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Showing papers on "genomic DNA published in 2005"


Book
01 Jan 2005
TL;DR: Experiments: looking at yeast cells isolation and characterization of auxotrophic, temperature-sensitive and UV-sensitive mutants meiotic mapping mitotic recombination and random spore analysis transformation of yeast synthetic lethal mutants gene replacement isolation of ras2 suppressors manipulating cell types isolating mutants by insertional mutagenesis two-hybrid protein interaction method.
Abstract: Experiments: looking at yeast cells isolation and characterization of auxotrophic, temperature-sensitive and UV-sensitive mutants meiotic mapping mitotic recombination and random spore analysis transformation of yeast synthetic lethal mutants gene replacement isolation of ras2 suppressors manipulating cell types isolating mutants by insertional mutagenesis two-hybrid protein interaction method. Techniques and protocols: high-efficiency transformation of yeast "Lazy Bones" plasmid transformation of yeast colonies yeast DNA isolations yeast DNA miniprep (40 ml) yeast DNA miniprep (5 ml) a ten-minute DNA preparation from yeast yeast genomic DNA - glass bead preparation yeast protein extracts yeast RNA isolation hydroxylamine mutagenesis of plasmid DNA assay of beta-galactosidase in yeast plate assay for carboxypeptidase Y random spore analysis yeast vital stains yeast immunofluorescence actin staining in fixed cells PCR protocol for PCR-mediated gene disruption yeast colony PCR protocol measuring yeast cell density by spectrophotometry cell synchrony chromatin immunoprecipitation flow cytometry of yeast DNA logarithmic growth EMS mutagenesis tetrad dissection making a tetrad dissection needle picking zygotes determining plating efficiency. Appendices: media stock preservation yeast genetic and physical maps templates for making streak plates electrophoretic karyotypes of strains for southern blot mapping strains counting yeast cells with a standard hemocytometer chamber tetrad scoring sheet trademarks suppliers.

2,088 citations


Journal ArticleDOI
TL;DR: A novel robust method that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction is described and used to detect aberrant methylation in DNA samples of patients with Prader–Willy syndrome, Angelman syndrome or acute myeloid leukemia.
Abstract: Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA-probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA-probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader-Willy syndrome, Angelman syndrome or acute myeloid leukemia.

386 citations


Journal ArticleDOI
TL;DR: A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA by the multiple displacement amplification (MDA) reaction, using φ 29 DNA polymerase.
Abstract: Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using φ 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying nonculturable microorganisms.

307 citations


Journal ArticleDOI
TL;DR: It is determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of ∼1/7,000 bases, indicating that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells.
Abstract: Long interspersed element 1 (LINE-1 or L1) is an abundant retrotransposon that comprises ∼17% of human DNA (43, 69). Most L1s are retrotransposition defective because they are 5′ truncated, contain internal rearrangements, or harbor mutations within their open reading frames (25, 43). However, the average human genome is estimated to contain ∼80 to 100 retrotransposition-competent L1s (RC-L1s), and approximately 10% of these elements are classified as highly active or “hot” (6, 63). Human RC-L1s are ∼6.0 kb and contain a 5′ untranslated region (UTR), two nonoverlapping open reading frames (ORF1 and ORF2), and a 3′ UTR that ends in a poly(A) tail (Fig. ​(Fig.1A)1A) (13, 53, 66). ORF1 encodes a 40-kDa nucleic acid binding protein (30, 31, 33), whereas ORF2 has the potential to encode a 150-kDa protein with demonstrated endonuclease (L1 EN) and reverse transcriptase (L1 RT) activities (15, 19, 22, 51). ORF2p also contains a cysteine-rich domain (CX3CX7HX4C) of unknown function (17, 54). Both proteins are required for retrotransposition in cis (54), which most probably occurs by a mechanism termed “target site primed reverse transcription” (TPRT) (19, 47, 54, 72). However, how L1 integration is completed remains a mystery. FIG. 1. Simple sequence alterations at the 5′ genomic DNA/L1 junction. A. Rationale of the assay. The 3′ UTR of a human RC-L1 was tagged with a reporter cassette designed to detect retrotransposition events. Open rectangles indicate L1 ORF1 and ... We recently developed a plasmid-based rescue system that allows the recovery of L1 insertions in cultured human HeLa cells with minimal influence from selective pressures that occur during genome evolution. We found that L1 retrotransposition is associated with various forms of genetic instability and that the nascent L1 cDNA can undergo recombination with endogenous L1 elements, resulting in the formation of chimeric L1s. Consistent findings by Symer et al., using a colon cell line (HCT116) with an essentially normal karyotype, have led to the hypothesis that L1 retrotransposition can lead to various types of genomic instability (21, 72). Here, we describe the analysis of 100 L1 retrotransposition events in HeLa cells derived from four previously characterized RC-L1s (L1.2A, LRE-2, L1.3, and L1RP). Consistent with previous studies, we have found that retrotransposition is associated with the generation of intrachromosomal deletions, the creation of chimeric L1 elements, and the addition of non-L1 nucleotides at the 5′ insertion junction (21, 56, 72). In addition, we have observed novel rearrangements, including the mobilization of U6 small uracil-rich nuclear RNA (U6 snRNA) to a new genomic location, the formation of intrachromosomal duplications, intra-L1 rearrangements, and the generation of a possible interchromosomal translocation. Finally, we have determined that the L1 RT can faithfully replicate its own transcript and has a base misincorporation error rate of ∼1/7,000 bases. Together, these data indicate that the resolution of L1 retrotransposition intermediates in transformed human cell lines can lead to a variety of genomic rearrangements and lead us to propose that host processes act to restrict L1 retrotransposition during integration, limiting the number of full-length L1s in the genome.

270 citations


Journal ArticleDOI
TL;DR: IT sequence-based identification is reliable and provides a promising tool for elucidation of the clinical significance of the different species of the A. baumannii complex and was confirmed for a subset of strains by amplified rRNA gene restriction analysis and genomic DNA analysis by AFLP analysis.
Abstract: The species Acinetobacter calcoaceticus, A. baumannii, genomic species 3, and genomic species 13TU included in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex are genetically highly related and difficult to distinguish phenotypically. Except for A. calcoaceticus, they are all important nosocomial species. In the present study, the usefulness of the 16S-23S rRNA gene intergenic spacer (ITS) sequence for the differentiation of (genomic) species in the A. calcoaceticus-A. baumannii complex was evaluated. The ITSs of 11 reference strains of the complex and 17 strains of other (genomic) species of Acinetobacter were sequenced. The ITS lengths (607 to 638 bp) and sequences were highly conserved for strains within the A. calcoaceticus-A. baumannii complex. Intraspecies ITS sequence similarities ranged from 0.99 to 1.0, whereas interspecies similarities varied from 0.86 to 0.92. By using these criteria, 79 clinical isolates identified as A. calcoaceticus (18 isolates) or A. baumannii (61 isolates) with the API 20 NE system (bioMerieux Vitek, Marcy l'Etoile, France) were identified as A. baumannii (46 isolates), genomic species 3 (19 isolates), and genomic species 13TU (11 isolates) by ITS sequencing. An identification rate of 96.2% (76 of 79 isolates) was obtained by using ITS sequence analysis for identification of isolates in the A. calcoaceticus-A. baumannii complex, and the accuracy of the method was confirmed for a subset of strains by amplified rRNA gene restriction analysis and genomic DNA analysis by AFLP analysis by using libraries of profiles of reference strains. In conclusion, ITS sequence-based identification is reliable and provides a promising tool for elucidation of the clinical significance of the different species of the A. calcoaceticus-A. baumannii complex.

263 citations


Journal ArticleDOI
TL;DR: The chromosome microarray analysis (CMA) results were entirely consistent with previous cytogenetic and FISH findings and is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype.

258 citations


Journal ArticleDOI
TL;DR: It is shown that DNMT deficiency in human cancer cells results in constitutive genomic instability manifested by chromosomal translocations, meeting the formal definition of genomic instability.
Abstract: DNA methyltransferase 1 (DNMT1)-deficient mice are tumor-prone, and this has been proposed to result from the induction of genomic instability. To address whether loss of DNMT1, or the related protein DNMT3b, results in genomic instability in human cancer cells, we used a near-diploid human colorectal cancer cell line, HCT116, in which one or both DNMT genes were disrupted by homologous recombination. Array-based comparative genomic hybridization analyses indicated that double, but not single, DNMT knock-out cells display two specific alterations in regional DNA copy number, suggesting that DNMT deficiency and genomic DNA hypomethylation are not associated with widespread genomic amplifications or deletions in human cancer cells. However, spectral karyotype analyses revealed that DNMT-deficient HCT116 cells are highly unstable with respect to large-scale chromosomal alterations; furthermore, this effect is characterized by a high degree of individual cell heterogeneity. The induction of chromosomal alterations in DNMT-deficient cells was evidenced both by aneuploidy and by large increases in the number of novel chromosomal translocations. Studies of double knock-out cells indicated that the generation of chromosomal alterations is spontaneous and persistent in vitro, meeting the formal definition of genomic instability. In summary, we show that DNMT deficiency in human cancer cells results in constitutive genomic instability manifested by chromosomal translocations.

255 citations


Journal ArticleDOI
TL;DR: A method, methylated-CpG island recovery assay (MIRA) that does not depend on the use of sodium Bisulfite but has similar sensitivity and specificity as bisulfite-based approaches.

222 citations


Journal ArticleDOI
TL;DR: A novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2'-deoxycytidine levels following enzymatic hydrolysis of genomic DNA is reported.
Abstract: Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2‘-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2‘-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2‘-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2‘-deoxycytidine to 2‘-deoxyguanosine and was determined directly using 2‘-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we va...

222 citations


Journal ArticleDOI
TL;DR: The data indicate CGH arrays can be used to detect monosomies and trisomies, to predict the sites of chromosome breaks, and to identify chromosomal aberrations that have not been detected with other approaches in C. albicans strains, and highlight the high level of genome instability in laboratory strains exposed to the stress of transformation and counterselection on 5‐fluoro‐orotic acid.
Abstract: Summary Clinical strains of Candida albicans are highly tolerant of aneuploidies and other genome rearrangements. We have used comparative genome hybridization (CGH), in an array format, to analyse the copy number of over 6000 open reading frames (ORFs) in the genomic DNA of C. albicans laboratory strains carry- ing one (CAI-4) to three (BWP17) auxotrophies. We find that during disruption of the HIS1 locus all genes telomeric to HIS1 were deleted and telomeric repeats were added to a 9 nt sequence within the transforming DNA. This deletion occurred in ~ 10% of transformants analysed and was stably maintained through two addi- tional rounds of transformation and counterselection of the transformation marker. In one example, the dele- tion was repaired, apparently via break-induced repli- cation. Furthermore, all CAI-4 strains tested were trisomic for chromosome 2 although this trisomy appears to be unstable, as it is not detected in strains subsequently derived from CAI-4. Our data indicate CGH arrays can be used to detect monosomies and trisomies, to predict the sites of chromosome breaks, and to identify chromosomal aberrations that have not been detected with other approaches in C. albicans strains. Furthermore, they highlight the high level of genome instability in C. albicans laboratory strains exposed to the stress of transformation and coun- terselection on 5-fluoro-orotic acid.

167 citations


Journal ArticleDOI
TL;DR: This work presents for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction and demonstrates its suitability for multipleX SNP profiling at the ‘point of care’.
Abstract: Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the 'point of care'.

Journal ArticleDOI
TL;DR: It is concluded that UVA1 induces promutagenic CPDs and oxidative DNA damage at both the genomic and nucleotide resolution level in normal human skin fibroblasts.
Abstract: The UV components of sunlight (UVA and UVB) are implicated in the etiology of human skin cancer. The underlying mechanism of action for UVB carcinogenicity is well defined; however, the mechanistic involvement of UVA in carcinogenesis is not fully delineated. We investigated the genotoxicity of UVA1 versus UVB in the overall genome and in the p53 tumor suppressor gene in normal human skin fibroblasts. Immuno-dot blot analysis identified the cis-syn cyclobutane pyrimidine-dimer (CPD) as a distinctive UVB-induced lesion and confirmed its formation in the genomic DNA of UVA1-irradiated cells dependent on radiation dose. HPLC/tandem MS analysis showed an induction of 8-oxo-7,8-dihydro-2′-deoxyguanosine in the genomic DNA of UVA1-irradiated cells only. Mapping of DNA damages by terminal transferase-dependent PCR revealed preferential, but not identical, formation of polymerase-blocking lesions and/or strand breaks along exons 5-8 of the p53 gene in UVB- and UVA1-irradiated cells. The UVB-induced lesions detected by terminal transferase-PCR were almost exclusively mapped to pyrimidine-rich sequences; however, the UVA1-induced lesions were mapped to purine- and pyrimidine-containing sequences along the p53 gene. Cleavage assays with lesion-specific DNA repair enzymes coupled to ligation-mediated PCR showed preferential, but not identical, formation of CPDs along the p53 gene in UVB- and UVA1-irradiated cells. Additionally, dose-dependent formation of oxidized and ring-opened purines and abasic sites was established in the p53 gene in only UVA1-irradiated cells. We conclude that UVA1 induces promutagenic CPDs and oxidative DNA damage at both the genomic and nucleotide resolution level in normal human skin fibroblasts.

Journal ArticleDOI
TL;DR: It is demonstrated that APOBEC3G is capable of deaminating genomic cytosines in Saccharomyces cerevisiae and postulate that the APOBec3-dependent innate cellular defense constitutes a tightly regulated arm of a conserved mobile nucleic acid restriction mechanism that is poised to limit internal as well as external assaults.
Abstract: Human cells harbor a variety of factors that function to block the proliferation of foreign nucleic acid. The APOBEC3G enzyme inhibits the replication of retroviruses by deaminating nascent retroviral cDNA cytosines to uracils, lesions that can result in lethal levels of hypermutation. Here, we demonstrate that APOBEC3G is capable of deaminating genomic cytosines in Saccharomyces cerevisiae. APOBEC3G expression caused a 20-fold increase in frequency of mutation to canavanine-resistance, which was further elevated in a uracil DNA glycosylase-deficient background. All APOBEC3G-induced base substitution mutations mapped to the nuclear CAN1 gene and were exclusively C/G → T/A transition mutations within a 5′-CC consensus. The APOBEC3G preferred sites were found on both strands of the DNA duplex, but were otherwise located in hotspots nearly identical to those found previously in retroviral cDNA. This unique genetic system further enabled us to show that expression of APOBEC3G or its homolog APOBEC3F was able to inhibit the mobility of the retrotransposon Ty1 by a mechanism that involves the deamination of cDNA cytosines. Thus, these data expand the range of likely APOBEC3 targets to include nuclear DNA and endogenous retroelements, which have pathological and physiological implications, respectively. We postulate that the APOBEC3-dependent innate cellular defense constitutes a tightly regulated arm of a conserved mobile nucleic acid restriction mechanism that is poised to limit internal as well as external assaults.

Journal ArticleDOI
TL;DR: The results show that the salting‐out method for DNA extraction is simple, fast, safe, and cost‐effective, and can be used in medical la‐ boratories and research centers.
Abstract: Different approaches have been used to extract DNA from whole blood. In most of these methods enzymes (such as proteinase K and RNAse A) or toxic organic solvents (such as phenol or guanidine isothiocyanate) are used. Since these enzymes are expensive, and most of the materials that are used routinely are toxic, it is desirable to apply an efficient DNA extraction procedure that does not require the use of such materials. In this study, genomic DNA was extracted by the salting-out method, but instead of using an analytical-grade enzyme and chemical detergents, as normally used for DNA isolation, a common laundry powder was used. Different concentrations of the powder were tested, and proteins were precipitated by NaCl-saturated distilled water. Finally, DNA precipitation was performed with the use of 96% ethanol. From the results, we conclude that the optimum concentration of laundry powder for the highest yield and purity of isolated DNA is 30 mg/mL. The procedure was optimized, and a final protocol is suggested. Following the same protocol, DNA was extracted from 100 blood samples, and their amounts were found to be >50 microg/mL of whole blood. The integrity of the DNA fragments was confirmed by agarose gel electrophoresis. Furthermore, the extracted DNA was used as a template for PCR reaction. The results obtained from PCR showed that the final solutions of extracted DNA did not contain any inhibitory material for the enzyme used in the PCR reaction, and indicated that the isolated DNA was of good quality. These results show that this method is simple, fast, safe, and cost-effective, and can be used in medical laboratories and research centers.

Journal ArticleDOI
TL;DR: This study shows that array-based CGH provides high resolution mapping of chromosomal aberrations in HCC, and demonstrates the feasibility of correlating array CGH data with gene expression data to identify novel oncogenes and tumor suppressor genes.
Abstract: Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide. We have previously characterized global gene expression patterns in HCC using microarrays. Here, we report the analysis of genomic DNA copy number among 49 HCC samples using BAC array-based comparative genomic hybridization (CGH). We observed recurrent and characteristic chromosomal aberrations, including frequent DNA copy number gains of 1q, 6p, 8q and 20q, and losses of 4q, 8p, 13q, 16q and 17p. We correlated gene expression with array CGH data, and identified a set of genes whose expression levels correlated with common chromosomal aberrations in HCC. Especially, we noticed that high expression of Jab1 in HCC significantly correlated with DNA copy number gain at 8q. Quantitative microsatellite analysis further confirmed DNA copy number gain at the Jab1 locus. Overexpression of Jab1 in HCC was also validated using real-time RT-PCR, and Jab1 protein levels were studied by immunohistochemistry on tissue microarrays. Functional analysis in HCC cell lines demonstrated that Jab1 may regulate HCC cell proliferation, thereby having a potential role in HCC development. In conclusion, this study shows that array-based CGH provides high resolution mapping of chromosomal aberrations in HCC, and demonstrates the feasibility of correlating array CGH data with gene expression data to identify novel oncogenes and tumor suppressor genes.

Journal ArticleDOI
TL;DR: It is shown that the EST-SSR markers developed in cultivated barley are polymorphic in wild and cultivated varieties and produced high quality markers, providing a direct estimate of functional biodiversity.
Abstract: Genetic variation present in wild and cultivated barley populations was investigated using two sources of microsatellite also known as simple sequence repeat (SSR) markers. EST-SSRs are derived from expressed sequences and genomic SSRs are isolated from genomic DNA. Genomic SSR markers detected a higher level of polymorphism than those derived from ESTs. Polymorphism information content was higher in genomic SSRs than EST-derived SSRs. This study showed that the EST-SSR markers developed in cultivated barley are polymorphic in wild and cultivated varieties and produced high quality markers. Ten of these functional markers were polymorphic across the accessions studied. EST markers indicated clearer separation between wild and cultivated barley than genomic SSRs. The EST-SSRs are a valuable source of new polymorphic markers and should be highly applicable to barley genetic resources, providing a direct estimate of functional biodiversity.

Journal ArticleDOI
TL;DR: In segregants from a cross between two unrelated strains of Saccharomyces cerevisiae, high-quality genotype data can also be obtained when mRNA is hybridized to an oligonucleotide-expression array, demonstrating how simultaneous genotyping and gene-expression profiling can reveal cis-regulatory variation.
Abstract: Oligonucleotide microarrays provide a high-throughput method for exploring genomes. In addition to their utility for gene-expression analysis, oligonucleotide-expression arrays have also been used to perform genotyping on genomic DNA. Here, we show that in segregants from a cross between two unrelated strains of Saccharomyces cerevisiae, high-quality genotype data can also be obtained when mRNA is hybridized to an oligonucleotide-expression array. We were able to identify and genotype nearly 1000 polymorphisms at an error rate close to 3% in segregants and at an error rate of 7% in diploid strains, a performance comparable to methods using genomic DNA. In addition, we demonstrate how simultaneous genotyping and gene-expression profiling can reveal cis-regulatory variation by screening hundreds of genes for allele-specific expression. With this method, we discovered 70 ORFs with evidence for preferential expression of one allele in a diploid hybrid of two S. cerevisiae strains.

Journal ArticleDOI
TL;DR: It is established that Cel-I-mediated TILLING can be used to efficiently obtain mutations in genes of interest in Drosophila.
Abstract: The availability of the full Drosophila genomic DNA sequence prompts the development of a method to efficiently obtain mutations in genes of interest identified by their sequence homologies or biochemically. To date, molecularly characterized mutations have been generated in around 6000 of the ∼15,000 annotated fly genes, of which around one-third are essential for viability. To obtain mutations in essential and nonessential genes of interest, we took a reverse genetics approach, based on the large-scale detection of point mutations by Cel-I-mediated heteroduplex cleavage. A library of genomic DNA from 2086 EMS-mutagenized lines was established. The library was screened for mutations in three genes. A total of 6.1 Mb were screened, and 44 hits were found in two different mutagenesis conditions. Optimal conditions yielded an average of one mutation every 156 kb. For an essential gene tested, five of 25 mutations turned out to cause lethality, confirming that EMS mutagenesis leads to high frequency of gene inactivation. We thereby established that Cel-I-mediated TILLING can be used to efficiently obtain mutations in genes of interest in Drosophila.

Journal ArticleDOI
TL;DR: It is shown that the coinjection of mRNA encoding φC31 integrase with plasmid DNA encoding the green fluorescent protein (GFP) can be used to generate transgenic X. laevis embryos.
Abstract: Bacteriophage φC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA. Here we show that the coinjection of mRNA encoding φC31 integrase with plasmid DNA encoding the green fluorescent protein (GFP) can be used to generate transgenic X. laevis embryos. Despite integration into the genome, appropriate promoter expression required modification of the reporter plasmid by bracketing the GFP reporter gene with tandem copies of the chicken β-globin 5′ HS4 insulator to relieve silencing owing to chromatin position effects. These experiments demonstrate that the integration of insulated gene sequences using φC31 integrase can be used to efficiently create transgenic embryos in X. laevis and may increase the practical use of φC31 integrase in other systems as well.

Journal ArticleDOI
05 Feb 2005-Virology
TL;DR: Analysis of T5 early promoters suggests a probable motif AAA{3, 4 T}nTTGCTT{17, 18 n}TATAATA{12, 13 W}{10 R} for strong promoters that may strengthen the step modification of host RNA polymerase, and thus control transcription of phage DNA.

Journal ArticleDOI
TL;DR: This study identified new chromosomal regions and therefore potential candidate genes that may play a role in retinoblastoma by matrix‐based comparative genomic hybridization (matrix‐CGH).
Abstract: The genetic hallmark of retinoblastoma is mutation or deletion of the RB1 gene, whereas other genetic alterations that are also required are largely unknown. To screen for genomic imbalances on a genomewide level, we studied a series of 17 primary retinoblastomas by matrix-based comparative genomic hybridization (matrix-CGH). The matrix-CGH chip contained 6,000 immobilized genomic DNA fragments covering the human genome, with an average resolution of about 500 kb. The most frequent imbalances detected were gains on chromosome arms 1q (12 of 17), 6p (10 of 17), 2p (5 of 17), and 19q (4 of 17) and loss on 16q (7 of 17). Candidate regions could be narrowed to small intervals by the identified minimally overlapping regions on 1q22, 1q32.1q32.2, 2p24.1, and 6p21.33–p21.31. Furthermore, two as-yet-unknown high-level amplifications were detected, each in a single patient, on chromosome bands 1p34.2 and 1p33. Thus, this study identified new chromosomal regions and therefore potential candidate genes that may play a role in retinoblastoma. © 2005 Wiley-Liss, Inc.


Journal ArticleDOI
TL;DR: The majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

Journal ArticleDOI
TL;DR: A microarray was designed using 62,358 probes matched to both prokaryotic and eukaryotic small-subunit ribosomal RNA genes as mentioned in this paper, which categorized environmental DNA to specific phylogenetic clusters in under 9 hours.
Abstract: A microarray has been designed using 62,358 probes matched to both prokaryotic and eukaryotic small-subunit ribosomal RNA genes. The array categorized environmental DNA to specific phylogenetic clusters in under 9 h. To a background of DNA generated from natural outdoor aerosols, known quantities of rRNA gene copies from distinct organisms were added producing corresponding hybridization intensity scores that correlated well with their concentrations ( r = 0.917). Reproducible differences in microbial community composition were observed by altering the genomic DNA extraction method. Notably, gentle extractions produced peak intensities for Mycoplasmatales and Burkholderiales , whereas a vigorous disruption produced peak intensities for Vibrionales , Clostridiales , and Bacillales.

Journal ArticleDOI
TL;DR: The implementation of qPCR detection in clinical laboratories will address the need to replace complex, expensive and time consuming FISH screening to detect genomic microdeletions or duplications of clinical importance.
Abstract: Quantitative Polymerase Chain Reaction (qPCR) is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH) to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques. In this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS), 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1) had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2) were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11. In this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive DNA, whilst providing a level of genomic resolution greater than standard cytogenetic assays. The implementation of qPCR detection in clinical laboratories will address the need to replace complex, expensive and time consuming FISH screening to detect genomic microdeletions or duplications of clinical importance.

Journal ArticleDOI
TL;DR: An assay based on digestion of genomic DNA with methylation-sensitive restriction enzyme and multiplexed PCR with gene-specific primers that correctly identified the methylation status of genes analyzed by other techniques is described.
Abstract: Abnormal DNA methylation is observed in certain promoters of neoplastic cells, although the likelihood of methylation for each individual promoter varies. Simultaneous analysis of many promoters in the same sample can allow use of statistical methods for identification of neoplasia. Here we describe an assay for such analysis, based on digestion of genomic DNA with methylation-sensitive restriction enzyme and multiplexed PCR with gene-specific primers (MSRE-PCR). MSRE-PCR includes extensive digestion of genomic DNA (uncut fragments cannot be identified by PCR), can be applied to dilute samples (<1 pg/microl), requires limited amount of starting material (42 pg or genomic equivalent of seven cells) and can identify methylation in a heterogeneous mix containing <2% of cells with methylated fragments. When applied to 53 promoters of breast cancer cell lines MCF-7, MDA-MB-231 and T47D, MSRE-PCR correctly identified the methylation status of genes analyzed by other techniques. For selected genes results of MSRE-PCR were confirmed by methylation-specific PCR and bisulfite sequencing. The assay can be configured for any number of desired targets in any user-defined set of genes.

Journal ArticleDOI
TL;DR: A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed and may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.
Abstract: A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system. The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.

Journal ArticleDOI
TL;DR: House dust mite DNA contains evidence of Bartonella and other Gram-negative species that are likely to be the sources of the endotoxin found in mite allergenic extracts.
Abstract: Background Allergen extracts prepared from Dermatophagoides farinae contain significantly more endotoxin than Dermatophagoides pteronyssinus extracts, and extracts from both mite extracts contain more endotoxin than pollen extracts. Attempts to culture bacteria from mite cultures have failed to establish the sources of the endotoxin. Objective To determine the bacterial sources of endotoxin in mite extracts. Methods Live mites of both species were obtained from 2 sources, DNA was extracted from the mites, and DNA encoding bacterial 16S ribosomal RNA was amplified by using specific primers. The amount of bacterial DNA in each mite DNA sample was determined by quantitative PCR using an internal standard, and sequence homologies were determined from amplifications performed by using a high-fidelity DNA polymerase. Results DNA from D farinae appeared to contain between 11-fold and 24-fold more 16S ribosomal gene copies than the genomic DNA from D pteronyssinus ( P ≤ .003). Sequence analysis indicated the dominant presence of at least 3 phylogenetic clusters of Bartonella species ( henselae , quintana , vinsonii , and grahamii ), as well as uncharacterized α-proteobacteria, from both D farinae and D pteronyssinus . In a few clones, sequences from Escherichia coli , Pseudomonas species, and Acinetobacter species were also identified. Conclusion House dust mite DNA contains evidence of Bartonella and other Gram-negative species. These Gram-negative species are likely to be the sources of the endotoxin found in mite allergenic extracts.

Journal Article
TL;DR: The advent of forensic DNA analysis methods greatly increased the ability to positively identify previously unknown skeletal remains by a comparative genetic analysis with presumptive relatives.
Abstract: Aim was to present twelve-year (1993-2005) experience in identification of human remains found in mass graves in Croatia and Bosnia and Herzegovina (BH), as well as remains that presumably belonged to Croatian citizens given by Serbia and Montenegro. The unique experience of identification of more than a thousand of skeletal samples is valuable for better organization of post-mortem identifications. Standard forensic methods and methods based on DNA analysis were used for identification of human remains from mass graves. DNA was isolated using standard phenol/chloroform/isoamyl alcohol extraction. In some cases, decalcification and repurification were used prior to the extraction to overcome inhibition of amplification process. Different DNA systems were used for DNA quantitation and amplification (AluQuant, short tandem repeats (STR) commercial systems, Y chromosome STRs, and mitochondrial DNA [mtDNA]). Typing of PCR products was performed on AmpliType (R) PM and AmpliType (R) DQA1 DNA probe strips, ABI PRISM (R) 310 Genetic Analyzer and immobilized sequence-specific oligonucleotide (SSO) probes. Up-to-date analysis of 1, 155 skeletal samples resulted in 703 positively identified bodies: 577 using standard forensic methods, 109 by DNA typing, and 17 by combination of these two methods. The majority of identifications from 1993 to 1999 was, as usual, achieved by standard forensic methods. Later on, these methods were not sufficient and DNA analysis was requested. It was performed in 42% of all cases in 12 years. The crucial step in DNA analysis is extraction of genomic DNA. Standard phenol/chloroform/isoamyl alcohol extraction, complemented with other methods and modifications, proved as the most successful method for this step. In certain cases, the quality and/or quantity of nDNA was not satisfying and the analysis of the mtDNA was performed. Our experience demonstrated that the advent of forensic DNA analysis methods greatly increased our ability to positively identify previously unknown skeletal remains by a comparative genetic analysis with presumptive relatives.

Journal ArticleDOI
TL;DR: A piezoelectric biosensor for direct detection of sequences in nonamplified genomic DNA is described, which relies on real-time and label-free detection of the hybridization reaction between an immobilized probe and the complementary sequence in solution.
Abstract: Label-free and real-time DNA sequence detection in PCR-amplified DNA samples can now be achieved by different approaches. On the contrary, only few works have been reported dealing with direct sequence detection in nonamplified genomic DNA. Here, a piezoelectric biosensor for direct detection of sequences in nonamplified genomic DNA is described. The system relies on real-time and label-free detection of the hybridization reaction between an immobilized probe and the complementary sequence in solution. The DNA probe is immobilized on the sensing surface (10 MHz quartz crystals), while the complementary sequence is present in the genomic DNA, previously fragmented with restriction enzymes.