Characterisation of Rhizobium isolates by amplification of DNA polymorphisms using random primers

1992 ◽  
Vol 38 (10) ◽  
pp. 1009-1015 ◽  
Author(s):  
Stephen P. Harrison ◽  
Lance R. Mytton ◽  
Leif Skøt ◽  
Malcolm Dye ◽  
Ann Cresswell
Keyword(s):  
Rhizobium Leguminosarum ◽  
Gc Content ◽  
Dna Amplification ◽  
Thermal Reaction ◽  
Dna Polymorphisms ◽  
Sequence Information ◽  
Target Sequence ◽  
Random Primers ◽  
Random Priming ◽  
Genetic Structures

The use of single random primers, selected in the absence of target sequence information, has been shown to be effective in producing DNA amplifications that provide fingerprints which are unique to individual organisms. DNA amplification by random priming was applied to the DNA from isolates of Rhizobium leguminosarum biovar trifolii. Amplification products were produced using a number of primers, and the resulting fingerprints allowed strain differentiation. However, the effectiveness of primers was dependent upon length and GC content. It was also possible to amplify DNA directly from cells in culture and in nodule tissue. Lysis of these cells was achieved simply through heat applied in the initial DNA denaturation stage of the thermal reaction. The ability to produce varied amplification patterns from different Rhizobium isolates, especially directly from nodules, gives this method potential for use in examining genetic structures and relationships in Rhizobium populations. Key words: Rhizobium, DNA amplification, random primers.

Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species

Genome ◽  
2004 ◽  
Vol 47 (2) ◽  
pp. 281-291 ◽  
Author(s):  
Mauro Bardini ◽  
David Lee ◽  
Paolo Donini ◽  
Anna Mariani ◽  
Silvia Gianì ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Gene Family ◽  
Dna Amplification ◽  
Plant Genome ◽  
Dna Polymorphisms ◽  
Sequence Information ◽  
Genome Origins ◽  
Primary Amino ◽  
Cultivated Species ◽  
Β Tubulin

TBP (tubulin-based polymorphism) is a new molecular marker based tool that relies on the presence of intron-specific DNA polymorphisms of the plant β-tubulin gene family. The multifunctional and essential role of the tubulin proteins is reflected in the conservation of regions within their primary amino acid sequence. The ubiquitous nature of this gene family can be exploited using primers that amplify the first intron of different β-tubulin isotypes, revealing specific fingerprints. The method is rapid, simple, and reliable and does not require preliminary sequence information of the plant genome of interest. The ability of TBP to discriminate between accessions and species in oilseed rape, coffee, and lotus is shown. In all cases, TBP was able to detect specific genetic polymorphisms in the context of a simplified and readily appreciable pattern of DNA amplification. The application of TBP for assessing genetic diversity and genome origins in disseminated plant landraces rather than in highly inbred cultivated species is also discussed.Key words: β-tubulin, Brassica napus, Coffea, Lotus, SSRs.

scholarly journals Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR

2016 ◽  
Vol 82 (10) ◽  
pp. 3022-3031 ◽  
Author(s):  
Ayako Fujiwara ◽  
Katsuhiro Kawato ◽  
Saori Kato ◽  
Kiyoshi Yasukawa ◽  
Ryota Hidese ◽  
...  
Keyword(s):  
Noise Reduction ◽  
Dna Polymerase ◽  
Biological Activities ◽  
Genetic Diagnosis ◽  
Gc Content ◽  
Dna Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Content Type ◽  
Thermococcus Kodakarensis

ABSTRACTDNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, theEuryarchaeota-specific helicase EshA (TK0566) from the hyperthermophilic archaeonThermococcus kodakarensis(Tk-EshA) was obtained as a recombinant form, and its enzymatic properties were examined.Tk-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5′ overhung, 3′ overhung, and blunt end) at 50°C.Tk-EshA unwound forked and 3′ overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers whenTk-EshA was added to a PCR mixture. To examine the effect ofTk-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence ofTk-EshA. In contrast, noise DNAs were eliminated in the presence ofTk-EshA. Noise reduction byTk-EshA was also confirmed whenTaqDNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, α type) were used for PCR. Misamplified bands were also eliminated duringtoxAgene amplification fromPseudomonas aeruginosaDNA, which possesses a high GC content (69%).Tk-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs duringtoxAamplification.Tk-EshA is a useful tool to reduce noise DNAs for accurate PCR.IMPORTANCEPCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostableEuryarchaeota-specific helicase (Tk-EshA) was included in the PCR mixture. The addition ofTk-EshA has reduced noise DNAs in PCR.

Phylogenetic grouping and identification of Rhizobium isolates on the basis of random amplified polymorphic DNA profiles

1993 ◽  
Vol 39 (7) ◽  
pp. 665-673 ◽  
Author(s):  
John J. Dooley ◽  
Stephen P. Harrison ◽  
Lance R. Mytton ◽  
Malcolm Dye ◽  
Ann Cresswell ◽  
...  
Keyword(s):  
Rhizobium Leguminosarum ◽  
Rhizobium Meliloti ◽  
Random Amplified Polymorphic Dna ◽  
Dna Amplification ◽  
Distinct Group ◽  
Principal Coordinate Analysis ◽  
The Other ◽  
Phylogenetic Grouping ◽  
Dna Profiles ◽  
Selection Of

Through the use of a single, random 15mer as a primer, between 1 and 12 DNA amplification products were obtained per strain from a selection of 84 Rhizobium and Bradyrhizobium isolates. A principal-coordinate analysis was used to analyse the resulting amplified DNA profiles and it was possible to assign isolates to specific groupings. Within the species Rhizobium leguminosarum, the biovar phaseoli formed a distinct group from the other biovars of the species, viciae and trifolii, which grouped together. Isolates of Rhizobium meliloti and Bradyrhizobium species formed their own clear, specific groups. Although it was possible to identify individual isolates on the basis of differences in their amplified DNA profiles, there was evidence that some amplified segments were conserved among individuals at the biovar and species levels.Key words: Rhizobium, DNA amplification, random primers.

A stydy of Rhizobium leguminosarum biovar trifolii populations from soil extracts using randomly amplified polymorphic DNA profiles

1995 ◽  
Vol 41 (4-5) ◽  
pp. 336-344 ◽  
Author(s):  
Malcolm Dye ◽  
Leif Sket ◽  
Lance R. Mytton ◽  
Ann Cresswell ◽  
Stephen P. Harrison ◽  
...  
Keyword(s):  
Rhizobium Leguminosarum ◽  
Rapd Analysis ◽  
Dna Amplification ◽  
Differential Centrifugation ◽  
Randomly Amplified Polymorphic Dna ◽  
Phenotypic Characteristics ◽  
Soil Extracts ◽  
Dna Profiles ◽  
Rapd Fragment ◽  
Rhizobium Leguminosarum Biovar Trifolii

This study has shown that isolates of Rhizobium leguminosarum biovar trifolii can be grouped on the basis of their randomly amplified polymorphic DNA (RAPD) fragment patterns. Evidence is presented that these groups are not entirely arbitrary but are consistent with other genetic and phenotypic characteristics. RAPD analysis has been used to assess the efficiency of a dispersion and differential centrifugation procedure used to extract bacteria from soil. Whilst the major groups of Rhizobium isolates in soils were also found in the extracts, some of the others were missing. This is also reflected in the finding, made by measuring abundance of organisms, that as many Rhizobium isolates are left in the residue as appear in the supernatant; more dispersion steps, possibly with different dispersants, are needed to maximize extraction. The technique has also demonstrated that dispersing soil by simply shaking it in water not only underestimates the numbers of Rhizobium isolates present but also masks much of their diversity.Key words: Rhizobium, DNA amplification, random primers, bacterial extraction.

scholarly journals Custom hereditary breast cancer gene panel selectively amplifies target genes for reliable variant calling

2018 ◽  
Author(s):  
Setor Amuzu ◽  
Timothée Revil ◽  
William D. Foulkes ◽  
Jiannis Ragoussis
Keyword(s):  
Breast Cancer ◽  
Hereditary Breast Cancer ◽  
Target Genes ◽  
Gc Content ◽  
Variant Calling ◽  
Read Depth ◽  
Target Sequence ◽  
Target Enrichment ◽  
Target Capture ◽  
Cancer Panel

AbstractBackground: Target enrichment coupled with next generation sequencing provide high-throughput approaches for screening several genes of interest. These approaches facilitate screening a panel of genes for mutations associated with inherited breast cancer for research, diagnostic, and genetic counseling applications.Objective: To evaluate the performance of our custom 13 gene breast cancer panel, based on singleplex PCR, developed by WaferGen BioSystems. The panel was evaluated using patient-derived DNA samples, in terms of target enrichment efficiency, off-target enrichment, uniformity of target capture, effect of GC content of target regions on coverage depth, and concordance with validated variant calls.Results: At least 90% of target sequence for each gene was captured at 30x or greater. We evaluated uniformity of target capture across samples by calculating the percentage of samples with at least 90% of total target captured at 100x or greater and found 92% (33/36 samples) uniformity for our panel. Off-target enrichment ranges between 7.2% and 22.3%. We found perfect concordance between our custom panel and the Qiagen human breast cancer panel for functionally annotated variant calls in high read depth shared target regions. Altogether, there was agreement between the panels for 779 variants at 41 loci. We also confirmed 10 pathogenic mutations, initially discovered by Sanger sequencing, in the appropriate samples following target enrichment using our custom WaferGen panel.Conclusion: Our custom hereditary breast cancer panel is sensitive to the desired target genes and facilitates deep sequencing for reliable variant calling.

scholarly journals Evaluation and Application of an Efficient Plant DNA Extraction Protocol in Laboratory and Field Testing

2020 ◽  
Author(s):  
Qi Wang ◽  
Xiaoxia Shen ◽  
Tian Qiu ◽  
Wei Wu ◽  
Zhian Wang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Molecular Identification ◽  
Filter Paper ◽  
Biological Samples ◽  
Gc Content ◽  
Dna Amplification ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Paper Strip ◽  
Cellulose Filter

Abstract Background CTAB has been considered as the standard protocol for DNA extraction. But the complex and time-consuming procedures can’t meet the needs of rapid molecular identification. The method of using cellulose filter paper strips to transfer the DNA in the plant tissue lysate to the nucleic acid amplification system shortening the DNA extraction time to 30 s. However, cellulose filter paper strips have some shortcomings that cannot be put into widespread use. And the data supporting the rapidly purification of DNA by cellulose filter paper is not sufficient.ResultsIn the study, the published filter paper strip was modified by sticking the filter paper on the PVC sheet. This modified method is named EZ-D, for easy DNA extraction. Compared with the original method, the DNA extracted by EZ-D is more efficient in PCR amplification. We also came up with a new DNA extraction buffer, which exhibited higher DNA extraction efficiency. When compared with classic CTAB, EZ-D also showed great advantages for higher efficiency, easier protocol and lower cost. PCR analyses showed that DNA extracted from several types of plants by EZ-D were appropriate for specific identification of biological samples. PCR using DNA extracted by EZ-D was sensitive enough to detect 0.1 ng/μL. Evaluation of the EZ-D showed that the DNA extracts can be successfully amplified by PCR reaction for the DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved in an equipment-free way. Conclusion Combined with DNA amplification technology, EZ-D protocol is a rapid, specific and sensitive method for molecular identification of plant samples. In addition, EZ-D method is the first application of cellulose filter paper in the identification of genetically modified crops and traditional Chinese medicine ultra-fine powder. In terms of practicability, EZ-D has realized the popularization of cellulose filter paper for rapid DNA purification in laboratories and markets.

DNA microarrays: a bridge between genome sequence information and biological understanding

2002 ◽  
Vol 10 (3) ◽  
pp. 389-408 ◽  
Author(s):  
KEITH HARSHMAN ◽  
CARLOS MARTÍNEZ-A
Keyword(s):  
Large Scale ◽  
Dna Microarrays ◽  
Expression Patterns ◽  
Dna Polymorphisms ◽  
Model Organisms ◽  
Sequence Information ◽  
Single Experiment ◽  
Cellular Processes ◽  
Large Scale Analysis ◽  
Sequence Variations

The development, refinement and increasingly widespread use of DNA microarrays have been important responses to the explosion of sequence information produced by genome science. The high sample densities possible with DNA microarrays, coupled with the complete or nearly complete genome sequences available for humans and model organisms, provide a powerful analytical method to measure both qualitative and quantitative variations in RNA and DNA. Principal among the applications of microarrays is the large-scale analysis of RNA expression, often referred to as expression profiling. The power of this application lies in its ability to determine the expression patterns of tens of thousands of genes in a single experiment. Additionally, the ability to detect DNA polymorphisms makes microarrays useful in studies designed to correlate DNA sequence variations with variations in phenotype. The unprecedented scale on which microarrays allow both experimentation and generation of results should make possible a more complete and comprehensive understanding of cells and cellular processes.

scholarly journals A random priming amplification method for whole genome sequencing of SARS-CoV-2 and H1N1 influenza A virus.

2021 ◽  
Author(s):  
Klaudia Chrzastek ◽  
Chandana Tennakoon ◽  
Dagmara Bialy ◽  
Graham L Freimanis ◽  
John Flannery ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Assembly ◽  
Influenza A ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Sequence Information ◽  
Whole Genome ◽  
Random Priming ◽  
Influenza A H1n1

Background: Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. Methods: The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. Results: The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of COVID that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. Conclusions: The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.

scholarly journals Comparativein silicoanalysis offtsZgene from different bacteria reveals the preference for core set of codons in coding sequence structuring and secondary structural elements determination

2019 ◽  
Author(s):  
Ayon Pal ◽  
Barnan Kr Saha ◽  
Jayanti Saha
Keyword(s):  
Bacterial Species ◽  
Gc Content ◽  
Gene Organization ◽  
Sequence Information ◽  
Antibacterial Drug ◽  
Bacterial Cell Division ◽  
Bacterial Gene ◽  
Coding Sequence ◽  
Ftsz Protein ◽  
Core Set

AbstractThe deluge of sequence information in the recent times provide us with an excellent opportunity to compare organisms on a large genomic scale. In this study we have tried to decipher the variation in the gene organization and structuring of a vital bacterial gene calledftsZwhich codes for an integral component of the bacterial cell division, the FtsZ protein. FtsZ is homologous to tubulin protein and has been found to be ubiquitous in eubacteria. FtsZ is showing increasing promise as a target for antibacterial drug discovery. Our study offtsZprotein from 143 different bacterial species spanning a wider range of morphological and physiological type demonstrates that theftsZgene of about ninety three percent of the organisms involved in our analyses show relatively biased codon usage profile and significant GC deviation from their genomic GC content. We have also detected a tendency among the different organisms to utilize a core set of codons in structuring theftsZcoding sequence. Our meticulous analysis of theftsZgene linked with the corresponding FtsZ protein show that there is a bias towards the use of specific synonymous codons particularly in the helix and strand regions of the multi-domain FtsZ protein. Overall our findings suggest that in an indispensable and vital protein such as FtsZ, there is an inherent tendency to maintain form and structure for optimized performance in spite of the extrinsic variability in coding features.

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