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PubMedDate not supplied

Diagnosis of trisomy 21 in preimplantation embryos by single-cell DNA fingerprinting.

Many couples presenting for preimplantation genetic diagnosis (PGD) for a single gene disorder are of advanced reproductive age (>35 years) and have a greater chance of producing embryos with chromosomal aneuploidies. The most common chromosomal aneuploidy observed in newborns is trisomy 21, or Down's syndrome. Consequently, the availability of a highly reliable system that simultaneously detects the heritable gene disorder and trisomy 21 would be beneficial to couples at specific risk. A pentaplex chromosome 21 (Ch 21) single-cell DNA fingerprinting system was developed in a multiplex fluorescence polymerase chain reaction (FL-PCR) on single cells. High reliability and accuracy rates were observed, together with low allele dropout (ADO) and preferential amplification rates on diploid buccal cells, trisomy 21 buccal cells and blastomeres derived from Ch 21 aneuploid embryos. A combined multiplex FL-PCR format was optimized with the common cystic fibrosis delta F508 mutation and validated on single buccal cells from a carrier of the cystic fibrosis delta F508 mutation. This new test is a very powerful technique, which also allows confirmation of the embryo parentage and the identification of extraneous DNA contamination that could cause a misdiagnosis in PGD cases.

Blastocyst↗

PubMed1991

Utility of the polymerase chain reaction (PCR) for prenatal diagnosis of genetic disease.

Gene amplification by the polymerase chain reaction (PCR) has been applied to prenatal diagnosis for alpha and beta thalassemias (1 and 5 cases respectively), Hemoglobin (Hb) Lepore/beta thalassemia (1 case) and cystic fibrosis (14 cases). Chorionic villus samples were obtained in the tenth week of pregnancy and DNA analysed in parallel with conventional gene mapping. Direct diagnosis of the common Mediterranean beta-thalassemia mutations (IVS-1-110 and codon 39), Hb Lepore, and the delta F508 mutation causing cystic fibrosis was achieved by hybridization of amplified material with pairs of allele-specific oligonucleotide (ASO) probes or by restriction enzyme digestion of PCR products. Results were confirmed by DNA mapping. Definitive diagnosis or exclusion of an affected fetus was possible in 17 of 21 cases thus examined. PCR reduces the time required for prenatal diagnosis. DNA contamination is a potential source of error.

Alleles↗

PubMed1976

FSH stimulation of DNA synthesis in Sertoli cells in culture.

The incorporation of [2H]thymidine into nuclear DNA was investigated in cultured Sertoli cells prepared from testes of 20-day-old rats. Addition of follicle-stimulating hormone (FSH) or dibutyryl cyclic, 3',5'-adenosine monophosphate (DBCAMP) to the culture medium greatly increased incorporation, expressed either as total amounts of [3H]thymidine incorporated per mug DNA or as the percentage of Sertoli cells with labeled nuclear DNA. No stimulation was observed in cells cultured in the presence of testosterone, insulin or cyclic 3',5'-GMP (cGMP). Light and electron microscopic autoradiographic analysis was employed to establish the identity of Sertoli cells having labeled nuclear DNA. Contaminating spermatogonia, which also took up labeled [3H]thymidine, were excluded from cell counts. In addition, Sertoli cells prepared from testes of irradiated 20-day-old germinal cell depleted rats were also observed to incorporate more [3H]thymidine into nuclear DNA when cultured in a chemically defined medium in the presence of FSH. DNA synthesis was abolished by prior treatment of cells with cytosine arabinoside. In separate experiments, the incorporation of [3H]thymidine into DNA of peritubular myoid cells was shown to be independent of FSH or dbcAMP.

Animals↗

PubMed1993

[Polymerase chain reaction: use in microbiological diagnosis].

The discovery of the polymerase chain reaction (PCR) enables the detection of just a few target gene copies present in almost every kind of tissue. The PCR technique uses two separated specific DNA-sequences for identification of a desired genetic sequence. This identification is followed by an almost unlimited production of the specific target gene sequence by a heat stable DNA-polymerase. The PCR technique will revolutionize several diagnostic areas, and especially the identification of virus, fungi and slowly growing bacteria will benefit from this new genetic technology. The impressive sensitivity is, however, the greatest pitfall of the technique, as just a few contaminating DNA fragments can initiate a false positive result. Great care is therefore needed when designing a PCR laboratory, as well as high demands upon the motivation and technical skills of the personnel involved. PCR is so far limited to research, but is expected to be released for routine diagnostic purposes in 1993.

Bacterial Infections↗

PubMed1994

Experience with cloned mitochondrial DNA analysis to forensic practice.

The sequencing method of the D-loop region of cloned mitochondrial DNA (mtDNA) together with multilocus and single locus DNA probe patterns was applied to identification of the dismembered and putrefied remains of a pregnant woman. The multilocus DNA fingerprinting was useful to identify whether or not 4 dismembered remains were from the body of the same person, whereas the single locus DNA analysis was useful to decide that the suspect was the father of the fetus. On the other hand, the sequencing method of the D-loop region of cloned mtDNA after amplification by polymerase chain reaction (PCR) was particularly advantageous to highly degraded and contaminated DNA specimens.

Base Sequence↗

PubMed1999

Background DNA damage for endogenous and unavoidable exogenous carcinogens: a basis for spontaneous cancer incidence?

It is undisputed today that DNA is constantly being damaged in the absence of any exposure to genotoxic carcinogens by a specific treatment or a particular habit. Damaging processes include endogenous and unavoidable exogenous sources. The list comprises chemical DNA instability (eg., depurination), spontaneous errors during DNA replication and repair, endogenous reactive chemicals (aldehydes, S-andenosylmethionine), numerous reactive oxygen species and products generated in consequence (e.g., lipid peroxides). Exogenous sources are ionizing and UV radiation, naturally occurring radioisotopes, and numerous genotoxic chemicals present in diet and air, both naturally or as contaminants. DNA repair and DNA replicative synthesis result in a constant reduction of the level of damage, but a certain steady-state level is measurable as a background at all times. The quantitative relationship between the background DNA damage and the spontaneous rates of mutation and cancer remains largely unknown. The quantitative relationship between the background DNA damange and the spontaneous rates of mutationa and caner remains largley unknown. In order to establish better correlations, more data have to be accumulated. This includes (i) measuring artefact-free levels of background DNA damage, (ii) investigating the mutagenic potency of the various lesions, (iii) including types of DNA damage other than adducts, and (iv) answer the question of organ-specific and cell type-specific +requirements for the primary DNA damage to be expressed as heritable genetic changes. The manuscripts put together for this Special Issue of Mutation Research describe the state of the art for these aspects.

Animals↗

PubMed1994

Immunochemiluminescent Southern blot assay for polymerase chain reaction detection of human parvovirus B19 DNA.

Human parvovirus B19 is not only an acute self-limited infection causing erythema infectiosum, transient aplastic crisis, foetal hydrops and arthritis but can also be a chronic infection causing chronic anaemia and associated with chronic neuropathy and vasculitis. Serologic studies have proven to be the most sensitive way to detect acute infection in the immunologically normal patient while polymerase chain reaction (PCR) assays for B19 DNA are the most sensitive way to detect chronic infection. The ability to detect B19 in clinical specimens can be further increased with a second amplification step using nested primers. However, nested PCR is both time consuming and enhances the risk of false-positive results due to contaminating DNA. In this study, we developed a sensitive immunochemiluminescent Southern blot assay for detecting PCR amplified B19 DNA with a digoxigenin labelled primer. The sensitivity and specificity of this assay were comparable to nested PCR and at least 100-fold more sensitive than a single PCR amplification.

Blotting, Southern↗

PubMed2005

LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications.

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged LacI protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Bacterial Proteins↗

PubMed2002

Molecular characterisation of the nucleic acids recovered from aged forensic samples.

The molecular composition of the genetic substrate recovered from seven aged forensic samples has been extensively investigated. A simple enzymatic test based on DNAseI incubation of the extracts showed that the UV-fluorescent material from the forensic specimens is composed of nucleic acids, with the DNA fraction representing at least 90% of the total amount. Since spectrophotometric determinations of the extracts showed unreliable results due to anomalous OD(260)/OD(280) ratios, quantification of the nuclease-sensitive genetic material was performed by a slightly modified agarose plate method. The first quantitative data on exogenous contamination in aged forensic samples are provided by slot-blot hybridisation of the extracts to human, bacterial and fungal probes. Only limited amounts of human and contaminant DNA were detected in the samples. The molecular integrity of the primary structure of these aged DNA samples was analysed by reversed-phase HPLC/MS. The data show a good correlation between the degree of chemical damage and the ability to hybridise to molecular probes. The ability to achieve specific genetic profiles was assessed by multiplex PCR amplification of STR loci. Our data show that accurate determination of the molecular composition of the DNA recovered from forensic samples can be extremely useful for a reliable evaluation of the PCR typing results.

Age Factors↗

PubMed1991

Quantitation of DNA and protein impurities in biopharmaceuticals.

The development of drugs and biologicals for human injection generated from recombinant DNA and hybridoma technologies has resulted in new standards for product purity. We discuss the regulatory position relative to impurities in these biopharmaceuticals, focusing on the analytical goals for quantitation. Current methods for making these measurements are reviewed, and a new system designed for improved analysis is described. Assay results for both contaminating DNA and proteins are presented.

Chemistry, Pharmaceutical↗

PubMed1991

Detection of Chlamydia trachomatis by the polymerase chain reaction in swabs and urine from men with non-gonococcal urethritis.

A polymerase chain reaction (PCR) was developed for Chlamydia trachomatis in which a 380 base pair DNA fragment was amplified. Amplification occurred with the DNA from the 15 serovars but not with that from other Chlamydia spp or with DNA from a variety of other organisms. Chlamydial DNA (10(-16) g) could be detected and the PCR seemed to be able to detect single organisms. Urethral swabs were obtained from 37 men with acute non-gonococcal urethritis (NGU), 18 (49%) of whom were positive for C trachomatis by MicroTrak. As a result of clinical re-examinations 65 urethral swabs were available for analysis by the PCR. In comparison with MicroTrak, PCR had a sensitivity of 95%, a specificity of 94%, a positive predictive value of 86% and a negative predictive value of 98%. The PCR was apparently less sensitive (82%) in tests on urine samples. Overall, however, values of sensitivity and specificity of the PCR compared favourably with those of MicroTrak. The PCR for C trachomatis is likely to be a valuable technique for research, but problems of DNA contamination suggest that it should not be recommended for routine diagnosis.

Base Sequence↗

PubMed2000

Purification and characterization of a common soil component which inhibits the polymerase chain reaction.

DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.

Canada↗

PubMed1995

Enzymatic amplification of specific deoxyribonucleic acid sequences from single cells: evaluation of a simplified and rapid method for use in preimplantation genetic diagnosis.

OBJECTIVE: To develop a simplified polymerase chain reaction (PCR) protocol on single cells for the purpose of preimplantation genetic diagnosis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene, La Jolla, CA), for reducing the time to complete PCR amplification. DESIGN: PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model. The assay was tested in human fetal cells, single lymphocytes and single human blastomeres. RESULTS: Amplification of the 149 bp segment using fetal cells was 100% correct. Results on single lymphocytes were concordant in all but one of the 15 male cases. However, 2 of the 25 female cases were identified as male suggesting the occurrence of DNA contamination. Analysis of 61 blastomeres were concordant in 57 cases (93%); results for male blastomeres showed 12% of false negatives. No false positives were detected for female cells. Amplification using the simplified PCR protocol in combination with the RoboCycler was completed in 2 hours. CONCLUSION: These data show that this PCR assay performed directly, without DNA extraction or purification and without re-amplification is a practical and effective approach for amplification of specific DNA sequences in single cells. Furthermore, the simplified PCR protocol significantly reduced the time to complete DNA amplification. The reduced time is expected to facilitate the management of a routine program for preimplantation genetic diagnosis.

Base Sequence↗

PubMed1994

Molecular taxonomy using single-strand conformation polymorphism (SSCP) analysis of mitochondrial ribosomal DNA genes.

Single-strand conformation polymorphism (SSCP) analysis detects single point mutations in DNA molecules. We demonstrate that SSCP analysis of mitochondrial ribosomal DNA (rDNA) genes is a sensitive taxonomic tool because these genes often differ at numerous sites among closely related species. Using conserved primers, portions of the 12S or 16S rDNA genes were amplified using the polymerase chain reaction (PCR) in congeneric species of ticks, leafhoppers, mosquitoes, and closely related endoparasitic wasps. SSCP was performed and products were visualized with silver staining. Species-specific patterns were observed in all taxa. Intraspecific variation at the level of single nucleotide substitutions was detected. SSCP diagnostics are less expensive and time consuming to develop than PCR with species-specific primers, and, unlike PCR with arbitrary primers, there is minimal concern with DNA contamination from non-target organisms.

Aedes↗

PubMed1997

Incognito rRNA and rDNA in databases and libraries.

Both ribosomal DNA (rDNA) and ribosomal RNA (rRNA) are over-represented in the starting material for genomic and cDNA libraries; thus, their sequences have the potential of repeatedly entering the various databases. When DNA (both transcribed and intergenic spacer regions) is used as query sequence, a great number of matches are found in the databases, particularly in the EST database, and to a lesser extent among genomic sequences and STSs, which are not identified as rDNA. We discuss the following explanations for the widespread occurrence of rDNA in cDNA and genomic DNA libraries: pseudogenes of rRNA in other genomic locations, mRNA-derived pseudogenes that reside in rDNA, cDNAs derived from rRNA [either by self-priming or by internal oligo(dT) priming], cDNAs derived from actual transcripts of the rDNA intergenic spacer, and genomic DNA contamination of RNA preparations. Because so many database entries contain unidentified rDNA, we recommend that all sequence submissions be checked (by the submitters) for the presence of structural RNAs in addition to repetitive sequences.

DNA, Ribosomal↗

PubMed1998

Purification of plasmids by triplex affinity interaction.

Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.

DNA, Bacterial↗

PubMed1985

A method for assessing damage to mitochondrial DNA caused by radiation and epichlorohydrin.

This paper describes a rapid and reliable method for quantification of damage to mitochondrial DNA (mtDNA), especially strand breaks. The degree of damage to mtDNA is assessed by the proportion of physical forms (i.e., supercoiled versus open-circular and linear forms) upon agarose gel electrophoresis, blotting, and visualization by hybridization with [32P]mtDNA probes. The use of a radiolabeled probe is a crucial step in the procedure because it provides both a means to quantify by radioautography and to obtain the mtDNA specificity required to eliminate misinterpretation due to nuclear DNA contamination. To demonstrate the utility of this technique, X-irradiation and epichlorohydrin are shown to damage both isolated mtDNA and mtDNA in whole cells in a dose-dependent fashion.

Animals↗

PubMed2004

Genotyping from semen of wild Japanese macaques (Macaca fuscata).

The noninvasive collection of animal cells is crucial for DNA analyses in wild populations that cannot be disturbed by capture. We describe the collection of 68 semen samples following copulation and masturbation events in wild habituated and nonhabituated troops of Japanese macaques on the protected island of Yakushima. We used this DNA to amplify 390 base pairs (bp) of the mitochondrial DNA control region in 16 individuals from eight troops, and found a monomorphic pattern in agreement with the low variability imposed by geographic isolation and female philopatry. We also amplified two microsatellite loci from samples collected after the resident males of a focal troop had copulated with different females. We found several different allele combinations in samples collected after the observed mating of a single male, indicating the presence of contaminant DNA, presumably from males that had previously mated with the same female. This discovery made it impossible to assign a given sample to a specific male except when the samples were recovered after masturbation events. Thus, it was not possible to test for kinship or estimate allele frequencies from the semen samples. The mixing of semen, and the pattern of sample collection observed in morphologically identified individuals support the notion that strong mating and sperm competition exists among resident and nonresident males.

Animals↗