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Genome Amplification (genome + amplification)
Kinds of Genome Amplification Selected AbstractsSimultaneous detection of genetically modified organisms by multiplex ligation-dependent genome amplification and capillary gel electrophoresis with laser-induced fluorescenceELECTROPHORESIS, Issue 13 2010Virginia García-Cañas Abstract In this work, an innovative method useful to simultaneously analyze multiple genetically modified organisms is described. The developed method consists in the combination of multiplex ligation-dependent genome dependent amplification (MLGA) with CGE and LIF detection using bare-fused silica capillaries. The MLGA process is based on oligonucleotide constructs, formed by a universal sequence (vector) and long specific oligonucleotides (selectors) that facilitate the circularization of specific DNA target regions. Subsequently, the circularized target sequences are simultaneously amplified with the same couple of primers and analyzed by CGE-LIF using a bare-fused silica capillary and a run electrolyte containing 2-hydroxyethyl cellulose acting as both sieving matrix and dynamic capillary coating. CGE-LIF is shown to be very useful and informative for optimizing MLGA parameters such as annealing temperature, number of ligation cycles, and selector probes concentration. We demonstrate the specificity of the method in detecting the presence of transgenic DNA in certified reference and raw commercial samples. The method developed is sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 1% of GA21, 1% of MON863, and 1% of MON810 in maize samples with signal-to-noise ratios for the corresponding DNA peaks of 15, 12, and 26, respectively. These results demonstrate, to our knowledge for the first time, the great possibilities of MLGA techniques for genetically modified organisms analysis. [source] Genome-wide amplification and allelotyping of sporadic pituitary adenomas identify novel regions of genetic lossGENES, CHROMOSOMES AND CANCER, Issue 3 2003D. J. Simpson Through the use of a candidate gene approach, several previous studies have identified loss of heterozygosity (LOH) at putative tumor-suppressor gene (TSG) loci in sporadic pituitary tumors. This study reports a genome-wide allelotyping by use of 122 microsatellite markers in a large cohort of tumors, consisting of somatotrophinomas and non-functioning adenomas. Samples were first subject to prior whole genome amplification by primer extension pre-amplification (PEP) to circumvent limitations imposed by insufficient DNA for whole-genome analysis with this number of microsatellite markers. The overall mean frequency of loss in invasive tumors was significantly higher than that in their non-invasive counterparts (7 vs. 3% somatotrophinomas; 6 vs. 3% non-functioning adenomas, respectively). Analysis of the mean frequency of LOH, across all markers to individual chromosomal arms, identified 13 chromosomal arms in somatotrophinomas and 10 in non-functioning tumors, with LOH greater than the 99% upper confidence interval calculated for the rate of overall random allelic loss. In the majority of cases, these losses were more frequent in invasive tumors than in their non-invasive counterparts, suggesting these to be markers of tumor progression. Other regions showed similar frequencies of LOH in both invasive and non-invasive tumors, implying these to be early changes in pituitary tumorigenesis. This genome-wide study also revealed chromosomal regions where losses were frequently associated with an individual marker, for example, chromosome arm 1q (LOH > 30%). In some cases, these losses were subtype-specific and were found at a higher frequency in invasive tumors than in their non-invasive counterparts. Identification of these regions of loss provides the first preliminary evidence for the location of novel putative TSGs involved in pituitary tumorigenesis that are, in some cases, subtype-specific. This investigation provides an unbiased estimate of global aberrations in sporadic pituitary tumors as assessed by LOH analysis. The identification of multiple "hotspots" throughout the genome may be a reflection of an unstable chromatin structure that is susceptible to a deletion or epigenetic-mediated gene-silencing events. © 2003 Wiley-Liss, Inc. [source] Chromosome 9 deletions are more frequent than FGFR3 mutations in flat urothelial hyperplasias of the bladderINTERNATIONAL JOURNAL OF CANCER, Issue 5 2006Johanna M.M. van Oers Abstract Flat urothelial hyperplasias (FUHs) in patients with papillary bladder tumours frequently show deletions of chromosome 9, suggesting that FUH could be the first neoplastic step in the development of papillary bladder cancer. FGFR3 mutations are frequent in non-invasive papillary tumours with low risk of progression. Our aim was to investigate the frequency of FGFR3 mutations and deletions of chromosomes 9p/q and 8p/q in FUH. Thirty FUH and 9 simultaneous or consecutive tumours were detected by 5-ALA-based photodynamic cystoscopy. DNA was isolated from frozen sections and whole genome amplification was done by I-PEP-PCR, followed by LOH analysis on chromosomes 8p/q and 9p/q. FGFR3 mutations were detected by SNaPshot analysis. LOH analysis on FUH revealed deletions at 9p/q (11/30, 37%) and 8p/q (3/30, 10%). FGFR3 mutations were found in 7/30 FUH (23%). Only 2 FUH showed an FGFR3 mutation without deletions of chromosome 9. In contrast, 6 FUH revealed chromosome 9 deletions but wild type FGFR3 (p = 0.03). These results suggest that chromosome 9 deletions are the earliest genetic alterations in bladder cancer. The detection of FGFR3 mutations in FUH further supports the role of this lesion as precursor of papillary bladder cancer. © 2006 Wiley-Liss, Inc. [source] Prevalence, whole genome characterization and phylogenetic analysis of hepatitis B virus in captive orangutan and gibbonJOURNAL OF MEDICAL PRIMATOLOGY, Issue 6 2008Pattaratida Sa-nguanmoo Abstract Background, Hepatitis B virus (HBV) is a public health problem worldwide and apart from infecting humans, HBV has been found in non-human primates. Methods, We subjected 93 non-human primates comprising 12 species to ELISA screening for the serological markers HBsAg, antiHBs and antiHBc. Subsequently, we detected HBV DNA, sequenced the whole HBV genome and performed phylogenetic analysis. Results, HBV infection was detected in gibbon (4/15) and orangutan (7/53). HBV DNA isolates from two gibbons and seven orangutans were chosen for complete genome amplification. We aligned the Pre-S/S, Pre-C/C and entire genomes with HBV sequences and performed phylogenetic analysis. The gibbon and orangutan viruses clustered within their respective groups. Conclusions, Both geographic location and host species influence which HBV variants are found in gibbons and orangutans. Hence, HBV transmission between humans and non-human primates might be a distinct possibility and additional studies will be required to further investigate this potential risk. [source] PGD for monogenic disorders: aspects of molecular biology,PRENATAL DIAGNOSIS, Issue 1 2009Claudia Spits Abstract Preimplantation genetic diagnosis (PGD) for monogenic diseases has known a considerable evolution since its first application in the early 1990s. Especially the technical aspects of the genetic diagnosis itself, the single-cell genetic analysis, has constantly evolved to reach levels of accuracy and efficiency nearing those of genetic diagnosis on regular DNA samples. In this review, we will focus on the molecular biological techniques that are currently in use in the most advanced centers for PGD for monogenic disorders, including multiplex polymerase chain reaction (PCR) and post-PCR diagnostic methods, whole genome amplification (WGA) and multiple displacement amplification (MDA). As it becomes more and more clear that when it comes to ethically difficult indications, PGD goes further than prenatal diagnosis (PND), we will also briefly discuss ethical issues. Copyright © 2008 John Wiley & Sons, Ltd. [source] Preimplantation genetic diagnosis of Morquio diseasePRENATAL DIAGNOSIS, Issue 10 2008Wafa Qubbaj Abstract Objectives Morquio syndrome is an autosomal recessive disease and mutations in the N -acetylgalactosamine 6-sulfate sulfatase (GALNS) gene cause Morquio type A disease. Preimplantation genetic diagnosis (PGD), an early form of prenatal diagnosis for couples at risk of transmitting inherited diseases, was applied to prevent transmission of this disease. Methods A couple with three affected children, having homozygous W159C (p. Trp 159 Cys) mutation in GALNS gene, underwent in vitro fertilization (IVF) treatment and PGD. Mutation analyses from the embryos were performed following whole genome amplification of single blastomeres using multiple displacement amplification (MDA). Results Three embryos were diagnosed as normal and two were transferred on day 4. The cycle resulted in a pregnancy and a live birth of a carrier male infant. Genetic haplotyping analysis of the infant and the leftover MDA samples enabled us to determine which embryo was implanted. The discrepancy in results was explained by allele dropout (ADO) of the mutant allele from the MDA product. Conclusions A feasible strategy for PGD of Morquio disease including whole genome amplification by MDA and the use of preimplantation genetic haplotyping is described. MDA product archiving will be useful for future investigations if needed. Copyright © 2008 John Wiley & Sons, Ltd. [source] Whole genome amplification from a single cell: a new era for preimplantation genetic diagnosisPRENATAL DIAGNOSIS, Issue 4 2007Serdar Coskun Abstract Preimplantation genetic diagnosis (PGD) is a technique used for determining the genetic status of a single cell biopsied from embryos or oocytes. Genetic analysis from a single cell is both rewarding and challenging, especially in PGD. The starting material is very limited and not replaceable, and the diagnosis has to be made in a very short time. Different whole genome amplification (WGA) techniques have been developed to specifically increase the DNA quantities originating from clinical samples with limited DNA contents. In this review, currently available WGA techniques are introduced and, among them, multiple displacement amplification (MDA) is discussed in detail. MDA generates abundant assay-ready DNA to perform broad panels of genetic assays through its ability to rapidly amplify genomes from single cells. The utilization of MDA for single-cell molecular analysis is expanding at a high rate, and MDA is expected to soon become an integral part of PGD. Copyright © 2007 John Wiley & Sons, Ltd. [source] Multiple genotyping in bovine pre-implantation embryos with whole genome amplificationANIMAL SCIENCE JOURNAL, Issue 5 2008Hiroki HIRAYAMA ABSTRACT This study examined genetic diagnosis using whole genome amplification (WGA) in bovine embryos. The first experiment was conducted to compare the WGA efficiency of primer extension preamplification-PCR (PEP-PCR) and multiple displacement amplification (MDA), and to optimize the DNA extraction method. The sensitivity of SRY -specific PCR from MDA products increased when DNA of fibroblasts was extracted by a NaOH treatment instead of the conventional method (heat treatment). The detectability of SRY from PEP-PCR products was lower than that in MDA regardless of the DNA extraction method (proteinase K or NaOH treatment). Sexing and genotyping were performed using MDA products from embryo biopsy. The accuracy of PCR-based and LAMP-based sexing was 100% regardless of the amounts of biopsy. Genotyping of CL16, BND3, SCD and F11 in MDA products from 10 to 20% of trophectoderm was successful 97, 97, 95 and 95% of the time, respectively, but reduced biopsy amount (<10% of trophectoderm) decreased the accuracies (33,83%). Microsatellite markers were analyzed using MDA products from 10 to 20% of trophectoderm. In eight out of 16 microsatellites, genotypes were not contradictory among the dam, sire and embryos. In the other eight microsatellites, the inconsistency rates were 17,83%. These results indicate that MDA is useful for multiple genetic diagnoses in bovine embryos. [source] DNA Cards: Determinants of DNA Yield and Quality in Collecting Genetic Samples for Pharmacogenetic StudiesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 2 2007Sergi Mas The aims of this study were two-fold: to compare the amount and quality of DNA obtained from two different DNA cards (IsoCode Cards® or FTA Classic Cards®, Whatman plc, Brentford, Middlesex, UK); and to evaluate the effects of time and storage temperature, as well as the influence of anticoagulant ethylenediaminetetraacetic acid on the DNA elution procedure. The samples were genotyped by several methods typically used in pharmacogenetic studies: multiplex PCR, PCR-restriction fragment length polymorphism, single nucleotide primer extension, and allelic discrimination assay. In addition, they were amplified by whole genome amplification to increase genomic DNA mass. Time, storage temperature and ethylenediaminetetraacetic acid had no significant effects on either DNA card. This study reveals the importance of drying blood spots prior to isolation to avoid haemoglobin interference. Moreover, our results demonstrate that re-isolation protocols could be applied to increase the amount of DNA recovered. The samples analysed were accurately genotyped with all the methods examined herein. In conclusion, our study shows that both DNA cards, IsoCode Cards® and FTA Classic Cards®, facilitate genetic and pharmacogenetic testing for routine clinical practice. [source] Whole genome amplification using single-primer PCRBIOTECHNOLOGY JOURNAL, Issue 3 2008Kaiqin Lao Dr. Abstract Comprehensive genomic molecular analyses require relatively large DNA amounts that are often not available from forensic, clinical and other crucial biological samples. Numerous methods to amplify the whole genome have been proposed for cancer, forensic and taxonomic research. Unfortunately, when using truly random primers for the initial priming step, all of these procedures suffer from high background problems for sub-nanogram quantities of input DNA. Here we report an approach to eliminate this problem for PCR-based methods even at levels of DNA approaching that of a single cell. [source] 4261: FNA biopsies for genomic analysis and adjuvant therapy for uveal melanomaACTA OPHTHALMOLOGICA, Issue 2010L DESJARDINS Purpose Recent changes in the management of uveal melanoma include the use of biopsies for genomic analysis and the identification of patients with a high risk of metastasis. We wish to describe our first experience with fine needle aspiration (FNA), genome profiling and adjuvant therapy protocol for high risk patients Methods we have started a multicentric adjuvant phase III trial of intravenous fotemustine (FOTEADJ) for high risk uveal melanoma patients. Patients with tumor of 15 mm or more in diameter with retinal detachment or extrascleral extension, patients with tumors of 18 mm or more in diameter and patients with loss of chromosome 3 and gain of entire 8q were considered high risk and eligible. Tumour genome profiling was achieved by array-CGH on a NimbleGen 72K microarray, after whole genome amplification (WGA) in cases of FNAs Local treatment consisted in enucleation or proton beam radiotherapy. FNA was offered to patients treated by radiotherapy for a tumor of 5 mm of thickness or more. Results Between May 2009 and May 2010, 74 patients were offered to participate. Only 16 patients were included because of various reasons: technical problems with the biopsy (13 samples evaluable out of 26 FNA), refusal of the biopsy or the protocol or non inclusion criteria. There has been some improvement in our results since the introduction of WGA for FNA specimens Conclusion Proposing fine needle aspiration biopsy and obtaining sufficient material is not always easy. Including patients in randomized protocols is always a challenge. During the first year for FOTEADJ, only 22% of the eligible patients were enrolled but this percentage is greatly improving with time and experience . [source] | |||||||||||||