Automated High-Throughput Purification of PCR Products Using Wizard®MagneSil™ Paramagnetic Particles

2002 ◽  
Vol 7 (6) ◽  
pp. 120-124
Author(s):  
P Otto
Keyword(s):  
High Throughput ◽  
Pcr Products

scholarly journals High-Throughput Sequencing Reveals Cyclamen persicum Mill. as a Natural Host for Fig Mosaic Virus

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 684 ◽  
Author(s):  
Toufic Elbeaino ◽  
Armelle Marais ◽  
Chantal Faure ◽  
Elisa Trioano ◽  
Thierry Candresse ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Small Scale ◽  
Rt Pcr ◽  
Cyclamen Persicum ◽  
Sequence Variations ◽  
Pcr Products ◽  
Complete Sequences ◽  
Fig Mosaic Virus

In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen (Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum, a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.

scholarly journals High-Throughput Transposon Mutagenesis of Corynebacterium glutamicum and Construction of a Single-Gene Disruptant Mutant Library

2006 ◽  
Vol 72 (5) ◽  
pp. 3750-3755 ◽  
Author(s):  
Nobuaki Suzuki ◽  
Naoko Okai ◽  
Hiroshi Nonaka ◽  
Yota Tsuge ◽  
Masayuki Inui ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
High Throughput ◽  
Single Gene ◽  
Dna Amplification ◽  
Open Reading Frames ◽  
Thermal Asymmetric Interlaced Pcr ◽  
Transposon Insertion ◽  
Cellular Life ◽  
Pcr Products ◽  
Tail Pcr

ABSTRACT A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

High-throughput sequencing of kDNA amplicons for the analysis ofLeishmaniaminicircles and identification of Neotropical species

Parasitology ◽  
2017 ◽  
Vol 145 (5) ◽  
pp. 585-594 ◽  
Author(s):  
ARTHUR KOCHER ◽  
SOPHIE VALIÈRE ◽  
ANNE-LAURE BAÑULS ◽  
JÉRÔME MURIENNE
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Pcr Primers ◽  
Genomic Region ◽  
Kinetoplast Dna ◽  
Sequence Comparisons ◽  
Conserved Sequence ◽  
Species Complexes ◽  
Pcr Products ◽  
Minicircle Sequence

SUMMARYLeishmaniakinetoplast DNA contains thousands of small circular molecules referred to as kinetoplast DNA (kDNA) minicercles. kDNA minicircles are the preferred targets for sensitiveLeishmaniadetection, because they are present in high copy number and contain conserved sequence blocks in which polymerase chain reaction (PCR) primers can be designed. On the other hand, the heterogenic nature of minicircle networks has hampered the use of this peculiar genomic region for strain typing. The characterization ofLeishmaniaminicirculomes used to require isolation and cloning steps prior to sequencing. Here, we show that high-throughput sequencing of single minicircle PCR products allows bypassing these laborious laboratory tasks. The 120 bp long minicircle conserved region was amplified by PCR from 18Leishmaniastrains representative of the major species complexes found in the Neotropics. High-throughput sequencing of PCR products enabled recovering significant numbers of distinct minicircle sequences from each strain, reflecting minicircle class diversity. Minicircle sequence analysis revealed patterns that are congruent with current hypothesis ofLeishmaniarelationships. Then, we show that a barcoding-like approach based on minicircle sequence comparisons may allow reliable identifications ofLeishmaniaspp. This work opens up promising perspectives for the study of kDNA minicercles and a variety of applications inLeishmaniaresearch.

scholarly journals Metagenomic Analysis of Subgingival Microbiota Following Non-Surgical Periodontal Therapy: a Pilot Study

2012 ◽  
Vol 6 (1) ◽  
pp. 255-261 ◽  
Author(s):  
Theresia Laksmana ◽  
Weerayuth Kittichotirat ◽  
Yanyan Huang ◽  
Weizhen Chen ◽  
Michael Jorgensen ◽  
...  
Keyword(s):  
16S Rdna ◽  
High Throughput ◽  
Pcr Amplification ◽  
Aggressive Periodontitis ◽  
Periodontal Therapy ◽  
Oral Microbiome ◽  
Metagenomic Analysis ◽  
Ribosomal Database Project ◽  
Pcr Products ◽  
Subgingival Microbiota

This study tested the feasibility of a high throughput metagenomic approach to analyze the pre- and posttreatment of subgingival plaque in two subjects with aggressive periodontitis. DNA was extracted from subgingival samples and subjected to PCR amplification of the c2-c4 regions of the 16S rDNA using primers with bar codes to identify individual samples. The PCR products were pooled and sequenced for the v4 region of the 16S rDNA using the 454 FLX standard platform. The results were analyzed for species/phylotypes in the Human Oral Microbiome Database (HOMD) and Ribosomal Database Project (RDP) database. The sequencing of the amplicons resulted in 24,673 reads and identified 208 species/phylotypes. Of those, 129 species/phylotypes were identified in both patients but their proportions varied. While >120 species/phylotypes were identified in all samples, 28-42 species/phylotypes cumulatively represent 90% of all subgingival bacteria in each sample. The remaining species/phylotypes each constituted ≤0.2% of the total subgingival bacteria. In conclusion, the subgingival microbiota are characterized by high species richness dominated by a few species/ phylotypes. The microbiota changed after periodontal therapy. High throughput metagenomic analysis is applicable to assess the complexity and changes of the subgingival microbiota.

High-Throughput Genetic Profiling of Acute Myeloid Leukemias: Novel Mutations Detected in Signal Transduction Pathway Genes by a Strategy Utilizing Endonuclease-Based Heteroduplex Scanning and DNA Sequencing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1447-1447
Author(s):  
Stan L. Lilleberg ◽  
Jill Hempel ◽  
Jeff Durocher ◽  
Damien Foster ◽  
Christopher Hartman ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Dna Sequencing ◽  
High Throughput ◽  
Somatic Mutations ◽  
Signal Transduction Pathway ◽  
Single Type ◽  
Beta Catenin ◽  
Transduction Pathway ◽  
Mutation Scanning ◽  
Pcr Products

Abstract The Raf/MEK/ERK, Wnt/beta-catenin, JAK/STAT and PI3K/Akt signal transduction pathways have key roles in regulation of cell cycle progression and apoptosis, and are current focal points of therapeutic development and intervention strategies for hematopoietic neoplasias. Although mutations in several pathway-associated genes known to contribute to the malignant phenotype have been described, the mutation status of genes encoding components of these pathways as a whole remains to be determined. This has yet to be achieved in even a single type of cancer. In this study, we applied a novel mutation scanning strategy (HTMS) that utilizes SURVEYOR Endouclease heteroduplex cleavage analysis of patient derived PCR products, along with selective fluorescent DNA sequencing in a combinatorial fashion to achieve high-throughput, accuracy, and sensitivity. Comprehensive screening of the protein coding portions of 33 signal transduction pathway genes was performed on PCR products from 192 AML samples and 48 controls. Target genes included receptor kinases (FLT3, KIT, CSF1R, FGFR3, NOTCH1), cytoplasmic kinases (ABL1, SRC, PIK3CA, JAK2), GTPases (KRAS, NRAS, HRAS), transcription factors (MYC, GATA1, CEBPA) and tumor suppressors (PTEN, P53). The Raf/MEK/ERK (genes: BRAF, ARAF, CRAF, PTPN11, MEK1/2, ERK1/2) and Wnt/beta-catenin (genes: APC, CTNNB1, CDH1, AXIN1, AXIN2, GSK3B, PP2A) pathways were emphasized. Using this high-throughput mutation scanning (HTMS) methodology, over 10 million base pairs from 192 cancer genomes and 48 control genomes was analyzed for somatic alterations and inherited polymorphisms in these genes. In the AML sample set, we identified 393 somatic mutations (2.0 mutations/AML), of which 54 (13.7%) have not been previously reported. These novel variants included point mutations in key functional domains of FLT3, KIT, NRAS, BRAF, ARAF, PTPN11, ABL1, FGFR3, MYC, NOTCH1, APC, CTNNB1, and GSK3B. At least one mutation was found in 100% of the AML samples. The relevance of these somatic mutations to the AML pathogenesis awaits detailed functional studies. The approach demonstrated in this study represents an effective in-depth mining strategy for high-throughput mutation analysis, compared to the standard re-sequencing approach for profiling of complex genetic diseases like cancer. The HTMS approach alleviates the current bottleneck of rigorous, manual sequence analysis that is required to identify somatic mutations. By integrating mutational profiling with comparative genomic hybridization, high-density SNP screening, and RNA profiling, many genetic changes relevant to cancer diagnosis, treatment and patient management, will surely be discovered, along with new targets for therapeutic intervention.

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