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PubMed1995

Evaluation by polymerase chain reaction on the effect of beta-propiolactone and binary ethyleneimine on DNA.

Inactivating treatments for viruses such as pasteurization or alkylation by beta-propiolactone or binary ethyleneimine were tested for their capacity to modify nucleic acids. The modification of a nucleic acid was measured as the decrease in spot intensity in Southern blots after polymerase chain reaction (PCR) amplification. The inactivating treatments were applied to cellular and viral genomic material from a human lymphoblastoid cell line immortalized by Epstein Barr Virus (EBV), which produced a monoclonal antibody. Pasteurization did not modify the ability to amplify and detect cellular or viral DNA. Binary ethyleneimine strongly reduced the amount of detectable DNA and beta-propiolactone under particular conditions of incubation abolished all trace of DNA.

Alkylating Agents↗

PubMed1991

Identification of the skeletal remains of a murder victim by DNA analysis.

There is considerable anthropological and forensic interest in the possibility of DNA typing skeletal remains. Trace amounts of DNA can be recovered even from 5,500-year-old bones and multicopy human mitochondrial DNA sequences can frequently be amplified from such DNA using the polymerase chain reaction (PCR). But given the sensitivity of PCR, it is very difficult to exclude contaminating material. We now report the successful identification of the 8-year-old skeletal remains of a murder victim, by comparative typing of nuclear microsatellite markers in the remains and in the presumptive parents of the victim. This analysis establishes the authenticity of the bone DNA and the feasibility of bone DNA typing in forensic investigations.

Adolescent↗

PubMed1984

Copper-phenanthroline-induced site-specific oxygen-radical damage to DNA. Detection of loosely bound trace copper in biological fluids.

Copper(II) ions, in the presence of 1,10-phenanthroline, O2 and a reducing agent, degrade DNA with the release of thiobarbituric-acid-reactive material. This reaction, dependent on the formation of oxygen radicals, was made the basis of a sensitive and specific assay for loosely bound copper in body fluids. When applied to certain extracellular fluids, trace amounts of copper could be detected in the lower micromolar range.

Binding Sites↗

PubMed1989

Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae.

Naturally elaborated membrane bleb material is frequently observed in cultures of Neisseria gonorrhoeae. This material was purified and analyzed for protein, lipopolysaccharide, and nucleic acid content. The electrophoretic protein profiles of two bleb-rich fractions, called BI and BII, were distinct, with only BII containing lipopolysaccharide and outer membrane proteins I and III. Both fractions contained RNA, circular DNA, and linear DNA. Exogenous pancreatic DNase I appeared to hydrolyze all bleb-associated DNA in fraction BI and the linear DNA in fraction BII. The circular DNA molecules associated with fraction BII resisted digestion. Electron microscopy of the bleb fractions verified their DNA content. Fixing blebs with glutaraldehyde before mounting them for microscopy prevented release of internal DNA. Such fixation produced little change in the micrographs of BI; however, only traces of DNA were observed in fixed BII preparations. Incubation of wild-type gonococci in mixtures of DNase and blebs purified from antibiotic-resistant strains resulted in efficient exchange of penicillinase-specifying R plasmids. Recipients incorporated plasmids independently of endogenous and exogenous chromosomal streptomycin resistance markers. These in vitro results suggest that bleb formation by N. gonorrhoeae may serve to transfer plasmids intercellularly in vivo, perhaps constituting a previously unexplored genetic exchange mechanism in these bacteria.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

[Clarification of a forced entry theft based on a beverage bottle left at the scene and PCR DNA typing of superficial saliva traces].

This paper describes the first case in Austria where a suspect was been convicted and condemned because of salivary traces found on an empty bottle which were successfully typed by PCR-DNA-analysis. This was the only evidence in that case. In contrast to cigarette butts, which are already relatively routine for PCR-DNA-typing, bottles found on crime scenes usually contain much less material which can be investigated. As the result of permanent refinement of the methods used, it is demonstrated that it should be possible to solve other similar cases in the future.

Adult↗

PubMed2002

Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases.

The nucleotide-binding site in a variety of DNA polymerases was probed by analyzing incorporation of dideoxy and acyclic chain terminators. Family B archaeon DNA polymerases Vent, Deep Vent, 9 degrees N and Pfu incorporated acyclic in preference to dideoxy terminators, while the Family A DNA polymerases Taq and Klenow preferred dideoxy terminators. These divergent biases suggest that significant differences exist in sugar recognition in these two classes of polymerases. Mutants of Vent (A488L) and Taq (F667Y) that increase incorporation of dideoxy terminators maintained the acyclic/dideoxy bias of the parent enzyme, while more efficiently incorporating both dideoxy and acyclic terminators. The preference of archaeon DNA polymerases for acyclic analogs was exploited in chain terminator DNA sequence and genotype analysis. This technology was additionally aided by identification of specific dye-modified bases that improve terminator incorporation over that of the unmodified terminator. These three enhancing effects, (i) acyclic analogs, (ii) archaeon variants and (iii) specific dyes, appear to act additively and independently to increase terminator incorporation efficiency, collectively enhancing incorporation approximately 8000-fold over the wild-type incorporation of dideoxynucleotides. Fluorescent chain terminator DNA sequence traces demonstrate the applicability of these advances in improving terminator incorporation, as required in DNA sequence and genotype determinations.

Amino Acid Substitution↗

PubMed1997

Increasing consensus accuracy in DNA fragment assemblies by incorporating fluorescent trace representations.

We present a new method for determining the consensus sequence in DNA fragment assemblies. The new method, Trace-Evidence, directly incorporates aligned ABI trace information into consensus calculations via our previously described representation, Trace-Data Classifications. The new method extracts and sums evidence indicated by the representation to determine consensus calls. Using the Trace-Evidence method results in automatically produced consensus sequences that are more accurate and less ambiguous than those produced with standard majority-voting methods. Additionally, these improvements are achieved with less coverage than required by the standard methods-using Trace-Evidence and a coverage of only three, error rates are as low as those with a coverage of over ten sequences.

Algorithms↗

PubMed1988

Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant.

The principal objectives of this study were to analyze the structure and coding potential of a long segment of DNA missing from a previously isolated (B. Moss, E. Winters, and J. A. Cooper (1981) J. Virol. 40, 387-395) attenuated variant of vaccinia virus strain WR and to examine the precise changes in the genome accompanying the deletion. The sequences of a 14.5-kbp region located at the left end of the standard vaccinia virus genome, extending from within the inverted terminal repetition (ITR) of the HindIII C fragment to the end of the HindIII N fragment, and of a 3-kbp segment from a corresponding region of the variant genome were determined. A comparison of these sequences revealed that the variant contained a deletion of 12 kbp and an insertion of 2.1 kbp. The origin of the inserted DNA was traced to the HindIII B region by using oligonucleotide probes indicating that a transposition of unique DNA located adjacent to the right ITR had occurred. Structural analysis indicated no extensive homologies, nucleotide substitutions, additions, or deletions at the boundaries of the transposed DNA. Examination of the right end of the variant genome indicated that a copy of the transposed DNA was still present and, therefore, the length of the ITR had been increased by 2.1 kbp. The variant genome could have formed by a mechanism that resulted in the replacement of a 22-kbp left-terminal fragment with a 12-kbp right-terminal fragment. The DNA missing from the variant and contained within the standard vaccinia virus WR genome contains 17 contiguous open reading frames (ORFs), all of which are directed leftward and apparently not required for replication in cultured cells. One deleted ORF has a 60% sequence similarity to another gene encoding a 42,000-Da protein present within the ITR suggesting that duplications have previously occurred during the evolution of vaccinia virus. Another deleted ORF has a 39% sequence similarity to a complement 4b binding protein. The transposed DNA contains two complete ORFs one of which has a 40% identity to a cowpox gene and a 30% identity to a family of plasma serine protease inhibitors.

Amino Acid Sequence↗

PubMed2006

An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex.

In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form in solution raises the possibility of other pathways for clamp loading. In this study, an alternative clamp loading sequence is evaluated in which gp44/62 first recognizes the DNA substrate and then sequesters the clamp from solution and loads it onto DNA. This pathway differs in terms of the initial formation of a gp44/62-DNA complex that is capable of loading gp45. In this work, we demonstrate ATP-dependent DNA binding by gp44/62. Among various DNA structures that were tested, gp44/62 binds specifically to primer-template DNA but not to single-stranded DNA or blunt-end duplex DNA. By tracing the dynamic clamp closing with pre-steady-state FRET measurements, we show that the clamp loader-DNA complex is functional in clamp loading. Furthermore, pre-steady-state ATP hydrolysis experiments suggest that 1 equiv of ATP is hydrolyzed when gp44/62 binds to DNA, and additional ATP hydrolysis is associated with the completion of the clamp loading process. We also investigated the detailed kinetics of binding of MANT-nucleotide to gp44/62 through stopped-flow FRET and demonstrated a conformational change as the result of ATP, but not ADP binding. The collective kinetic data allowed us to propose and evaluate a sequence of steps describing this alternative pathway for clamp loading and holoenzyme formation.

Adenosine Diphosphate↗

PubMed1994

Evaluation of the efficacy and safety of in vitro, adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA.

Cystic fibrosis (CF) is a common, fatal recessive disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene manifested by abnormalities in the regulation of chloride ion (Cl-) secretion across the apical membrane of epithelial cells throughout the body. Adenovirus-mediated delivery of the normal CFTR cDNA and correction of the CF epithelial cell Cl- secretory phenotype suggests the feasibility of gene therapy for CF lung disease. Few studies, however, have focused on the evaluation of the safety of the adenovirus-mediated gene transfer approach. This study presents in vitro data on the efficacy and safety of adenovirus-mediated transfer of the human CFTR cDNA using Av1Cf2. Av1Cf2-mediated transfer of the human CFTR cDNA complemented the abnormal cAMP-regulated Cl- permeability of cells with the CF epithelial phenotype. Av1 vectors did not replicate infectious virus in HeLa cells infected in vitro, although trace vector DNA synthesis was observed at very high multiplicity of infection. Expression of the adenoviral late gene for the hexon capsid protein was observed at trace levels in Av1 vector-infected HeLa cells, but not in freshly isolated human bronchial epithelial cells, consistent with the pattern of DNA synthesis observed in these different target cells. Although, these observations support the efficacy and safety of use of Av1Cf2 for treatment of the fatal pulmonary component of CF.

Adenoviruses, Human↗

PubMed1999

DNA typing of fingerprints using capillary electrophoresis: effect of dactyloscopic powders.

DNA typing is a useful tool in crime solving, not only for blood samples, sperm, or saliva but also for traces of DNA left on tools or pieces of clothing used in burglaries or thefts. On these kinds of samples, the sources of DNA are extremely small amounts of skin debris left after gripping tools. When a sensitive technique such as polymerase chain reaction (PCR) coupled with capillary electrophoresis is used, it is possible to get a profile from these low amounts of DNA. The classic technique in such cases, used in forensic sciences, is to reveal fingerprints by different dactyloscopic powders. Therefore, DNA profiling was performed on physical fingerprints left on glass and wooden plates, in order to establish eventual problems or interferences involved by using both techniques simultaneously. Eleven dactyloscopic powders were investigated on their influence on DNA typing. The results show that some can be used together with DNA profiling but that serious precautions have to be taken to avoid contamination.

Artifacts↗

PubMed2002

Epidemiology of tuberculosis in Hamburg, Germany: long-term population-based analysis applying classical and molecular epidemiological techniques.

To determine a detailed picture of tuberculosis (TB) epidemiology in Hamburg, Germany, 423 Mycobacterium tuberculosis complex isolates from 77.0% of all patients with culture-confirmed TB diagnosed from 1997 to 1999 in Hamburg were analyzed by IS6110 DNA fingerprinting. IS6110 restriction fragment length polymorphism (RFLP) clusters were assumed to have arisen from recent transmission. Results of contact tracing and additional patient interviews were used for further epidemiological analyses. Of the 423 cases, 398 were included in the cluster analysis, of which 135 (33.9%) were classified into 35 clusters ranging from 2 to 23 patients. Epidemiological links verifying recent transmission could be confirmed for 87 of the 135 clustered patients. Risk factors for recent transmission were calculated by a two-step procedure: first, based on patients with clustered isolates; and second, based on patients with clustered isolates and transmission links. In both analyses, alcohol abuse appeared to be the strongest predictor for recent transmission, followed by a history of previous contact tracing and unemployment. Homelessness, foreign ethnicity, sex, drug addiction, and human immunodeficiency virus positivity were not independent risk factors for clustering in multivariate analyses. Classical contact tracing performed prior to IS6110 RFLP analysis identified only 24 of the 135 clustered patient. In conclusion, recent transmission seems to be frequent in Hamburg and was found to be strongly associated with alcohol abuse. Conventional contact tracing appears to be insufficient for the detection of recent transmission chains. The data presented also indicate that improved TB control strategies, including the use of RFLP for the detection of transmission chains, are needed for TB control in the setting of countries with a low incidence of TB.

Adolescent↗

PubMed2004

Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle, sheep, goat, roe buck and deer by PCR-RFLP.

BACKGROUND: As an alternative to direct DNA sequencing of PCR products, random PCR-RFLP is an efficient technique to discriminate between species. The PCR-RFLP-method is an inexpensive tool in forensic science, even if the template is degraded or contains only traces of DNA from various species. RESULTS: Interspecies-specific DNA sequence polymorphisms in the mitochondrial cytochrome b gene were analyzed using PCR-RFLP technology to determine the source (i.e., species) of blood traces obtained from a leaf. CONCLUSIONS: The method presented can be used for the discrimination of cattle (Bos taurus), sheep (Ovis aries), goat (Capra hircus), roe buck (Capreolus capreolus) and red deer (Cervus elaphus).

Animals↗

PubMedDate not supplied

[DNA synthesis on the heterogeneous nuclear RNA template catalysed by DNA polymerase of avian myeloblastosis virus].

Template activity of nuclear pre-mRNA has been investigated in DNA-polymerase reaction. Active synthesis of DNA was demonstrated on pre-mRNA as a template in the absence of primer. A part of synthetic activity may be attributed to the traces of DNA present in the pre-mRNA preparation. Addition of oligo(dT)10 to the template stimulated the synthesis of DNA product due to transcription of heteropolymeric regions near the poly(A). The rate of DNA synthesis was different depending on the fraction of template used: the RNA extracted by hot phenol at 85 degrees showed higher template activity without adding of primer than the 65 degrees C fraction. On the contrary 65 degrees C pre-mRNA which is known to contain greater quantity of molecules with poly(A) at the 3'-end is more strongly stimulated by addition of oligo(dT). The nuclear RNA corresponding to the precursors of rRNAs extracted at 40 degrees C were not transcribed by the reverse transcriptase. The size of the DNA-product (about 7-8S in alkaline sucrose gradient) did not depend on the size of the template neither on the presence of oligo(dT)10 primer. The inhibition of the second DNA strand synthesis with actinomycin D had also no influence on the size of DNA-product.

Avian Leukosis Virus↗

PubMed1985

Initiation of DNA synthesis by the calf thymus DNA polymerase-primase complex.

The calf thymus DNA polymerase-alpha-primase complex purified by immunoaffinity chromatography catalyzes the synthesis of RNA initiators on phi X174 single-stranded viral DNA that are efficiently elongated by the DNA polymerase. Trace amounts of ATP and GTP are incorporated into products that are full length double-stranded circular DNAs. When synthetic polydeoxynucleotides are used as templates, initiation and DNA synthesis occurs with both poly(dT) and poly(dC), but neither initiation nor DNA synthesis was observed with poly(dA) and poly(dI) templates. Nitrocellulose filter binding and sucrose gradient centrifugation studies show that the DNA polymerase-primase complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Using d(pA)-50 with 3'-oligo(dC) tails and d(pI)-50 with 3'-oligo(dT) tails, initiator synthesis and incorporation of deoxynucleotide can be demonstrated when the average pyrimidine sequence lengths are 8 and 4, respectively. These results suggest that purine polydeoxynucleotides are used as templates by the DNA polymerase only after initiation has occurred on the oligodeoxypyrimidine sequence and that the pyrimidine stretch required by the primase activity is relatively short. Analysis of initiator chain length with poly(dC) as template showed a series of oligo(G) initiators of 19-27 nucleotides in the absence of dGTP, and 5-13 nucleotides in the presence of dGTP. The chain length of initiators synthesized by the complex when poly(dT) or oligodeoxythymidylate-tailed poly(dI) was used can be as short as a dinucleotide. Analysis of the products of replication of oligo(dC)-tailed poly(dA) shows that initiator with chain length as low as 4 can be used for initiation by the polymerase-primase complex.

Animals↗

PubMed1991

Genes encoding the small subunit of RUBISCO belong to two highly conserved subfamilies in Nicotianeae.

The sequences of seven complementary DNAs or genes encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase oxygenase (RUBISCO) in several Nicotianeae were examined. Two new SSU genes isolated from Nicotiana sylvestris were included. Both sequence comparisons and Southern analyses with specific probes reveal that SSU genes fall into two homogeneous subfamilies that are highly conserved in Nicotianeae and are also present in other Solanaceae. Additional criteria such as number of introns and level of expression fitted to this classification. Homogeneity must have been maintained by gene conversion and/or an unusually high fidelity of DNA replication, whereas traces of slippage-stranded DNA mispairing and/or transposition probably explain local changes. Taken as a whole, these results show that the divergence between the two subfamilies predated the divergence between genera inside the Solanaceae, but that Nicotianeae retained the most simple SSU gene family structure.

Base Sequence↗

PubMed1975

Purification of S1 muclease from Takadiastase by affinity chromatography on single-stranded DNA-acrylamide columns.

When S1 nuclease from Takadiastase was partially purified according to previously reported methods, it showed a 10 to 15 fold increase in specificactivity. Although such preparations were highly active on single-stranded DNA, they had traces of activity on native DNA and were contaminated by T1-RNase. The S1 enzyme was further purified by a single step of affinity chromatography on single-stranded DNA-acrylamide column to a final purification of 275-fold. This preparation was free of T1-RNase and had an absolute specificity for single-stranded DNA.

Acrylamides↗

PubMed2003

Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresis.

Recent developments in capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) using long-wavelength (maximum excitation wavelength>500 nm) dyes are reviewed. These dyes are particularly of interest when conducting the analyses of biopolymers by CE-LIF using He-Ne lasers. These systems are benefited from low background, low costs, easy maintenance, and compactness. Derivatizations of DNA and proteins with fluorescent or nonfluorescent chemicals can be carried out prior to, during, or after separations. With the advantages of sensitivity, rapidity, and high efficiency, the applications of CE-LIF to the analysis of polymerase chain reaction products, DNA sequencing, trace analysis of proteins, and single cell analysis have been presented.

DNA↗