however, with only two distinct phases and varying intensity. By Fourier analysis, we show that our new alignment has enhanced periodicity and sequence identity compared with center alignment. The significance of the nucleosomal DNA sequence alignment is evaluated by comparing it with that obtained using the same model on non-nucleosomal sequences.

Algorithms↗
PubMed1989

Mitochondrial NADH-ubiquinone reductase: complementary DNA sequences of import precursors of the bovine and human 24-kDa subunit.

The 24-kDa subunit of mitochondrial NADH-ubiquinone reductase (complex I) is an iron-sulfur protein that is present in the flavoprotein or NADH dehydrogenase II subcomplex. It is a nuclear gene product and is imported into the organelle. A group of human patients with mitochondrial myopathy have been shown to have reduced levels of subunits of complex I in skeletal muscle mitochondria, and in one patient the 24-kDa subunit appears to be absent (Schapira et al., 1988). To investigate the genetic basis of this type of myopathy, cDNA clones have been isolated from a bovine library derived from heart and liver mRNA by hybridization with two mixtures of 48 synthetic oligonucleotides 17 bases in length that were designed on the basis of known protein sequences. The recombinant DNA sequence has been determined, and it encodes a precursor of the mature 24-kDa protein. The N terminus of the mature protein is preceded by a presequence of 32 amino acids that has properties that are characteristic of mitochondrial import sequences. The sequence of the mature protein deduced from the cDNA contains a segment of nine amino acids that was not determined in an earlier partial protein sequence analysis. The bovine clone has been employed as a hybridization probe to identify cDNA clones of the human homologue of the 24-kDa protein. Its DNA sequence has also been determined, and it codes for a protein that is closely related to the bovine protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1981

Revertant seedlings from crown gall tumors retain a portion of the bacterial Ti plasmid DNA sequences.

BT37 is a crown gall teratoma incited on tobacco by Agrobacterium tumefaciens containing pTi-T37, a nopaline-type Ti plasmid. Treatment of this cloned tumor tissue with kinetin at 1 mg/liter results in the formation of relatively normal-appearing shoots. These shoots can be induced to root and set viable seed. In contrast to BT37 tissue, the derived tissues are not phytohormone independent and do not produce nopaline. The reverted plants, like normal tobacco plants, are susceptible to infection by A. tumefaciens. This loss of tumorous traits is accompanied by the loss of most of the Ti plasmid sequences (T-DNA) found in BT37 DNA. Southern blot analysis indicates that the revertant tissues have lost the central portion of the T-DNA, which contains the "common DNA" sequences, a highly conserved region of the Ti plasmid that has been found to be incorporated into all tumors studied. Thus, these sequences appear necessary for oncogenicity and tumor maintenance and their loss is probably directly related to tumor reversal. The reverted plants as well as the plants obtained from seed, however, do retain sequences homologous to the ends of the T-DNA present in the parental teratoma. The persistence of foreign DNA sequences during the process of meiosis and seed formation has important implications for the possibility of the genetic engineering of plants.

Journal Article↗
PubMed1989

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence.

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

Animals↗
PubMed1982

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.

Animals↗
PubMed1992

Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.

Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.

Autoradiography↗
PubMed1981

Quantitative in situ hybridization reveals extent of sequence homology between related DNA sequences in Drosophila melanogaster.

Cloned DNA from the larval serum protein one (LSP-1) genes was hybridized to polytene chromosomes of D. melanogaster. The ratio of grains deposited over any two of the three LSP-1 genes with any one LSP-1 subunit probe was constant. Varying the gene dose of any one LSP-1 subunit relative to the others by up to six fold gave a linear relationship of grain ratios to gene ratios. We show that these constant ratios closely reflect the extent of sequence homology between the genes as determined by heteroduplex mapping (Smith et al., 1981) and thermal denaturation studies. The results obtained demonstrate that the LSP-1 subunit genes are present in equal copies in the genome.

Animals↗
PubMed1997

Direct DNA sequence determination from total genomic DNA.

It is possible to perform a combined amplification and sequencing reaction ('DEXAS') directly from complex DNA mixtures by using two thermostable DNA polymerases, one that favours the incorporation of deoxynucleotides over dideoxynucleotides, and one which has a decreased ability to discriminate between these two nucleotide forms. During cycles of thermal denaturation, annealing and extension, the former enzyme primarily amplifies the target sequence whereas the latter enzyme primarily performs a sequencing reaction. This method allows the determination of single-copy nuclear DNA sequences from amounts of human genomic DNA comparable to those used to amplify nucleotide sequences by the polymerase chain reaction. Thus, DNA sequences can be easily determined directly from total genomic DNA.

Base Sequence↗
PubMed1983

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses.

Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.

Journal Article↗
PubMed2001

Detection of rifampin-resistant Mycobacterium tuberculosis in sputa by nested PCR-linked single-strand conformation polymorphism and DNA sequencing.

Either PCR-mediated single strand conformation polymorphism (SSCP) analysis or DNA sequencing of rpoB DNA (157 bp) can be used as a rapid screening method for the detection of mutations related to the rifampin resistance of Mycobacterium tuberculosis. However, due to the nonspecific amplification of rpoB DNA from nontuberculous mycobacteria these methods cannot be directly applied to clinical specimens such as sputa. We developed a nested PCR method that can specifically amplify the rpoB DNA of M. tuberculosis on the basis of rpoB DNA sequences of 44 mycobacteria. Nested PCR-linked SSCP analysis and the DNA sequencing method were applied directly in order to detect M. tuberculosis and determine its rifampin susceptibility in 56 sputa. The results obtained by nested PCR-SSCP and DNA sequencing were concordant with those of conventional drug susceptibility testing and DNA sequencing performed with culture isolates.

Antibiotics, Antitubercular↗
PubMed1989

DNA sequence of a class I pseudogene from the Tla region of the murine MHC: recombination at a B2 alu repetitive sequence.

We have determined the DNA sequence of a BALB/c Tla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1c gene from the Tlac genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1c gene contains sequences that induced expression of a serologically reactive Tla gene in the genome of the recipient L cell. The T1c gene is structurally related to the previously sequenced T13c gene that encodes a serologically defined TL antigen. The 3' half of the T1c gene including exons 4, 5, 6, and the 3' untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13c gene; however, the 5' half of the T1c gene has little homology with the T13c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event.

Animals↗
PubMed1987

Asynchronous distance between homologous DNA sequences.

The distance between homologous DNA sequences of two species is proposed to be -1/4 ln[det(P)], where P is the conditional probability matrix specifying the proportions of the various nucleotides in the second sequence, corresponding to each of the four nucleotides in the first sequence. A probability model is described which supports this choice of distance. Distance measures based on a constant evolutionary rate assumption are described and compared with the proposed measure. Sampling properties of both types of distance are examined and we conclude by applying the distance measures to mitochondrial DNA sequences of the hominoids.

Base Sequence↗
PubMed1988

Determinations of the DNA sequence of the mreB gene and of the gene products of the mre region that function in formation of the rod shape of Escherichia coli cells.

The 6.5-kilobase mre region at 71 min in the Escherichia coli chromosome map, where genes involved in formation of a rod-shaped cell form a gene cluster, was analyzed by in vivo protein synthesis in a maxicell system and by base sequencing of DNA. An open reading frame that may code for a protein with an Mr of about 37,000 on sodium dodecyl sulfate-polyacrylamide gels was found and was correlated with the mreB gene. N-terminal amino acid sequencing of the hybrid mreB-lacZ protein confirmed the production by mreB of a protein of 347 amino acid residues with a molecular weight of 36,958. The amino acid sequence of this protein deduced from the DNA sequence showed close similarity with that of a protein of the ftsA gene which is involved in cell division of E. coli. Three other contiguous genes that formed three proteins with Mrs of about 40,000, 22,000, and 51,000, respectively, were detected downstream of the mreB gene by in vivo protein synthesis. The mreB protein and some of these three proteins may function together in determination of cell shape.

Amino Acid Sequence↗
PubMed1995

Latent periodicity of DNA sequences from some human gene regions.

The mutual information is used to reveal of DNA sequences latent periodicity. Latent periodicity of DNA sequence is periodic. ity with low level of homology between any two periods inside DNA sequence. The mutual information between artificial numerical sequence and DNA sequence is calculated. The length of artificial sequence period is changed from 2 to 250. High level of mutual information between artificial and DNA sequences allows to find any type of latent periodicity of DNA sequence. The latent periodicity of some DNA coding regions is considered. For example, 24 exon of Apo B-100 gene from HSAP821 clone contains latent period 84 bases long. The IGF-I receptor gene from HSIGFIRR clone contains the region with latent period 57 bases long. Possible significance of latent periodicity is discussed.

Apolipoprotein B-100↗
PubMed2005

Molecular engineering approaches for DNA sequencing and analysis.

High-throughput DNA sequencing development for mutation screening and identification is essential to realize the goal of pharmacogenomics and personalized medicine, which will lead to a new era in clinical medicine and healthcare. Molecular engineering approaches to modify the building blocks of DNA by introducing functional groups for purification and detection has led to the development of high-throughput genetic analysis technologies. This review is focused on the following two DNA sequencing approaches. The first approach is based on the use of molecular affinity and mass spectrometry to perform quick and highly accurate mutation screening, heterozygote identification and insertion/deletion detection. The second approach is based on a sequencing-by-synthesis platform that has the potential for generating DNA sequencing data in a massive, parallel manner. The basic principles, fundamental challenges and methods of implementation of these exciting new technologies will be discussed.

Animals↗
PubMed1999

An efficient DNA sequencing strategy based on the bacteriophage mu in vitro DNA transposition reaction.

A highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an artificial transposon with a chloramphenicol acetyltransferase (cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10.3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasmids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells that were then selected on appropriate selection plates. Plasmids from individual clones were isolated and used as templates for DNA sequencing, each with two primers specific for the transposon sequence but reading the sequence into opposite directions, thus creating a minicontig. By combining the information from overlapping minicontigs, the sequence of the entire 10,288-bp region of mouse genome including six exons of mouse Kcc2 gene was obtained. The results indicated that the described methodology is extremely well suited for DNA sequencing projects in which considerable sequence information is on demand. In addition, massive DNA sequencing projects, including those of full genomes, are expected to benefit substantially from the Mu strategy.

Amino Acid Sequence↗
PubMed1999

Functional analysis of DNA sequences located within a cluster of DNase I hypersensitive sites colocalizing with a MAR element at the upstream border of the chicken alpha-globin gene domain.

We have cloned and sequenced a genomic DNA fragment of chicken containing a cluster of DNase I hypersensitive sites (DHS) located 11-15 kb upstream from the first gene of the alpha-globin gene domain and including a constitutive DHS flanked by two erythroid-specific ones. A 1.2-kb subfragment of the DNA fragment under study located upstream to the constitutive DHS and colocalizing roughly with one of the erythroid-specific DHS was shown to possess the properties of a matrix association region (MAR). The cloned DNA sequences were tested for their ability to serve as promoters and/or influence transcription from the promoter of the alphaD globin gene. In the region studied, we did not find any promoters or enhancers that were active in erythroid cells. The whole DNase I hypersensitive region and some of its subfragments showed a silencing effect when placed downstream from the reporter gene. The expression of the reporter gene was completely abolished, however, when these DNA fragments were placed between the alphaD promoter and the reporter gene. Thus, they seem to act as transcription "terminators." Numerous polyadenylation signals (AATAAA) and an AT-rich palindrome were found within the sequenced DNA fragment. These observations are discussed within the frame of the hypothesis postulating that continuous transcription is essential for maintaining the active status of genomic domains. Furthermore, it is suggested that the DNA fragment studied contains a negative control element that keeps globin genes silent within the chromatin domain permanently open in nonerythroid cells.

Animals↗